Validation of a real-time reverse transcriptase-PCR assay for the detection of H7 avian influenza virus

USGS Publications Warehouse

Pedersen, J.; Killian, M.L.; Hines, N.; Senne, D.; Panigrahy, B.; Ip, Hon S.; Spackman, Erica

2010-01-01

This report describes the validation of an avian influenza virus (AIV) H7 subtype-specific real-time reverse transcriptasePCR (rRT-PCR) assay developed at the Southeast Poultry Research Laboratory (SEPRL) for the detection of H7 AI in North and South American wild aquatic birds and poultry. The validation was a collaborative effort by the SEPRL and the National Veterinary Services Laboratories. The 2008 H7 rRT-PCR assay detects 101 50% embryo infectious doses per reaction, or 103104 copies of transcribed H7 RNA. Diagnostic sensitivity and specificity were estimated to be 97.5% and 82.4%, respectively; the assay was shown to be specific for H7 AI when tested with >270 wild birds and poultry viruses. Following validation, the 2008 H7 rRT-PCR procedure was adopted as an official U.S. Department of Agriculture procedure for the detection of H7 AIV. The 2008 H7 assay replaced the previously used (2002) assay, which does not detect H7 viruses currently circulating in wild birds in North and South America. ?? 2010 American Association of Avian Pathologists.

What assay is optimal for the diagnosis of measles virus infection? An evaluation of the performance of a measles virus real-time reverse transcriptase PCR using the Cepheid SmartCycler(®) and antigen detection by immunofluorescence.

PubMed

Chua, Kyra Y L; Thapa, Kiran; Yapa, Chaturangi M; Somerville, Lucy K; Chen, Sharon C-A; Dwyer, Dominic E; Sheppeard, Vicky; Kok, Jen

2015-09-01

Despite the World Health Organization (WHO)-reported elimination of measles in Australia, importation of cases especially in travellers from Asia continues in Sydney, Australia's largest city. Laboratory confirmation supports clinico-epidemiological evidence of measles virus infection, and is needed to establish elimination. To evaluate the performance of a random access real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay using the moderate complexity SmartCycler(®) platform, and measles antigen detection by immunofluorescence (IFA), for the detection of measles virus in patient samples. One hundred samples comprising nose and throat swabs, nasopharyngeal aspirates and urine, collected from patients with suspected measles were tested in parallel using IFA and nucleic acid testing using the SmartCycler(®) and LightCycler(®) RT-PCR platforms. The LightCycler(®) RT-PCR was used as the reference assay against which the SmartCycler(®) RT-PCR and IFA were compared. Using the LightCycler(®) RT-PCR, measles virus was detected in 35 clinical samples. There was 100% concordance between the results of the SmartCycler(®) and the LightCycler(®)-based RT-PCR. Measles genotypes detected included B3, D8, and D9. Testing urine in addition to NTS did not improve diagnostic yield. In contrast, the sensitivity and specificity of IFA compared to the reference LightCycler(®) RT-PCR was 34.3% and 96.7%, respectively. The performance of the SmartCycler(®) is comparable to the LightCycler(®) for the detection of measles virus. However, IFA had poor sensitivity and should not be used to confirm measles virus infection where nucleic acid testing is available. Copyright © 2015 Elsevier B.V. All rights reserved.

Measurement of gene expression in archival paraffin-embedded tissues: development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay.

PubMed

Cronin, Maureen; Pho, Mylan; Dutta, Debjani; Stephans, James C; Shak, Steven; Kiefer, Michael C; Esteban, Jose M; Baker, Joffre B

2004-01-01

Throughout the last decade many laboratories have shown that mRNA levels in formalin-fixed and paraffin-embedded (FPE) tissue specimens can be quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) techniques despite the extensive RNA fragmentation that occurs in tissues so preserved. We have developed RT-PCR methods that are sensitive, precise, and that have multianalyte capability for potential wide use in clinical research and diagnostic assays. Here it is shown that the extent of fragmentation of extracted FPE tissue RNA significantly increases with archive storage time. Probe and primer sets for RT-PCR assays based on amplicons that are both short and homogeneous in length enable effective reference gene-based data normalization for cross comparison of specimens that differ substantially in age. A 48-gene assay used to compare gene expression profiles from the same breast cancer tissue that had been either frozen or FPE showed very similar profiles after reference gene-based normalization. A 92-gene assay, using RNA extracted from three 10- micro m FPE sections of archival breast cancer specimens (dating from 1985 to 2001) yielded analyzable data for these genes in all 62 tested specimens. The results were substantially concordant when estrogen receptor, progesterone receptor, and HER2 receptor status determined by RT-PCR was compared with immunohistochemistry assays for these receptors. Furthermore, the results highlight the advantages of RT-PCR over immunohistochemistry with respect to quantitation and dynamic range. These findings support the development of RT-PCR analysis of FPE tissue RNA as a platform for multianalyte clinical diagnostic tests.

Comparison of Real-Time PCR, Reverse Transcriptase Real-Time PCR, Loop-Mediated Isothermal Amplification, and the FDA Conventional Microbiological Method for the Detection of Salmonella spp. in Produce ▿ †

PubMed Central

Zhang, Guodong; Brown, Eric W.; González-Escalona, Narjol

2011-01-01

Contamination of foods, especially produce, with Salmonella spp. is a major concern for public health. Several methods are available for the detection of Salmonella in produce, but their relative efficiency for detecting Salmonella in commonly consumed vegetables, often associated with outbreaks of food poisoning, needs to be confirmed. In this study, the effectiveness of three molecular methods for detection of Salmonella in six produce matrices was evaluated and compared to the FDA microbiological detection method. Samples of cilantro (coriander leaves), lettuce, parsley, spinach, tomato, and jalapeno pepper were inoculated with Salmonella serovars at two different levels (105 and <101 CFU/25 g of produce). The inoculated produce was assayed by the FDA Salmonella culture method (Bacteriological Analytical Manual) and by three molecular methods: quantitative real-time PCR (qPCR), quantitative reverse transcriptase real-time PCR (RT-qPCR), and loop-mediated isothermal amplification (LAMP). Comparable results were obtained by these four methods, which all detected as little as 2 CFU of Salmonella cells/25 g of produce. All control samples (not inoculated) were negative by the four methods. RT-qPCR detects only live Salmonella cells, obviating the danger of false-positive results from nonviable cells. False negatives (inhibition of either qPCR or RT-qPCR) were avoided by the use of either a DNA or an RNA amplification internal control (IAC). Compared to the conventional culture method, the qPCR, RT-qPCR, and LAMP assays allowed faster and equally accurate detection of Salmonella spp. in six high-risk produce commodities. PMID:21803916

The use of quantitative real-time reverse transcriptase PCR for 5' and 3' portions of ALK transcripts to detect ALK rearrangements in lung cancers.

PubMed

Wang, Rui; Pan, Yunjian; Li, Chenguang; Hu, Haichuan; Zhang, Yang; Li, Hang; Luo, Xiaoyang; Zhang, Jie; Fang, Zhaoyuan; Li, Yuan; Shen, Lei; Ji, Hongbin; Garfield, David; Sun, Yihua; Chen, Haiquan

2012-09-01

Approximately 3% to 7% of non-small cell lung cancers (NSCLC) harbor an ALK fusion gene, thus defining a tumor group that may be responsive to targeted therapy. The breakpoint in ALK consistently occurs at exon 20 and EML4 or other fusion partners, thus driving a strong expression of ALK kinase domain and resulting in an unbalanced expression in 5' and 3' portions of ALK transcripts. We have developed a rapid and accurate method by simultaneously detecting the expression in 5' and 3' portions of ALK mRNA. Quantitative real-time reverse transcriptase PCR (qRT-PCR) was used to examine expression levels of the 5' and 3' portions of ALK transcripts in177 NSCLCs, in which EGFR, KRAS, HER2, and BRAF mutations were absent. If unbalanced ALK mRNA expression was seen, ALK rearrangement was assumed to exist. ALK FISH was used to confirm the accuracy of qRT-PCR. RT-PCR and 5' RACE coupling sequencing identified the fusion variants. Real-time RT-PCR showed excellent sensitivity and specificity (100% and 100%, respectively) for detection of ALK rearrangements in resected specimens. In addition, six novel ALK fusion variants were identified, including one KIF5B-ALK (E17;A20) and five EML4-ALK variants (E6a;A19, E6a/b ins 18;A20, E17b ins 39;A20, E10a/b, E13;A20, and E17 ins 65;A20). Real-time RT-PCR is a rapid and accurate method for diagnosing ALK-rearranged lung cancers. Coupling of 5' RACE to this method should further facilitate rapid identification of novel ALK fusion genes. ©2012 AACR.

A duplex real-time RT-PCR assay for detecting H5N1 avian influenza virus and pandemic H1N1 influenza virus.

PubMed

Kang, Xiao-ping; Jiang, Tao; Li, Yong-qiang; Lin, Fang; Liu, Hong; Chang, Guo-hui; Zhu, Qing-yu; Qin, E-de; Qin, Cheng-feng; Yang, Yin-hui

2010-06-02

A duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was improved for simultaneous detection of highly pathogenic H5N1 avian influenza virus and pandemic H1N1 (2009) influenza virus, which is suitable for early diagnosis of influenza-like patients and for epidemiological surveillance. The sensitivity of this duplex real-time RT-PCR assay was 0.02 TCID50 (50% tissue culture infective dose) for H5N1 and 0.2 TCID50 for the pandemic H1N1, which was the same as that of each single-target RT-PCR for pandemic H1N1 and even more sensitive for H5N1 with the same primers and probes. No cross reactivity of detecting other subtype influenza viruses or respiratory tract viruses was observed. Two hundred and thirty-six clinical specimens were tested by comparing with single real-time RT-PCR and result from the duplex assay was 100% consistent with the results of single real-time RT-PCR and sequence analysis.

Comparison of four molecular assays for the detection of Tembusu virus.

PubMed

Tang, Yi; Yeh, Yin-Ting; Chen, Hao; Yu, Chunmei; Gao, Xuhui; Diao, Youxiang

2015-10-01

Tembusu virus (TMUV) belongs to the genus Flavivirus that may cause severe egg drop in ducks. In order to evaluate the most efficient TMUV detection method, the performances of a conventional RT-PCR (C-RT-PCR), a semi-nested PCR (SN-RT-PCR), a reverse-transcriptase real-time quantitative PCR (Q-RT-PCR), and a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) targeting the TMUV virus-specific NS5 gene were examined. In order to compare the sensitivity of these four techniques, two templates were used: (1) plasmid DNA that contained a partial region of the NS5 gene and (2) genomic RNA from TMUV-positive cell culture supernatants. The sensitivities using plasmid DNA detection by C-RT-PCR, SN-RT-PCR, Q-RT-PCR, and RT-LAMP were 2 × 10(4) copies/μL, 20 copies/μL, 2 copies/μL, and 20 copies/μL, respectively. The sensitivities using genomic RNA for the C-RT-PCR, SN-RT-PCR, Q-RT-PCR, and RT-LAMP were 100 pg/tube, 100, 10, and 100 fg/tube, respectively. All evaluated assays were specific for TMUV detection. The TMUV-specific RNA was detected in cloacal swabs from experimentally infected ducks using these four methods with different rates (52-92%), but not in the control (non-inoculated) samples. The sensitivities of RT-PCR, SN-RT-PCR, Q-RT-PCR, and RT-LAMP performed with cloacal swabs collected from suspected TMUV infected ducks within 2 weeks of severe egg-drop were 38/69 (55.1%), 52/69 (75.4%), 57/69 (82.6%), and 55/69 (79.7%), respectively. In conclusion, both RT-LAMP and Q-RT-PCR can provide a rapid diagnosis of TMUV infection, but RT-LAMP is more useful in TMUV field situations or poorly equipped laboratories.

Disappearance of AML1-MTG8 transcript by reverse transcriptase polymerase chain reaction in a patient in remission of acute myeloid leukemia (M2) after low-dose cytosine arabinoside.

PubMed

Sawada, H; Serino, Y; Wake, A; Yamasaki, Y; Izumi, Y

1998-09-01

It is well-known that low dose cytosine arabinoside (LDAC) has activity in elderly patients with acute myeloid leukemia (AML). Several studies have shown that AML patients with t(8;21) in long term complete remission (CR) following intensive chemotherapy or allogeneic bone marrow transplantation (BMT) still have persistence of AML1-MTG8 transcripts by reverse transcriptase polymerase chain reaction (RT-PCR) method. We report here a patient who has no evidence of residual disease detectable by RT-PCR after LDAC. A 69-year-old patient did not obtain CR after two courses of intensive chemotherapy with behenoyl-ara-C, daunorubicin, 6-mercaptopurine and prednisolone. He received subcutaneous LDAC 10 mg every 12 h and granulocyte colony-stimulating factor (G-CSF) for 29 days and achieved CR. He continued on a 21 to 28-day course of LDAC without G-CSF every 2 or 3 months and has remained well and in CR for 5 years without chimeric AMLI-MTG8 transcript by RT-PCR. LDAC therapy seems to be effective in eradicating the leukemic clone as post-induction or maintenance therapy in this patient. This is the first case report of the disappearance of AML1-MTG8 transcript by RT-PCR in a patient with t(8;21) in long-term remission after LDAC.

Comparison of FilmArray and Quantitative Real-Time Reverse Transcriptase PCR for Detection of Zaire Ebolavirus from Contrived and Clinical Specimens

PubMed Central

Southern, Timothy R.; Racsa, Lori D.; Albariño, César G.; Fey, Paul D.; Hinrichs, Steven H.; Murphy, Caitlin N.; Herrera, Vicki L.; Sambol, Anthony R.; Hill, Charles E.; Ryan, Emily L.; Kraft, Colleen S.; Campbell, Shelley; Sealy, Tara K.; Schuh, Amy; Ritchie, James C.; Lyon, G. Marshall; Mehta, Aneesh K.; Varkey, Jay B.; Ribner, Bruce S.; Brantly, Kent P.; Ströher, Ute; Iwen, Peter C.

2015-01-01

Rapid, reliable, and easy-to-use diagnostic assays for detection of Zaire ebolavirus (ZEBOV) are urgently needed. The goal of this study was to examine the agreement among emergency use authorization (EUA) tests for the detection of ZEBOV nucleic acids, including the BioFire FilmArray BioThreat (BT) panel, the FilmArray BT-E panel, and the NP2 and VP40 quantitative real-time reverse transcriptase (qRT) PCR assays from the Centers for Disease Control and Prevention (CDC). Specimens used in this study included whole blood spiked with inactivated ZEBOV at known titers and whole-blood, plasma, and urine clinical specimens collected from persons diagnosed with Ebola virus disease (EVD). The agreement for FilmArray and qRT-PCR results using contrived whole-blood specimens was 100% (6/6 specimens) for each ZEBOV dilution from 4 × 107 to 4 × 102 50% tissue culture infective dose (TCID50)/ml, as well as the no-virus negative-control sample. The limit of detection for FilmArray and qRT-PCR assays with inactivated ZEBOV, based on duplicate positive results, was determined to be 4 × 102 TCID50/ml. Rates of agreement between FilmArray and qRT-PCR results for clinical specimens from patients with EVD were 85% (23/27 specimens) for whole-blood specimens, 90% (18/20 specimens) for whole-blood specimens tested by FilmArray testing and matched plasma specimens tested by qRT-PCR testing, and 85% (11/13 specimens) for urine specimens. Among 60 specimens, eight discordant results were noted, with ZEBOV nucleic acids being detected only by FilmArray testing in four specimens and only by qRT-PCR testing in the remaining four specimens. These findings demonstrate that the rapid and easy-to-use FilmArray panels are effective tests for evaluating patients with EVD. PMID:26157148

VIRUS ISOLATION AND MOLECULAR DETECTION OF BLUETONGUE AND EPIZOOTIC HEMORRHAGIC DISEASE VIRUSES FROM NATURALLY INFECTED WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS).

PubMed

Kienzle, Clara; Poulson, Rebecca L; Ruder, Mark G; Stallknecht, David E

2017-10-01

Hemorrhagic disease in North America is caused by multiple serotypes of epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV). Diagnostic tests for detection of EHDV and BTV include virus isolation (VI), reverse transcriptase (RT)-PCR, and real-time RT-PCR (rRT-PCR). Our objective was to compare the diagnostic capabilities of three rRT-PCR protocols for detection of EHDV and BTV from naturally infected white-tailed deer (Odocoileus virginianus). We compared the effectiveness of these assays to traditional viral detection methods (e.g., VI) for historic and current clinical cases. Because of the variable nature of tissue collection and storage before diagnostic testing, an evaluation of viral persistence on multiple freeze-thaw events was also conducted. Two of the rRT-PCR assays provided for reliable detection of EHDV and BTV from 100% of clinically affected and VI-confirmed infected animals. Additionally, no significant change in viral titer was observed on multiple freeze-thaw events.

Serial detection of circulating tumour cells by reverse transcriptase-polymerase chain reaction assays is a marker for poor outcome in patients with malignant melanoma

PubMed Central

Palmieri, Giuseppe; Satriano, Sabrina MR; Budroni, Mario; Cossu, Antonio; Tanda, Francesco; Canzanella, Sergio; Caracò, Corrado; Simeone, Ester; Daponte, Antonio; Mozzillo, Nicola; Comella, Giuseppe; Castello, Giuseppe; Ascierto, Paolo A

2006-01-01

Background Detection of circulating malignant cells (CMCs) through a reverse transcriptase-polymerase chain reaction (RT-PCR) assay seems to be a demonstration of systemic disease. We here evaluated the prognostic role of RT-PCR assays in serially-taken peripheral blood samples from patients with malignant melanoma (MM). Methods One hundred forty-nine melanoma patients with disease stage ranging from I to III were consecutively collected in 1997. A multi-marker RT-PCR assay was used on peripheral blood samples obtained at time of diagnosis and every 6 months during the first two years of follow-up (total: 5 samples). Univariate and multivariate analyses were performed after 83 months of median follow-up. Results Detection of at least one circulating mRNA marker was considered a signal of the presence of CMC (referred to as PCR-positive assay). A significant correlation was found between the rate of recurrences and the increasing number of PCR-positive assays (P = 0.007). Presence of CMC in a high number (≥2) of analysed blood samples was significantly correlated with a poor clinical outcome (disease-free survival: P = 0.019; overall survival: P = 0.034). Multivariate analysis revealed that presence of a PCR-positive status does play a role as independent prognostic factors for overall survival in melanoma patients, adding precision to the predictive power of the disease stage. Conclusion Our findings indicated that serial RT-PCR assay may identify a high risk subset of melanoma patients with occult cancer cells constantly detected in blood circulation. Prolonged presence of CMCs seems to act as a surrogate marker of disease progression or a sign of more aggressive disease. PMID:17107608

Serial detection of circulating tumour cells by reverse transcriptase-polymerase chain reaction assays is a marker for poor outcome in patients with malignant melanoma.

PubMed

Palmieri, Giuseppe; Satriano, Sabrina M R; Budroni, Mario; Cossu, Antonio; Tanda, Francesco; Canzanella, Sergio; Caracò, Corrado; Simeone, Ester; Daponte, Antonio; Mozzillo, Nicola; Comella, Giuseppe; Castello, Giuseppe; Ascierto, Paolo A

2006-11-15

Detection of circulating malignant cells (CMCs) through a reverse transcriptase-polymerase chain reaction (RT-PCR) assay seems to be a demonstration of systemic disease. We here evaluated the prognostic role of RT-PCR assays in serially-taken peripheral blood samples from patients with malignant melanoma (MM). One hundred forty-nine melanoma patients with disease stage ranging from I to III were consecutively collected in 1997. A multi-marker RT-PCR assay was used on peripheral blood samples obtained at time of diagnosis and every 6 months during the first two years of follow-up (total: 5 samples). Univariate and multivariate analyses were performed after 83 months of median follow-up. Detection of at least one circulating mRNA marker was considered a signal of the presence of CMC (referred to as PCR-positive assay). A significant correlation was found between the rate of recurrences and the increasing number of PCR-positive assays (P = 0.007). Presence of CMC in a high number (> or =2) of analysed blood samples was significantly correlated with a poor clinical outcome (disease-free survival: P = 0.019; overall survival: P = 0.034). Multivariate analysis revealed that presence of a PCR-positive status does play a role as independent prognostic factors for overall survival in melanoma patients, adding precision to the predictive power of the disease stage. Our findings indicated that serial RT-PCR assay may identify a high risk subset of melanoma patients with occult cancer cells constantly detected in blood circulation. Prolonged presence of CMCs seems to act as a surrogate marker of disease progression or a sign of more aggressive disease.

Effect of saliva stabilisers on detection of porcine reproductive and respiratory syndrome virus in oral fluid by quantitative reverse transcriptase real-time PCR.

PubMed

Decorte, Inge; Van der Stede, Yves; Nauwynck, Hans; De Regge, Nick; Cay, Ann Brigitte

2013-08-01

This study evaluated the effect of extraction-amplification methods, storage temperature and saliva stabilisers on detection of porcine reproductive and respiratory syndrome virus (PRRSV) RNA by quantitative reverse transcriptase real-time PCR (qRT-PCR) in porcine oral fluid. The diagnostic performance of different extraction-amplification methods was examined using a dilution series of oral fluid spiked with PRRSV. To determine RNA stability, porcine oral fluid, with or without commercially available saliva stabilisers, was spiked with PRRSV, stored at 4°C or room temperature and tested for the presence of PRRSV RNA by qRT-PCR. PRRSV RNA could be detected in oral fluid using all extraction-amplification combinations, but the limit of detection varied amongst different combinations. Storage temperature and saliva stabilisers had an effect on the stability of PRRSV RNA, which could only be detected for 7 days when PRRSV spiked oral fluid was kept at 4°C or stabilised at room temperature with a commercial mRNA stabiliser. Copyright © 2013 Elsevier Ltd. All rights reserved.

Development of a pan-Simbu real-time reverse transcriptase PCR for the detection of Simbu serogroup viruses and comparison with SBV diagnostic PCR systems.

PubMed

Fischer, Melina; Schirrmeier, Horst; Wernike, Kerstin; Wegelt, Anne; Beer, Martin; Hoffmann, Bernd

2013-11-05

Schmallenberg virus (SBV), a novel orthobunyavirus of the Simbu serogroup, was first identified in October 2011 in dairy cattle in Germany, where it caused fever, diarrhea and a drop in milk yield. Since then, SBV additionally has been detected in adult sheep and goats. Although symptoms of acute infection were not observed, infection during a vulnerable phase of pregnancy caused congenital malformations and stillbirths. In view of the current situation and the possible emergence of further Simbu serogroup members, a pan-Simbu real-time reverse transcriptase (RT) PCR system for the reliable detection of Simbu serogroup viruses should be developed. In this study a pan-Simbu real-time RT-PCR system was established and compared to several SBV real-time RT-PCR assays. All PCR-systems were tested using a panel of different Simbu serogroup viruses as well as several field samples from diseased cattle, sheep and goats originating from all over Germany. Several pan-Simbu real-time RT-PCR products were sequenced via Sanger sequencing. Furthermore, in silico analyses were performed to investigate suitability for the detection of further orthobunyaviruses. All tested members of the Simbu serogroup (n = 14) as well as most of the field samples were successfully detected by the pan-Simbu real-time RT-PCR system. The comparison of this intercalating dye assay with different TaqMan probe-based assays developed for SBV diagnostics confirmed the functionality of the pan-Simbu assay for screening purposes. However, the SBV-TaqMan-assay SBV-S3 delivered the highest analytical sensitivity of less than ten copies per reaction for duplex systems including an internal control. In addition, for confirmation of SBV-genome detection the highly specific SBV-M1 assay was established. The pan-Simbu real-time RT-PCR system was able to detect all tested members of the Simbu serogroup, most of the SBV field samples as well as three tested Bunyamwera serogroup viruses with a suitable sensitivity. According to in silico analyses, this system seems to be able to detect a broad orthobunyavirus spectrum. As an additional feature of the pan-Simbu real-time RT-PCR system, subsequent species classification via sequencing is feasible. Regarding SBV diagnostics, the performance of the S-segment targeting SBV-S3 assay was superior with respect to the analytical sensitivity.

Prognostic role of a multigene reverse transcriptase-PCR assay in patients with node-negative breast cancer not receiving adjuvant systemic therapy.

PubMed

Esteva, Francisco J; Sahin, Aysegul A; Cristofanilli, Massimo; Coombes, Kevin; Lee, Sang-Joon; Baker, Joffre; Cronin, Maureen; Walker, Michael; Watson, Drew; Shak, Steven; Hortobagyi, Gabriel N

2005-05-01

To test the ability of a reverse transcriptase-PCR (RT-PCR) assay, based on gene expression profiles, to accurately determine the risk of recurrence in patients with node-negative breast cancer who did not receive systemic therapy using formalin-fixed, paraffin-embedded tissue. A secondary objective was to determine whether the quantitative RT-PCR data correlated with immunohistochemistry assay data regarding estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 status. We obtained archival paraffin-embedded tissue from patients with invasive breast cancer but no axillary lymph node involvement who had received no adjuvant systemic therapy and been followed for at least 5 years. RNA was extracted from three 10-microm-thick sections. The expression of 16 cancer-related genes and 5 reference genes was quantified using RT-PCR. A gene expression algorithm was used to calculate a recurrence score for each patient. We then assessed the ability of the test to accurately predict distant recurrence-free survival in this population. We identified 149 eligible patients. Median age at diagnosis was 59 years; mean tumor diameter was 2 cm; and 69% of tumors were estrogen receptor positive. Median follow-up was 18 years. The 5-year disease-free survival rate for the group was 80%. The 21 gene-based recurrence score was not predictive of distant disease recurrence. However, a high concordance between RT-PCR and immunohistochemical assays for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 status was noted. RT-PCR can be done on paraffin-embedded tissue to validate the large numbers of genes associated with breast cancer recurrence. However, further work needs to be done to develop an assay to identify the likelihood of recurrent disease in patients with node-negative breast cancer who do not receive adjuvant tamoxifen or chemotherapy.

Touch-down reverse transcriptase-PCR detection of IgV(H) rearrangement and Sybr-Green-based real-time RT-PCR quantitation of minimal residual disease in patients with chronic lymphocytic leukemia.

PubMed

Peková, Sona; Marková, Jana; Pajer, Petr; Dvorák, Michal; Cetkovský, Petr; Schwarz, Jirí

2005-01-01

Patients with chronic lymphocytic leukemia (CLL) can relapse even after aggressive therapy and autografts. It is commonly assumed that to prevent relapse the level of minimal residual disease (MRD) should be as low as possible. To evaluate MRD, highly sensitive quantitative assays are needed. The aim of the study was to develop a robust and sensitive method for detection of the clonal immunoglobulin heavy-chain variable (IgV(H)) rearrangement in CLL and to introduce a highly sensitive and specific methodology for MRD monitoring in patients with CLL who undergo intensive treatment. As a prerequisite for MRD detection, touch-down reverse transcriptase (RT)-PCR using degenerate primers were used for the diagnostic identification of (H) gene rearrangement(s). For quantitative MRD detection in 18 patients, we employed a real-time RT-PCR assay (RQ-PCR) making use of patient-specific primers and the cost-saving Sybr-Green reporter dye (SG). For precise calibration of RQ-PCR, patient-specific IgV(H) sequences were cloned. Touch-down RT-PCR with degenerate primers allowed the successful detection of IgV(H) clonal rearrangement(s) in 252 of 257 (98.1%) diagnostic samples. Biallelic rearrangements were found in 27 of 252 (10.7%) cases. Degenerate primers used for the identification of clonal expansion at diagnosis were not sensitive enough for MRD detection. In contrast, our RQ-PCR assay using patient-specific primers and SG reached the sensitivity of 10(-)(6). We demonstrated MRD in each patient tested, including four of four patients in complete remission following autologous hematopoietic stem cell transplantation (HSCT) and three of three following allogeneic 'mini'-HSCT. Increments in MRD might herald relapse; aggressive chemotherapy could induce molecular remission. Our touch-down RT-PCR has higher efficiency to detect clonal IgV(H) rearrangements including the biallelic ones. MRD quantitation of IgV(H) expression using SG-based RQ-PCR represents a highly specific, sensitive, and economic alternative to the current quantitative methods.

Combined use of reverse transcriptase polymerase chain reaction and flow cytometry to study minimal residual disease in Philadelphia positive acute lymphoblastic leukemia.

PubMed

Muñoz, L; López, O; Martino, R; Brunet, S; Bellido, M; Rubiol, E; Sierra, J; Nomdedéu, J F

2000-07-01

The Philadelphia chromosome in acute lymphoblastic leukemia (Ph+ ALL) is associated with a poor prognosis given the high frequency of chemoresistance and leukemia relapse. Minimal residual disease (MRD) detection before cytogenetic and hematologic relapse could be useful in early therapy. The most suitable methods for detecting MRD in Ph+ ALL are flow cytometry (FC) and reverse transcriptase polymerase chain reaction (RT-PCR). However, since both techniques carry the risk of false-negative results the combined use of these two techniques could overcome this problem. We report our experience using this approach in 47 bone marrow samples obtained from 10 Ph+ ALL patients. Twenty-seven marrow aspirates were taken from patients in clinical remission (CR). The samples were considered positive for MRD by FC when two conditions were met: 1) detection of an abnormal B-cell differentiation pattern and 2) presence of more than 1x10(-3) cells coexpressing CD22/CD34/CD45 or CD66/CD34/CD10. After FC analysis, RNA was purified using standard methods. FC was positive in 23/27 samples in CR (sensitivity 85%). RT-PCR was successfully performed in 23 samples in CR. RT-PCR was positive in 18/23 samples (sensitivity 78%). There were 5 samples with discordant results. FC was positive in 3 samples with a negative RT-PCR and FC was negative in 2 samples with a positive RT. All the 10 patients relapsed and only 1 is currently alive after an allogeneic stem cell transplantation (alloSCT). The median (range) time from MRD detection to relapse in patients treated with chemotherapy was 42 (39-71) days. These data suggest that RT-PCR may be negative despite the presence of neoplastic cells identified by their immunophenotypic traits. We conclude that immunologic and molecular techniques can be used in tandem for monitoring MRD in Ph+ ALL.

Two Novel Real-Time Reverse Transcriptase PCR Assays for Rapid Detection of Bacterial Contamination in Platelet Concentrates

PubMed Central

Dreier, Jens; Störmer, Melanie; Kleesiek, Knut

2004-01-01

The incidence of platelet bacterial contamination is approximately 1 per 2,000 units and has been acknowledged as the most frequent infectious risk from transfusion. In preliminary studies, the sterility of platelet concentrates (PCs) was tested with an automated bacterial blood culturing system and molecular genetic assays. Two real-time reverse transcriptase PCR (RT-PCR) assays performed in a LightCycler instrument were developed and compared regarding specificity and sensitivity by the use of different templates to detect the majority of the clinically important bacterial species in platelets. Primers and probes specific for the conserved regions of the eubacterial 23S rRNA gene or the groEL gene (encoding the 60-kDa heat shock protein Hsp60) were designed. During the development of the 23S rRNA RT-PCR, problems caused by the contamination of reagents with bacterial DNA were noted. Treatment with 8-methoxypsoralen and UV irradiation reduced the level of contaminating DNA. The sensitivity of the assays was greatly influenced by the enzyme system which was used. With rTth DNA polymerase in a one-enzyme system, we detected 500 CFU of Escherichia coli or Staphylococcus epidermidis/ml. With a two-enzyme system consisting of Moloney murine leukemia virus RT and Taq DNA polymerase, we detected 16 CFU/ml. With groEL mRNA as the target of RT-PCR under optimized conditions, we detected 125 CFU of E. coli/ml, and no problems with false-positive results caused by reagent contamination or a cross-reaction with human nucleic acids were found. Furthermore, the use of mRNA as an indicator of viability was demonstrated. Here we report the application of novel real-time RT-PCR assays for the detection of bacterial contamination of PCs that are appropriate for transfusion services. PMID:15472337

Digital-Direct-RT-PCR: a sensitive and specific method for quantification of CTC in patients with cervical carcinoma

PubMed Central

Pfitzner, Claudia; Schröder, Isabel; Scheungraber, Cornelia; Dogan, Askin; Runnebaum, Ingo Bernhard; Dürst, Matthias; Häfner, Norman

2014-01-01

The detection of circulating tumour cells (CTC) in cancer patients may be useful for therapy monitoring and prediction of relapse. A sensitive assay based on HPV-oncogene transcripts which are highly specific for cervical cancer cells was established. The Digital-Direct-RT-PCR (DD-RT-PCR) combines Ficoll-separation, ThinPrep-fixation and one-step RT-PCR in a low-throughput digital-PCR format enabling the direct analysis and detection of individual CTC without RNA isolation. Experimental samples demonstrated a sensitivity of one HPV-positive cell in 500,000 HPV-negative cells. Spike-in experiments with down to 5 HPV-positive cells per millilitre EDTA-blood resulted in concordant positive results by PCR and immunocytochemistry. Blood samples from 3 of 10 CxCa patients each contained a single HPV-oncogene transcript expressing CTC among 5 to 15*105 MNBC. Only 1 of 7 patients with local but 2 of 3 women with systemic disease had CTC. This highly sensitive DD-RT-PCR for the detection of CTC may also be applied to other tumour entities which express tumour-specific transcripts. Abbreviations: CTC – circulating tumour cells, CxCa – cervical cancer, DD-RT-PCR – Digital-Direct Reverse Transcriptase PCR, HPV – Human Papilloma Virus, MNBC – mononuclear blood cells, ICC – immunocytochemistry. PMID:24496006

Detection of infectious bronchitis virus with the use of real-time quantitative reverse transcriptase-PCR and correlation with virus detection in embryonated eggs.

PubMed

Roh, Ha-Jung; Hilt, Deborah A; Jackwood, Mark W

2014-09-01

Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assays have been used to detect the presence of challenge virus when the efficacy of infectious bronchitis virus (IBV) vaccine against field viruses is being experimentally evaluated. However, federal guidelines for licensing IBV vaccines indicate that challenge-virus detection following vaccination is to be conducted in embryonated eggs. In this study, we examined qRT-PCR data with the use of universal and type-specific primers and probe sets for IBV detection and compared those data with challenge-virus detection in embryonated eggs to determine if the two methods of evaluating vaccine efficacy are comparable. In addition, we tested the qRT-PCR assays on thermocyclers from two different manufacturers. We found the universal IBV primers and probe set to be comparable to challenge-virus detection in embryonated eggs. However, for some IBV types (Mass41 and Conn on the SmartCycler II and Ark, Mass41, Conn, and GA98 on the ABI 7500) the qRT-PCR assay was more sensitive than virus detection in embryonated eggs. This may simply be due to the universal IBV qRT-PCR assay being more sensitive than virus detection in eggs or to the assay detecting nucleic acid from nonviable virus. This finding is important and needs to be considered when evaluating challenge-virus detection for vaccination and challenge studies, because qRT-PCR could potentially identify positive birds that would otherwise be negative by virus detection in embryonated eggs; thus it could lead to a more stringent measure of vaccine efficacy. We also found that the IBV type-specific primers and probe sets designed in this study were in general less sensitive than the universal IBV primers and probe set. Only the Ark-DPI-spedcific assay on the SmartCycler II and the Ark-DPI-, Mass41-, and DE072/GA98- (for detection of GA98 virus only) specific assays on the ABI 7500 were comparable in sensitivity to virus detection in eggs. We found that a number of variables, including the virus type examined, primers and probe efficiency and stability, and assay conditions, including thermocycler platform, can affect the data obtained from qRT-PCR assays. These results indicate that qRT-PCR assays can be used to detect IBV challenge virus, but each assay, including the assay conditions and thermocycler, should be individually evaluated if those data are expected to be comparable to virus detection in embryonated eggs.

Application of Reverse Transcriptase-PCR-DGGE as a rapid method for routine determination of Vibrio spp. in foods.

PubMed

Chahorm, Kanchana; Prakitchaiwattana, Cheunjit

2018-01-02

The aim of this research was to evaluate the feasibility of PCR-DGGE and Reverse Transcriptase-PCR-DGGE techniques for rapid detection of Vibrio species in foods. Primers GC567F and 680R were initially evaluated for amplifying DNA and cDNA of ten references Vibrio species by PCR method. The GC-clamp PCR amplicons were separated according to their sequences by the DGGE using 10% (w/v) polyacrylamide gel containing 45-70% urea and formamide denaturants. Two pair of Vibrio species, which could not be differentiated on the gel, was Vibrio fluvialis - Vibrio furnissii and Vibrio parahaemolyticus - Vibrio harveyi. To determine the detection limit, in the community of 10 reference strains containing the same viable population, distinct DNA bands of 3 species; Vibrio cholerae, Vibrio mimicus and Vibrio alginolyticus were consistently observed by PCR-DGGE technique. In fact, 5 species; Vibrio cholerae, Vibrio mimicus, Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio fluvialis consistently observed by Reverse Transcriptase-PCR-DGGE. In the community containing different viable population increasing from 10 2 to 10 5 CFU/mL, PCR-DGGE analysis only detected the two most prevalent species, while RT-PCR-DGGE detected the five most prevalent species. Therefore, Reverse Transcriptase-PCR-DGGE was also selected for detection of various Vibrio cell conditions, including viable cell (VC), injured cells from frozen cultures (IVC) and injured cells from frozen cultures with pre-enrichment (PIVC). It was found that cDNA band of all cell conditions gave the same migratory patterns, except that multiple cDNA bands of Plesiomonas shigelloides under IVC and PIVC conditions were found. When Reverse Transcriptase-PCR-DGGE was used for detecting Vibrio parahaemolyticus in the pathogen-spiked food samples, Vibrio parahaemolyticus could be detected in the spiked samples containing at least 10 2 CFU/g of this pathogen. The results obtained also corresponded to standard method (USFDA, 2004). In comparison with the detection of the Vibrio profiles in fourteen food samples using standard method, Reverse Transcriptase-PCR-DGGE resulted in 100%, 75% and 50% similarity in 3, 1 and 6 food samples, respectively. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of reference genes for RT-qPCR analysis in peach genotypes with contrasting chilling requirements.

PubMed

Marini, N; Bevilacqua, C B; Büttow, M V; Raseira, M C B; Bonow, S

2017-05-25

Selecting and validating reference genes are the first steps in studying gene expression by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). The present study aimed to evaluate the stability of five reference genes for the purpose of normalization when studying gene expression in various cultivars of Prunus persica with different chilling requirements. Flower bud tissues of nine peach genotypes from Embrapa's peach breeding program with different chilling requirements were used, and five candidate reference genes based on the RT-qPCR that were useful for studying the relative quantitative gene expression and stability were evaluated using geNorm, NormFinder, and bestKeeper software packages. The results indicated that among the genes tested, the most stable genes to be used as reference genes are Act and UBQ10. This study is the first survey of the stability of reference genes in peaches under chilling stress and provides guidelines for more accurate RT-qPCR results.

Effects of anticoagulant, processing delay, and assay method (branched DNA versus reverse transcriptase PCR) on measurement of human immunodeficiency virus type 1 RNA levels in plasma.

PubMed

Kirstein, L M; Mellors, J W; Rinaldo, C R; Margolick, J B; Giorgi, J V; Phair, J P; Dietz, E; Gupta, P; Sherlock, C H; Hogg, R; Montaner, J S; Muñoz, A

1999-08-01

We conducted two studies to determine the potential influence of delays in blood processing, type of anticoagulant, and assay method on human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma. The first was an experimental study in which heparin- and EDTA-anticoagulated blood samples were collected from 101 HIV-positive individuals and processed to plasma after delays of 2, 6, and 18 h. HIV-1 RNA levels in each sample were then measured by both branched-DNA (bDNA) and reverse transcriptase PCR (RT-PCR) assays. Compared to samples processed within 2 h, the loss (decay) of HIV-1 RNA in heparinized blood was significant (P < 0.05) but small after 6 h (bDNA assay, -0.12 log(10) copies/ml; RT-PCR, -0.05 log(10) copies/ml) and after 18 h (bDNA assay, -0.27 log(10) copies/ml; RT-PCR, -0.15 log(10) copies/ml). Decay in EDTA-anticoagulated blood was not significant after 6 h (bDNA assay, -0.002 log(10) copies/ml; RT-PCR, -0.02 log(10) copies/ml), but it was after 18 h (bDNA assay, -0.09 log(10) copies/ml; RT-PCR, -0.09 log(10) copies/ml). Only 4% of samples processed after 6 h lost more than 50% (>/=0.3 log(10) copies/ml) of the HIV-1 RNA, regardless of the anticoagulant or the assay that was used. The second study compared HIV-1 RNA levels in samples from the Multicenter AIDS Cohort Study (MACS; samples were collected in heparin-containing tubes in 1985, had a 6-h average processing delay, and were assayed by bDNA assay) and the British Columbia Drug Treatment Program (BCDTP) (collected in EDTA- or acid citrate dextrose-containing tubes in 1996 and 1997, had a 2-h maximum processing delay, and were assayed by RT-PCR). HIV-1 RNA levels in samples from the two cohorts were not significantly different after adjusting for CD4(+)-cell count and converting bDNA assay values to those corresponding to the RT-PCR results. In summary, the decay of HIV-1 RNA measured in heparinized blood after 6 h was small (-0.05 to -0.12 log(10) copies/ml), and the minor impact of this decay on HIV-1 RNA concentrations in archived plasma samples of the MACS was confirmed by the similarity of CD4(+)-cell counts and assay-adjusted HIV-1 RNA concentrations in the MACS and BCDTP.

Development of a duplex real-time RT-PCR for the simultaneous detection and differentiation of Theiler's murine encephalomyelitis virus and rat theilovirus.

PubMed

Yuan, Wen; Wang, Jing; Xu, Fengjiao; Huang, Bihong; Lian, Yuexiao; Rao, Dan; Yin, Xueqin; Wu, Miaoli; Zhu, Yujun; Zhang, Yu; Huang, Ren; Guo, Pengju

2016-10-01

Theiler's murine encephalomyelitis virus (TMEV) and rat theilovirus (RTV), the member of the genus Cardiovirus, are widespread in laboratory mice and rats, and are potential contaminants of biological materials. Cardioviruses infection may cause serious complications in biomedical research. To improve the efficiency of routine screening for Cardioviruses infection, a duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for simultaneous detection and differentiation of TMEV and RTV. The duplex assay was specific for reference strains of TMEV and RTV, and no cross-reaction was found with seven other rodent viruses. The limits of detection of both TMEV and RTV were 4×10(1) copies RNA/reaction. Reproducibility was estimated using standard dilutions, with coefficients of variation <3.1%. 439 clinical samples were evaluated by both duplex real-time RT-PCR and conventional RT-PCR. For 439 clinical samples，95 samples were positive for TMEV and 72 samples were positive for RTV using duplex real-time RT-PCR approach, whereas only 77 samples were positive for TMEV and 66 samples were positive for RTV when conventional RT-PCR was applied. Mixed infections were found in 20 samples when analyzed by conventional RT-PCR whereas 30 samples were found to be mixed infection when duplex real-time RT-PCR was applied. This duplex assay provides a useful tool for routine health monitoring and screening of contaminated biological materials of these two viruses. Copyright © 2016 Elsevier B.V. All rights reserved.

Use of alternative alkali chlorides in RT and PCR of polynucleotides containing G quadruplex structures.

PubMed

Ramos-Alemán, Fabiola; González-Jasso, Eva; Pless, Reynaldo C

2018-02-15

Several alkali chlorides were compared for their use in reverse transcription (RT) and PCR of different types of nucleic acid templates. On a test region of biological DNA incapable of forming G quadruplex (G4) structures, Taq DNA polymerase showed similar PCR performance with 50 mM KCl, CsCl, LiCl, and NaCl. In contrast, on a synthetic model polydeoxyribonucleotide prone to G4 formation, good PCR amplification was obtained with 50 mM CsCl, but little or none with LiCl or KCl. Similarly, in RT of a G4-prone model polyribonucleotide, MMLV reverse transcriptase produced a good yield with 50 mM CsCl, mediocre yields with LiCl or without added alkali chloride, and a poor yield with 50 mM KCl. The full RT-PCR assay starting from the G4-prone polyribonucleotide, showed good results with CsCl in both stages, poor results with LiCl, and no product formation with KCl. The model polynucleotides showed fast G quadruplex formation under PCR or RT conditions with 50 mM KCl, but not with CsCl or LiCl. The results argue for the use of CsCl instead of KCl for RT and PCR of G4-prone sequences. No advantage was observed when using the 7-deaza type nucleotide analog c 7 dGTP in PCR amplification of the G4-prone polydeoxyribonucleotide. Copyright © 2017 Elsevier Inc. All rights reserved.

Increased yield of PCR products by addition of T4 gene 32 protein to the SMART PCR cDNA synthesis system.

PubMed

Villalva, C; Touriol, C; Seurat, P; Trempat, P; Delsol, G; Brousset, P

2001-07-01

Under certain conditions, T4 gene 32 protein is known to increase the efficiency of different enzymes, such as Taq DNA polymerase, reverse transcriptase, and telomerase. In this study, we compared the efficiency of the SMART PCR cDNA synthesis kit with and without the T4 gene 32 protein. The use of this cDNA synthesis procedure, in combination with T4 gene 32 protein, increases the yield of RT-PCR products from approximately 90% to 150%. This effect is even observed for long mRNA templates and low concentrations of total RNA (25 ng). Therefore, we suggest the addition of T4 gene 32 protein in the RT-PCR mixture to increase the efficiency of cDNA synthesis, particularly in cases when low amounts of tissue are used.

TaqMan real-time RT-PCR detection of infectious salmon anaemia virus (ISAV) from formalin-fixed paraffin-embedded Atlantic salmon Salmo salar tissues.

PubMed

Godoy, M G; Kibenge, F S; Kibenge, M J; Olmos, P; Ovalle, L; Yañez, A J; Avendaño-Herrera, R

2010-05-18

The objective of this study was to evaluate the application of a TaqMan real-time reverse transcriptase PCR (RT-PCR) assay for the detection of infectious salmon anaemia virus (ISAV) in formalin-fixed paraffin-embedded (FFPE) fish tissues from Atlantic salmon Salmo salar with and without clinical signs of infection, and to compare it with histological and immunohistochemical (IHC) techniques. Sixteen fish samples obtained in 2007 and 2008 from 4 different farms in Chile were examined. The real-time RT-PCR allowed the detection of ISAV in FFPE samples from 9 of 16 fish, regardless of the organs analyzed, whereas 4 of the real-time RT-PCR negative fish were positive as indicated by histological examination and 3 of the real-time RT-PCR positive fish were negative as indicated by immunohistochemistry evaluation. The presence of ISAV in RT-PCR positive samples was confirmed by amplicon sequencing. This work constitutes the first report on the use of real-time RT-PCR for the detection of ISAV in FFPE sections. The assay is very useful for the examination of archival wax-embedded tissues, and allows for both prospective and retrospective evaluation of tissue samples for the presence of ISAV. However, the method only confirms the presence of the pathogen and should be used in combination with histopathology, which is a more precise tool. The combination of both techniques would be invaluable for confirmatory diagnosis of infectious salmon anaemia (ISA), which is essential for solving salmon farm problems.

Molecular cloning and expression analysis of annexin A2 gene in sika deer antler tip.

PubMed

Xia, Yanling; Qu, Haomiao; Lu, Binshan; Zhang, Qiang; Li, Heping

2018-04-01

Molecular cloning and bioinformatics analysis of annexin A2 ( ANXA2 ) gene in sika deer antler tip were conducted. The role of ANXA2 gene in the growth and development of the antler were analyzed initially. The reverse transcriptase polymerase chain reaction (RT-PCR) was used to clone the cDNA sequence of the ANXA2 gene from antler tip of sika deer ( Cervus Nippon hortulorum ) and the bioinformatics methods were applied to analyze the amino acid sequence of Anxa2 protein. The mRNA expression levels of the ANXA2 gene in different growth stages were examined by real time reverse transcriptase polymerase chain reaction (real time RT-PCR). The nucleotide sequence analysis revealed an open reading frame of 1,020 bp encoding 339 amino acids long protein of calculated molecular weight 38.6 kDa and isoelectric point 6.09. Homologous sequence alignment and phylogenetic analysis indicated that the Anxa2 mature protein of sika deer had the closest genetic distance with Cervus elaphus and Bos mutus . Real time RT-PCR results showed that the gene had differential expression levels in different growth stages, and the expression level of the ANXA2 gene was the highest at metaphase (rapid growing period). ANXA2 gene may promote the cell proliferation, and the finding suggested Anxa2 as an important candidate for regulating the growth and development of deer antler.

Clinical validation of 3 commercial real-time reverse transcriptase polymerase chain reaction assays for the detection of Middle East respiratory syndrome coronavirus from upper respiratory tract specimens.

PubMed

Mohamed, Deqa H; AlHetheel, AbdulKarim F; Mohamud, Hanat S; Aldosari, Kamel; Alzamil, Fahad A; Somily, Ali M

2017-04-01

Since discovery of Middle East respiratory syndrome coronavirus (MERS-CoV), a novel betacoronavirus first isolated and characterized in 2012, MERS-CoV real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assays represent one of the most rapidly expanding commercial tests. However, in the absence of extensive evaluations of these assays on positive clinical material of different sources, evaluating their diagnostic effectiveness remains challenging. We describe the diagnostic performance evaluation of 3 common commercial MERS-CoV rRT-PCR assays on a large panel (n = 234) of upper respiratory tract specimens collected during an outbreak episode in Saudi Arabia. Assays were compared to the RealStar® MERS-CoV RT-PCR (Alton Diagnostics, Hamburg, Germany) assay as the gold standard. Results showed i) the TIB MolBiol® LightMix UpE and Orf1a assays (TIB MolBiol, Berlin, Germany) to be the most sensitive, followed by ii) the Anyplex™ Seegene MERS-CoV assay (Seegene, Seoul, Korea), and finally iii) the PrimerDesign™ Genesig® HCoV_2012 assay (PrimerDesign, England, United Kingdom). We also evaluate a modified protocol for the PrimerDesign™ Genesig® HCoV_2012 assay. Copyright © 2017 Elsevier Inc. All rights reserved.

Rapid detection of duck hepatitis A virus genotype C using reverse transcription loop-mediated isothermal amplification.

PubMed

Li, Chuanfeng; Chen, Zongyan; Meng, Chunchun; Liu, Guangqing

2014-02-01

A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was used and optimized to develop a rapid and sensitive detection system for duck hepatitis A virus genotype C (DHAV-C) RNA. A set of four specific primers was designed against highly conserved sequences located within the 3D gene from DHAV (strain GX1201). Under optimal reaction conditions, the sensitivity of DHAV-C-specific RT-LAMP was 100-fold higher than that of reverse transcriptase-polymerase chain reaction (RT-PCR), with a detection limit of 0.3pg (6.59×10(4) copies) per reaction. No cross-reactivity was observed from the samples of other duck viruses, which is in good accordance with RT-PCR. Furthermore, a positive reaction can be visually inspected by observing turbidity or color change after the addition of SYBR green I dye. The DHAV-C-specific RT-LAMP assay was applied to the samples and compared with RT-PCR. The positive-sample ratios were 26.7% (12 of 45) by RT-LAMP and 20% (9 of 45) by RT-PCR. Therefore, the newly developed RT-LAMP assay is a rapid, specific, sensitive, and cost-effective method of DHAV-C detection. This assay has potential applications in both clinical diagnosis and field surveillance of DHAV-C infection. Copyright © 2013. Published by Elsevier B.V.

Use of a novel virus inactivation method for a multicenter avian influenza real-time reverse transcriptase-polymerase chain reaction proficiency study.

PubMed

Spackman, Erica; Suarez, David L

2005-01-01

Proficiency assessments are important elements in quality control for diagnostic laboratories. Traditionally, proficiency testing for polymerase chain reaction (PCR)-based assays has involved the use of clinical samples, samples "spiked" with live agents or DNA plasmids. Because of government regulations and biosecurity concerns, distribution of live high-consequence pathogens of livestock and poultry, such as avian influenza, is not possible, and DNA plasmids are not technically suitable for evaluating RNA virus detection. Therefore, a proficiency testing panel using whole avian influenza in a diluent containing a phenolic disinfectant that inactivates the virus while preserving the RNA for at least 8 weeks at -70 C was developed and used in a multicenter proficiency assessment for a type A influenza real-time reverse transcriptase (RT)-PCR test. The test, which was highly standardized, except for variation in the real-time RT-PCR equipment used, was shown to be highly reproducible by proficiency testing in 12 laboratories in the United States, Canada, and Hong Kong. Variation in cycle threshold values among 35 data sets and 490 samples was minimal (CV = 5.19%), and sample identifications were highly accurate (96.7% correct identifications) regardless of real-time PCR instrumentation.

Molecular diagnosis of lyssaviruses and sequence comparison of Australian bat lyssavirus samples.

PubMed

Foord, A J; Heine, H G; Pritchard, L I; Lunt, R A; Newberry, K M; Rootes, C L; Boyle, D B

2006-07-01

To evaluate and implement molecular diagnostic tests for the detection of lyssaviruses in Australia. A published hemi-nested reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of all lyssavirus genotypes was modified to a fully nested RT-PCR format and compared with the original assay. TaqMan assays for the detection of Australian bat lyssavirus (ABLV) were compared with both the nested and hemi-nested RT-PCR assays. The sequences of RT-PCR products were determined to assess sequence variations of the target region (nucleocapsid gene) in samples of ABLV originating from different regions. The nested RT-PCR assay was highly analytically specific, and at least as analytically sensitive as the hemi-nested assay. The TaqMan assays were highly analytically specific and more analytically sensitive than either RT-PCR assay, with a detection level of approximately 10 genome equivalents per microl. Sequence of the first 544 nucleotides of the nucleocapsid protein coding sequence was obtained from all samples of ABLV received at Australian Animal Health Laboratory during the study period. The nested RT-PCR provided a means for molecular diagnosis of all tested genotypes of lyssavirus including classical rabies virus and Australian bat lyssavirus. The published TaqMan assay proved to be superior to the RT-PCR assays for the detection of ABLV in terms of analytical sensitivity. The TaqMan assay would also be faster and cross contamination is less likely. Nucleotide sequence analyses of samples of ABLV from a wide geographical range in Australia demonstrated the conserved nature of this region of the genome and therefore the suitability of this region for molecular diagnosis.

First report of Cocksfoot mottle virus infecting wheat (Triticum aestivum) in Ohio

USDA-ARS?s Scientific Manuscript database

Cocksfoot mottle virus (CfMV) was discovered in Ohio wheat during a 2016 field survey utilizing RNA-Seq to identify virus-like sequences. Virus sequences were confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and Sanger sequencing, and CfMV was transmitted to orchardgrass and pas...

The role of pleural fluid MAGE RT-nested PCR in the diagnosis of malignant pleural effusion.

PubMed

Jeon, Eun Ju; Park, Hye Kyeong; Jeon, Kyeongman; Koh, Won-Jung; Suh, Gee Young; Chung, Man Pyo; Kim, Hojoong; Kwon, O Jung; Ki, Chang-Seok; Kim, Jong-Won; Shim, Young Mog; Um, Sang-Won

2012-11-01

  Melanoma antigen (MAGE) genes are expressed in tumor cells, the testis and the placenta. The purpose of this prospective study was to investigate the sensitivity, specificity, and accuracy of the carcinoembryonic antigen (CEA), MAGE reverse transcriptase-nested polymerase chain reaction (RT-nested PCR), and cytology of pleural fluid in the diagnosis of malignant pleural effusion.   Patients in whom unilateral pleural effusion was identified on chest radiography from January to December 2009 were included in the study. MAGE genes were analyzed by RT-nested PCR using MAGE A1-6 common primers.   Of 81 enrolled patients, 46 were diagnosed as malignant pleural effusion, and 24 were diagnosed as benign pleural effusion. The diagnoses of 11 patients were not confirmed in this study. The diagnostic sensitivity, specificity, and accuracy of MAGE RT-nested PCR were 61.4%, 95.7%, and 73.1%, respectively. The diagnostic sensitivities of cytology and CEA (>5 ng/mL) were 61.4% and 75.0%, respectively. Among 17 patients with negative cytology who had malignant pleural effusion, 12 and 10 patients were positive for CEA (>5.0 ng/mL) and MAGE RT-nested PCR, respectively. However, of five patients with malignant pleural effusion that was not recognized by cytology and CEA, MAGE RT-nested PCR correctly predicted a malignant etiology in only one additional patient (20%).   MAGE RT-nested PCR seems to add little on the combination of conventional methods in the diagnosis of malignant effusion. © 2012 Tianjin Lung Cancer Institute and Wiley Publishing Asia Pty. Ltd.

Detection of EML4-ALK fusion gene in Chinese non-small cell lung cancer by using a sensitive quantitative real-time reverse transcriptase PCR technique.

PubMed

Fu, Sha; Wang, Fang; Shao, Qiong; Zhang, Xu; Duan, Li-Ping; Zhang, Xiao; Zhang, Li; Shao, Jian-Yong

2015-04-01

Anaplastic lymphoma kinase (ALK) rearrangement is present in approximately 5% of lung adenocarcinoma. Clinical trials on ALK inhibitor phase I to III have shown an interesting disease control rate and acceptable tolerability in ALK rearrangement patients. In clinical application, the precise diagnostic strategy for identifying ALK rearrangements remains to be determined. In this study, ALK rearrangement was screened by using quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR), direct sequencing, 2 fluorescence in situ hybridization (FISH) assays, and immunohistochemistry in 173 lung adenocarcinomas. We identified 18 cases (10.4%) with EML4-ALK fusion-positive by qRT-PCR, and all were positive for EML4-ALK fusion gene validated by direct sequencing. The result was consistent with that of other methods. Furthermore, of the 18 EML4-ALK fusion-positive cases, 16 (9.2%) were positive by using EML4-ALK fusion probe FISH, and 15 (8.7%) were positive by using ALK break-apart probe FISH and immunohistochemistry staining. Of the 18 ALK fusion-positive lung adenocarcinomas, 8 cases (44.4%) were histologically diagnosed as subtypes of cribriform adenocarcinoma, 7 cases (38.9%) as cribriform adenocarcinoma mixed with papillary and/or mucinous pattern, 2 cases (11.1%) as papillary adenocarcinoma, and 1 case (5.6%) as mucinous adenocarcinoma. In the present study, the ALK rearrangement frequency detected by qRT-PCR in Chinese NSCLC patients was higher than that in the western populations. QRT-PCR is a rapid, sensitive technology that could be used as a screening tool for identifying EML4-ALK fusion-positive NSCLC patients who would be sensitive for receiving ALK inhibitor therapy.

Effects of Anticoagulant, Processing Delay, and Assay Method (Branched DNA versus Reverse Transcriptase PCR) on Measurement of Human Immunodeficiency Virus Type 1 RNA Levels in Plasma

PubMed Central

Kirstein, Lynn M.; Mellors, John W.; Rinaldo, Charles R.; Margolick, Joseph B.; Giorgi, Janis V.; Phair, John P.; Dietz, Edith; Gupta, Phalguni; Sherlock, Christopher H.; Hogg, Robert; Montaner, J. S. G.; Muñoz, Alvaro

1999-01-01

We conducted two studies to determine the potential influence of delays in blood processing, type of anticoagulant, and assay method on human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma. The first was an experimental study in which heparin- and EDTA-anticoagulated blood samples were collected from 101 HIV-positive individuals and processed to plasma after delays of 2, 6, and 18 h. HIV-1 RNA levels in each sample were then measured by both branched-DNA (bDNA) and reverse transcriptase PCR (RT-PCR) assays. Compared to samples processed within 2 h, the loss (decay) of HIV-1 RNA in heparinized blood was significant (P < 0.05) but small after 6 h (bDNA assay, −0.12 log10 copies/ml; RT-PCR, −0.05 log10 copies/ml) and after 18 h (bDNA assay, −0.27 log10 copies/ml; RT-PCR, −0.15 log10 copies/ml). Decay in EDTA-anticoagulated blood was not significant after 6 h (bDNA assay, −0.002 log10 copies/ml; RT-PCR, −0.02 log10 copies/ml), but it was after 18 h (bDNA assay, −0.09 log10 copies/ml; RT-PCR, −0.09 log10 copies/ml). Only 4% of samples processed after 6 h lost more than 50% (≥0.3 log10 copies/ml) of the HIV-1 RNA, regardless of the anticoagulant or the assay that was used. The second study compared HIV-1 RNA levels in samples from the Multicenter AIDS Cohort Study (MACS; samples were collected in heparin-containing tubes in 1985, had a 6-h average processing delay, and were assayed by bDNA assay) and the British Columbia Drug Treatment Program (BCDTP) (collected in EDTA- or acid citrate dextrose-containing tubes in 1996 and 1997, had a 2-h maximum processing delay, and were assayed by RT-PCR). HIV-1 RNA levels in samples from the two cohorts were not significantly different after adjusting for CD4+-cell count and converting bDNA assay values to those corresponding to the RT-PCR results. In summary, the decay of HIV-1 RNA measured in heparinized blood after 6 h was small (−0.05 to −0.12 log10 copies/ml), and the minor impact of this decay on HIV-1 RNA concentrations in archived plasma samples of the MACS was confirmed by the similarity of CD4+-cell counts and assay-adjusted HIV-1 RNA concentrations in the MACS and BCDTP. PMID:10405379

Development of a real-time RT-PCR assay for a novel influenza A (H1N1) virus.

PubMed

Jiang, Tao; Kang, Xiaoping; Deng, Yongqiang; Zhao, Hui; Li, Xiaofeng; Yu, Xuedong; Yu, Man; Qin, Ede; Zhu, Qingyu; Yang, Yinhui; Qin, Chengfeng

2010-02-01

A pandemic caused by a novel influenza A virus (H1N1) poses a serious public health threat. In this study, a real-time reverse transcriptase PCR (RT-PCR) assay based on the hemagglutinin gene was developed that discriminates the novel H1N1 from swine influenza virus, seasonal H1N1/H3N2 virus and the highly pathogenic H5N1 avian influenza virus. The sensitivity of this assay was 0.2 50% tissue culture infective dose of virus and 200 copies of in vitro-transcribed target RNA. Three hundred and forty-eight clinical specimens from suspected H1N1 patients were tested using this assay, and forty-two (12.07%) were found to be positive. Tests using the real-time PCR assay recommended by WHO and virus isolation gave identical results. This sensitive and specific real-time RT-PCR assay will contribute to the early diagnosis and control of the emerging H1N1 influenza pandemic. 2009 Elsevier B.V. All rights reserved.

A rapid method of accurate detection and differentiation of Newcastle disease virus pathotypes by demonstrating multiple bands in degenerate primer based nested RT-PCR.

PubMed

Desingu, P A; Singh, S D; Dhama, K; Kumar, O R Vinodh; Singh, R; Singh, R K

2015-02-01

A rapid and accurate method of detection and differentiation of virulent and avirulent Newcastle disease virus (NDV) pathotypes was developed. The NDV detection was carried out for different domestic avian field isolates and pigeon paramyxo virus-1 (25 field isolates and 9 vaccine strains) by using APMV-I "fusion" (F) gene Class II specific external primer A and B (535bp), internal primer C and D (238bp) based reverses transcriptase PCR (RT-PCR). The internal degenerative reverse primer D is specific for F gene cleavage position of virulent strain of NDV. The nested RT-PCR products of avirulent strains showed two bands (535bp and 424bp) while virulent strains showed four bands (535bp, 424bp, 349bp and 238bp) on agar gel electrophoresis. This is the first report regarding development and use of degenerate primer based nested RT-PCR for accurate detection and differentiation of NDV pathotypes by demonstrating multiple PCR band patterns. Being a rapid, simple, and economical test, the developed method could serve as a valuable alternate diagnostic tool for characterizing NDV isolates and carrying out molecular epidemiological surveillance studies for this important pathogen of poultry. Copyright © 2014 Elsevier B.V. All rights reserved.

A novel duplex real-time reverse transcriptase-polymerase chain reaction assay for the detection of hepatitis C viral RNA with armored RNA as internal control

PubMed Central

2010-01-01

Background The hepatitis C virus (HCV) genome is extremely heterogeneous. Several HCV infections can not be detected using currently available commercial assays, probably because of mismatches between the template and primers/probes. By aligning the HCV sequences, we developed a duplex real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay using 2 sets of primers/probes and a specific armored RNA as internal control. The 2 detection probes were labelled with the same fluorophore, namely, 6-carboxyfluorescein (FAM), at the 5' end; these probes could mutually combine, improving the power of the test. Results The limit of detection of the duplex primer/probe assay was 38.99 IU/ml. The sensitivity of the assay improved significantly, while the specificity was not affected. All HCV genotypes in the HCV RNA Genotype Panel for Nucleic Acid Amplification Techniques could be detected. In the testing of 109 serum samples, the performance of the duplex real-time RT-PCR assay was identical to that of the COBAS AmpliPrep (CAP)/COBAS TaqMan (CTM) assay and superior to 2 commercial HCV assay kits. Conclusions The duplex real-time RT-PCR assay is an efficient and effective viral assay. It is comparable with the CAP/CTM assay with regard to the power of the test and is appropriate for blood-donor screening and laboratory diagnosis of HCV infection. PMID:20529244

Quantitative Assessment of the Sensitivity of Various Commercial Reverse Transcriptases Based on Armored HIV RNA

PubMed Central

Okello, John B. A.; Rodriguez, Linda; Poinar, Debi; Bos, Kirsten; Okwi, Andrew L.; Bimenya, Gabriel S.; Sewankambo, Nelson K.; Henry, Kenneth R.; Kuch, Melanie; Poinar, Hendrik N.

2010-01-01

Background The in-vitro reverse transcription of RNA to its complementary DNA, catalyzed by the enzyme reverse transcriptase, is the most fundamental step in the quantitative RNA detection in genomic studies. As such, this step should be as analytically sensitive, efficient and reproducible as possible, especially when dealing with degraded or low copy RNA samples. While there are many reverse transcriptases in the market, all claiming to be highly sensitive, there is need for a systematic independent comparison of their applicability in quantification of rare RNA transcripts or low copy RNA, such as those obtained from archival tissues. Methodology/Principal Findings We performed RT-qPCR to assess the sensitivity and reproducibility of 11 commercially available reverse transcriptases in cDNA synthesis from low copy number RNA levels. As target RNA, we used a serially known number of Armored HIV RNA molecules, and observed that 9 enzymes we tested were consistently sensitive to ∼1,000 copies, seven of which were sensitive to ∼100 copies, while only 5 were sensitive to ∼10 RNA template copies across all replicates tested. Despite their demonstrated sensitivity, these five best performing enzymes (Accuscript, HIV-RT, M-MLV, Superscript III and Thermoscript) showed considerable variation in their reproducibility as well as their overall amplification efficiency. Accuscript and Superscript III were the most sensitive and consistent within runs, with Accuscript and Superscript II ranking as the most reproducible enzymes between assays. Conclusions/Significance We therefore recommend the use of Accuscript or Superscript III when dealing with low copy number RNA levels, and suggest purification of the RT reactions prior to downstream applications (eg qPCR) to augment detection. Although the results presented in this study were based on a viral RNA surrogate, and applied to nucleic acid lysates derived from archival formalin-fixed paraffin embedded tissue, their relative performance on RNA obtained from other tissue types may vary, and needs future evaluation. PMID:21085668

Impact of human immunodeficiency virus type 1 reverse transcriptase inhibitor drug resistance mutation interactions on phenotypic susceptibility.

PubMed

Trivedi, Vinod; Von Lindern, Jana; Montes-Walters, Miguel; Rojo, Daniel R; Shell, Elisabeth J; Parkin, Neil; O'Brien, William A; Ferguson, Monique R

2008-10-01

The role specific reverse transcriptase (RT) drug resistance mutations play in influencing phenotypic susceptibility to RT inhibitors in virus strains with complex resistance interaction patterns was assessed using recombinant viruses that consisted of RT-PCR-amplified pol fragments derived from plasma HIV-1 RNA from two treatment-experienced patients. Specific modifications of key RT amino acids were performed by site-directed mutagenesis. A panel of viruses with defined genotypic resistance mutations was assessed for phenotypic drug resistance. Introduction of M184V into several different clones expressing various RT resistance mutations uniformly decreased susceptibility to abacavir, lamivudine, and didanosine, and increased susceptibility to zidovudine, stavudine, and tenofovir; replication capacity was decreased. The L74V mutation had similar but slightly different effects, contributing to decreased susceptibility to abacavir, lamivudine, and didanosine and increased susceptibility to zidovudine and tenofovir, but in contrast to M184V, L74V contributed to decreased susceptibility to stavudine. In virus strains with the nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations K101E and G190S, the L74V mutation increased replication capacity, consistent with published observations, but replication capacity was decreased in strains without NNRTI resistance mutations. K101E and G190S together tend to decrease susceptibility to all nucleoside RT inhibitors, but the K103N mutation had little effect on nucleoside RT inhibitor susceptibility. Mutational interactions can have a substantial impact on drug resistance phenotype and replication capacity, and this has been exploited in clinical practice with the development of fixed-dose combination pills. However, we are the first to report these mutational interactions using molecularly cloned recombinant strains derived from viruses that occur naturally in HIV-infected individuals.

Impact of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Inhibitor Drug Resistance Mutation Interactions on Phenotypic Susceptibility

PubMed Central

Trivedi, Vinod; Von Lindern, Jana; Montes-Walters, Miguel; Rojo, Daniel R.; Shell, Elisabeth J.; Parkin, Neil; O'Brien, William A.

2008-01-01

Abstract The role specific reverse transcriptase (RT) drug resistance mutations play in influencing phenotypic susceptibility to RT inhibitors in virus strains with complex resistance interaction patterns was assessed using recombinant viruses that consisted of RT-PCR-amplified pol fragments derived from plasma HIV-1 RNA from two treatment-experienced patients. Specific modifications of key RT amino acids were performed by site-directed mutagenesis. A panel of viruses with defined genotypic resistance mutations was assessed for phenotypic drug resistance. Introduction of M184V into several different clones expressing various RT resistance mutations uniformly decreased susceptibility to abacavir, lamivudine, and didanosine, and increased susceptibility to zidovudine, stavudine, and tenofovir; replication capacity was decreased. The L74V mutation had similar but slightly different effects, contributing to decreased susceptibility to abacavir, lamivudine, and didanosine and increased susceptibility to zidovudine and tenofovir, but in contrast to M184V, L74V contributed to decreased susceptibility to stavudine. In virus strains with the nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations K101E and G190S, the L74V mutation increased replication capacity, consistent with published observations, but replication capacity was decreased in strains without NNRTI resistance mutations. K101E and G190S together tend to decrease susceptibility to all nucleoside RT inhibitors, but the K103N mutation had little effect on nucleoside RT inhibitor susceptibility. Mutational interactions can have a substantial impact on drug resistance phenotype and replication capacity, and this has been exploited in clinical practice with the development of fixed-dose combination pills. However, we are the first to report these mutational interactions using molecularly cloned recombinant strains derived from viruses that occur naturally in HIV-infected individuals. PMID:18844463

Estrogen- and progesterone-receptor status in ECOG 2197: comparison of immunohistochemistry by local and central laboratories and quantitative reverse transcription polymerase chain reaction by central laboratory.

PubMed

Badve, Sunil S; Baehner, Frederick L; Gray, Robert P; Childs, Barrett H; Maddala, Tara; Liu, Mei-Lan; Rowley, Steve C; Shak, Steven; Perez, Edith A; Perez, Edith D; Shulman, Lawrence J; Martino, Silvana; Davidson, Nancy E; Sledge, George W; Goldstein, Lori J; Sparano, Joseph A

2008-05-20

Central and local laboratory concordance for hormone receptor measurement is therapeutically important. This study compares estrogen receptor (ER) and progesterone receptor (PR) measured by local laboratory immunohistochemistry (IHC), central IHC, and central reverse-transcriptase polymerase chain reaction (RT-PCR) using a proprietary 21-gene assay. A case-control sample of 776 breast cancer patients from Eastern Cooperative Oncology Group (ECOG) study E2197 was evaluated. Central IHC Allred score for ER and PR was obtained using tissue microarrays and 1D5 ER antibody and 636 PR antibody. Quantitative RT-PCR for ER and PR in whole sections was performed using the 21-gene assay. For ER, the concordance between local and central IHC was 90% (95% CI, 88% to 92%), between local IHC and central RT-PCR was 91% (95% CI, 89% to 93%), and between central IHC and central RT-PCR was 93% (95% CI, 91% to 95%). For PR, the concordance between local IHC and central IHC was 84% (95% CI, 82% to 87%), between local IHC and central RT-PCR was 88% (95% CI, 85% to 90%), and between central IHC and central RT-PCR was 90% (95% CI, 88% to 92%). Although concordance was high, IHC ER-negative cases that were RT-PCR positive were more common than IHC ER-positive cases that were RT-PCR negative. In ER-positive patients, ER expression by central IHC Allred score was marginally associated with recurrence (P = .091), and ER expression by central RT-PCR was significantly associated with recurrence (P = .014). However, recurrence score, which incorporates additional genes/pathways, was a highly significant predictor of recurrence (P < .0001). There is a high degree of concordance among local IHC, central IHC, and central RT-PCR by the proprietary gene assay for ER and PR status. Although ER expression is marginally associated with relapse in ER-positive patients treated with chemohormonal therapy, recurrence score is a highly significant predictor of recurrence.

Field detection of avian influenza virus in wild birds: evaluation of a portable rRT-PCR system and freeze-dried reagents

USGS Publications Warehouse

Takekawa, John Y.; Iverson, Samuel A.; Schultz, Annie K.; Hill, Nichola J.; Cardona, Carol J.; Boyce, Walter M.; Dudley, Joseph P.

2010-01-01

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAIV) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds is often conducted in remote regions, but results are often delayed because of limited local analytical capabilities, difficulties with sample transportation and permitting, or problems keeping samples cold in the field. In response to these challenges, the performance of a portable real-time, reverse transcriptase-polymerase chain reaction (rRT-PCR) unit (RAPID(Registered), Idaho Technologies, Salt Lake City, UT) that employed lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies) was compared to virus isolation combined with real-time RT-PCR conducted in a laboratory. This study included both field and experimental-based sampling. Field samples were collected from migratory shorebirds captured in northern California, while experimental samples were prepared by spiking fecal material with an H6N2 AIV isolate. Results indicated that the portable rRT-PCR unit had equivalent specificity to virus isolation with no false positives, but sensitivity was compromised at low viral titers. Use of portable rRT-PCR with lyophilized reagents may expedite surveillance results, paving the way to a better understanding of wild bird involvement in HPAIV H5N1 transmission.

Characterization of the interferon genes in homozygous rainbow trout reveals two novel genes, alternate splicing and differential regulation of duplicated genes

USGS Publications Warehouse

Purcell, M.K.; Laing, K.J.; Woodson, J.C.; Thorgaard, G.H.; Hansen, J.D.

2009-01-01

The genes encoding the type I and type II interferons (IFNs) have previously been identified in rainbow trout and their proteins partially characterized. These previous studies reported a single type II IFN (rtIFN-??) and three rainbow trout type I IFN genes that are classified into either group I (rtIFN1, rtIFN2) or group II (rtIFN3). In this present study, we report the identification of a novel IFN-?? gene (rtIFN-??2) and a novel type I group II IFN (rtIFN4) in homozygous rainbow trout and predict that additional IFN genes or pseudogenes exist in the rainbow trout genome. Additionally, we provide evidence that short and long forms of rtIFN1 are actively and differentially transcribed in homozygous trout, and likely arose due to alternate splicing of the first exon. Quantitative reverse transcriptase PCR (qRT-PCR) assays were developed to systematically profile all of the rainbow trout IFN transcripts, with high specificity at an individual gene level, in na??ve fish and after stimulation with virus or viral-related molecules. Cloned PCR products were used to ensure the specificity of the qRT-PCR assays and as absolute standards to assess transcript abundance of each gene. All IFN genes were modulated in response to Infectious hematopoietic necrosis virus (IHNV), a DNA vaccine based on the IHNV glycoprotein, and poly I:C. The most inducible of the type I IFN genes, by all stimuli tested, were rtIFN3 and the short transcript form of rtIFN1. Gene expression of rtIFN-??1 and rtIFN-??2 was highly up-regulated by IHNV infection and DNA vaccination but rtIFN-??2 was induced to a greater magnitude. The specificity of the qRT-PCR assays reported here will be useful for future studies aimed at identifying which cells produce IFNs at early time points after infection. ?? 2008 Elsevier Ltd.

Detection of Respiratory Viruses in Sputum from Adults by Use of Automated Multiplex PCR

PubMed Central

Walsh, Edward E.; Formica, Maria A.; Falsey, Ann R.

2014-01-01

Respiratory tract infections (RTI) frequently cause hospital admissions among adults. Diagnostic viral reverse transcriptase PCR (RT-PCR) of nose and throat swabs (NTS) is useful for patient care by informing antiviral use and appropriate isolation. However, automated RT-PCR systems are not amenable to utilizing sputum due to its viscosity. We evaluated a simple method of processing sputum samples in a fully automated respiratory viral panel RT-PCR assay (FilmArray). Archived sputum and NTS samples collected in 2008-2012 from hospitalized adults with RTI were evaluated. A subset of sputum samples positive for 10 common viruses by a uniplex RT-PCR was selected. A sterile cotton-tip swab was dunked in sputum, swirled in 700 μL of sterile water (dunk and swirl method) and tested by the FilmArray assay. Quantitative RT-PCR was performed on “dunked” sputum and NTS samples for influenza A (Flu A), respiratory syncytial virus (RSV), coronavirus OC43 (OC43), and human metapneumovirus (HMPV). Viruses were identified in 31% of 965 illnesses using a uniplex RT-PCR. The sputum sample was the only sample positive for 105 subjects, including 35% (22/64) of influenza cases and significantly increased the diagnostic yield of NTS alone (302/965 [31%] versus 197/965 [20%]; P = 0.0001). Of 108 sputum samples evaluated by the FilmArray assay using the dunk and swirl method, 99 (92%) were positive. Quantitative RT-PCR revealed higher mean viral loads in dunked sputum samples compared to NTS samples for Flu A, RSV, and HMPV (P = 0.0001, P = 0.006, and P = 0.011, respectively). The dunk and swirl method is a simple and practical method for reliably processing sputum samples in a fully automated PCR system. The higher viral loads in sputa may increase detection over NTS testing alone. PMID:25056335

Use of FTA filter paper for the molecular detection of Newcastle disease virus.

PubMed

Perozo, Francisco; Villegas, Pedro; Estevez, Carlos; Alvarado, Iván; Purvis, Linda B

2006-04-01

The feasibility of using Flinders Technology Associates filter papers (FTA cards) to collect allantoic fluid and chicken tissue samples for Newcastle disease virus (NDV) molecular detection was evaluated. Trizol RNA extraction and one-step reverse transcriptase-polymerase chain reaction (RT-PCR) were used. FTA cards allowed NDV identification from allantoic fluid with a titre of 10(5.8) median embryo lethal doses/ml. The inactivated virus remained stable on the cards for 15 days. NDV was detected from FTA imprints of the trachea, lung, caecal tonsil and cloacal faeces of experimentally infected birds. RT-PCR detection from FTA cards was confirmed by homologous frozen-tissue RT-PCR and virus isolation. Direct nucleotide sequence of the amplified F gene allowed prediction of NDV virulence. No virus isolation was possible from the FTA inactivated samples, indicating viral inactivation upon contact. The FTA cards are suitable for collecting and transporting NDV-positive samples, providing a reliable source of RNA for molecular characterization and a hazard-free sample.

Dengue virus in bats from southeastern Mexico.

PubMed

Sotomayor-Bonilla, Jesús; Chaves, Andrea; Rico-Chávez, Oscar; Rostal, Melinda K; Ojeda-Flores, Rafael; Salas-Rojas, Mónica; Aguilar-Setien, Álvaro; Ibáñez-Bernal, Sergio; Barbachano-Guerrero, Arturo; Gutiérrez-Espeleta, Gustavo; Aguilar-Faisal, J Leopoldo; Aguirre, A Alonso; Daszak, Peter; Suzán, Gerardo

2014-07-01

To identify the relationship between landscape use and dengue virus (DENV) occurrence in bats, we investigated the presence of DENV from anthropogenically changed and unaltered landscapes in two Biosphere Reserves: Calakmul (Campeche) and Montes Azules (Chiapas) in southern Mexico. Spleen samples of 146 bats, belonging to 16 species, were tested for four DENV serotypes with standard reverse transcriptase polymerase chain reaction (RT-PCR) protocols. Six bats (4.1%) tested positive for DENV-2: four bats in Calakmul (two Glossophaga soricina, one Artibeus jamaicensis, and one A. lituratus) and two bats in Montes Azules (both A. lituratus). No effect of anthropogenic disturbance on the occurrence of DENV was detected; however, all three RT-PCR-positive bat species are considered abundant species in the Neotropics and well-adapted to disturbed habitats. To our knowledge, this study is the first study conducted in southeastern Mexico to identify DENV-2 in bats by a widely accepted RT-PCR protocol. The role that bats play on DENV's ecology remains undetermined. © The American Society of Tropical Medicine and Hygiene.

Factors Affecting Detection of Hepatitis E Virus on Canadian Retail Pork Chops and Pork Livers Assayed Using Real-Time RT-PCR.

PubMed

Wilhelm, B J; Leblanc, D; Avery, B; Pearl, D L; Houde, A; Rajić, A; McEwen, S A

2016-03-01

We collected 599 Canadian retail pork chops and 283 pork livers routinely (usually weekly) from April 2011 to March 2012 using the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) retail sampling platform. Samples were assayed using validated real-time (q) reverse transcriptase polymerase chain reaction (RT-PCR) and nested classical RT-PCR for the detection of hepatitis E virus (HEV), porcine enteric calicivirus (PEC) and rotavirus (RV). The presence of Escherichia coli, Salmonella spp. and Campylobacter spp. was measured on a subset of our samples. Exact logistic regression models were fitted for predictors for HEV detection, for each assay. For both assays, sample type (pork chop versus liver) was a significant predictor for HEV RNA detection. For nested classical RT-PCR but not qRT-PCR, region of sample collection was a significant predictor (P = 0.008) of HEV detection. Odds of HEV detection were greatest in spring relative to other seasons. E. coli was a significant predictor for HEV RNA detection using the qRT-PCR (P = 0.03). Overall, the prevalence of E. coli, Salmonella spp. and Campylobacter spp. was significantly greater than HEV, PEC or RV on our retail pork samples. Our sparse data set for the detection of PEC and RV precluded modelling of risk factors for the detection of these viruses. © 2015 Zoonoses and Public Health © 2015 Her Majesty the Queen in Right of Canada Reproduced with the permission of the Minister of the Public Health Agency of Canada.

Measurement of Gene Expression in Archival Paraffin-Embedded Tissues

PubMed Central

Cronin, Maureen; Pho, Mylan; Dutta, Debjani; Stephans, James C.; Shak, Steven; Kiefer, Michael C.; Esteban, Jose M.; Baker, Joffre B.

2004-01-01

Throughout the last decade many laboratories have shown that mRNA levels in formalin-fixed and paraffin-embedded (FPE) tissue specimens can be quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) techniques despite the extensive RNA fragmentation that occurs in tissues so preserved. We have developed RT-PCR methods that are sensitive, precise, and that have multianalyte capability for potential wide use in clinical research and diagnostic assays. Here it is shown that the extent of fragmentation of extracted FPE tissue RNA significantly increases with archive storage time. Probe and primer sets for RT-PCR assays based on amplicons that are both short and homogeneous in length enable effective reference gene-based data normalization for cross comparison of specimens that differ substantially in age. A 48-gene assay used to compare gene expression profiles from the same breast cancer tissue that had been either frozen or FPE showed very similar profiles after reference gene-based normalization. A 92-gene assay, using RNA extracted from three 10-μm FPE sections of archival breast cancer specimens (dating from 1985 to 2001) yielded analyzable data for these genes in all 62 tested specimens. The results were substantially concordant when estrogen receptor, progesterone receptor, and HER2 receptor status determined by RT-PCR was compared with immunohistochemistry assays for these receptors. Furthermore, the results highlight the advantages of RT-PCR over immunohistochemistry with respect to quantitation and dynamic range. These findings support the development of RT-PCR analysis of FPE tissue RNA as a platform for multianalyte clinical diagnostic tests. PMID:14695316

Modified concentration method for the detection of enteric viruses on fruits and vegetables by reverse transcriptase-polymerase chain reaction or cell culture.

PubMed

Dubois, Eric; Agier, Cécilia; Traoré, Ousmane; Hennechart, Catherine; Merle, Ghislaine; Crucière, Catherine; Laveran, Henri

2002-12-01

Fruits and vegetables may act as a vehicle of human enteric virus if they are irrigated with sewage-contaminated water or prepared by infected food handlers. An elution-concentration method was modified to efficiently detect, by reverse transcriptase-polymerase chain reaction (RT-PCR) or by cell culture, contamination by poliovirus, hepatitis A virus (HAV), and Norwalk-like virus (NLV) of various fresh and frozen berries and fresh vegetables. The protocol included washing the fruit or vegetable surface with 100 mM Tris-HCl, 50 mM glycine, and 3% beef extract, pH 9.5 buffer, which favors viral elution from acid-releasing berries, supplemented with 50 mM MgCl2 to reduce the decrease in viral infectivity during the process. The viral concentration method was based on polyethylene glycol precipitation. Copurified RT-PCR inhibitors and cytotoxic compounds were removed from viral concentrates by chloroform-butanol extraction. Viruses from 100 g of vegetal products could be recovered in volumes of 3 to 5 ml. Viral RNAs were isolated by a spin column method before molecular detection or concentrates were filtered (0.22-microm porosity) and inoculated on cell culture for infectious virus detection. About 15% of infectious poliovirus and 20% of infectious HAV were recovered from frozen raspberry surfaces. The percentage of viral RNA recovery was estimated by RT-PCR to be about 13% for NLV, 17% for HAV, and 45 to 100% for poliovirus. By this method, poliovirus and HAV RNA were detected on products inoculated with a titer of about 5 x 10(1) 50% tissue culture infectious dose per 100 g. NLV RNA was detected at an initial inoculum of 1.2 x 10(3) RT-PCR amplifiable units. This method would be useful for the viral analysis of fruits or vegetables during an epidemiological investigation of foodborne diseases.

Bioinformatic analysis of variability of Newcastle disease virus diagnostic primers and probes and the potential for false negative detection

USDA-ARS?s Scientific Manuscript database

The use of reverse transcriptase polymerase chain reaction (RT-PCR) or other molecular diagnostic methods is commonly used for the primary diagnosis of Newcastle disease virus (NDV). However, NDV in nature has a range of virulence, and the low virulence viruses must be differentiated from virulent ...

Evaluation of Cytokine Synthesis in Human Whole Blood by Enzyme Linked Immunoassay (ELISA), Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), and Flow Cytometry

DTIC Science & Technology

2007-05-08

deoxynucleotide triphosphates, from Sigma. Sequences for glyceraldehyde-3-phosphate dehydrogenase ( G3PDH ), IL-8,and TNF-a were amplified with primer...This was accomplished by normalizing all samples to the mRNA for the moderately expressed housekeeping function glyceraldehyde-3 -phosphate...without and with isolation of cells before reverse transcription and PCR. G3PDH mRNA target amplifies at 983 base pairs. The 630 base pair band is the

Development of a highly sensitive screen for influenza A in guano and its application in the search for ancient RNA preserved under Antarctic Adelie penguin colonies.

PubMed

Briggs, L C; Ashton, R M; Metcalf, P

2003-01-01

We have developed a reverse transcriptase polymerase chain reaction (RT-PCR)-based assay to detect influenza A in guano samples as part of our program to investigate ancient viral RNA from under Antarctic Adelie penguin (Pygoscelis adeliae) colonies. Of five extraction protocols tested (RNeasy, GTC TRIZOL, GTC Silica, Rnaid, and AGPC), AGPC proved to be the most consistent and sensitive to low concentrations of influenza, but further purification with commercial viral nucleic acid spin filter system was still required to remove remaining PCR inhibitors. RT-PCR was then performed on the eluent and 650 bases of the M1 gene were amplified. The assay was found to be able to detect as little as 100 microl of 0.1 hemagglutination units (HU)/ml influenza.

[Disappearance of residual disease confirmed by RT-PCR following induction chemotherapy in two hypoplastic leukemia patients with t(8;21)].

PubMed

Sawada, M; Tsurumi, H; Yamada, T; Hara, T; Oyama, M; Moriwaki, H

1999-04-01

Reverse transcriptase-polymerase chain reaction (RT-PCR) methods often detect the AML1/MTG8 fusion transcript even in acute myelogenous leukemia (AML) patients with t(8;21) who have been in long-term remission. We encountered 2 hypoplastic leukemia patients with t(8;21) who achieved cytogenetic remission with short-term conventional chemotherapy. Patient 1 was a 42-year-old woman. Chromosomal analysis detected t(8;21) (q22;q22) and PCR analysis (35 cycles PCR amplification; detection limit 1 x 10(-5) cells) detected the AML1/MTG8 fusion transcript. Complete remission was obtained with 1 course of chemotherapy consisting of low-dose cytarabine (20 mg x 14 days) and etoposide (50 mg x 14 days). After 2 courses of consolidation chemotherapy consisting of conventional-dose cytarabine and mitoxantrone, the RT-PCR findings were negative for the AML1/MTG8 fusion transcript. Patient 2 was a 67-year-old man. Cytogenetic analysis detected t(8;21) (q22;q22), and was positive for the AML1/MTG8 fusion transcript. After 2 courses of induction chemotherapy comprising low-dose cytarabine (20 mg x 14 days) and etoposide (50 mg x 14 days), and 3 courses of conventional consolidation chemotherapy, RT-PCR analysis confirmed the disappearance of the AML1/MTG8 fusion transcript.

Improved Detection of Norovirus and Hepatitis A Virus in Surface Water by Applying Pre-PCR Processing.

PubMed

Borgmästars, Emmy; Jazi, Mehrdad Mousavi; Persson, Sofia; Jansson, Linda; Rådström, Peter; Simonsson, Magnus; Hedman, Johannes; Eriksson, Ronnie

2017-12-01

Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) detection of waterborne RNA viruses generally requires concentration of large water volumes due to low virus levels. A common approach is to use dead-end ultrafiltration followed by precipitation with polyethylene glycol. However, this procedure often leads to the co-concentration of PCR inhibitors that impairs the limit of detection and causes false-negative results. Here, we applied the concept of pre-PCR processing to optimize RT-qPCR detection of norovirus genogroup I (GI), genogroup II (GII), and hepatitis A virus (HAV) in challenging water matrices. The RT-qPCR assay was improved by screening for an inhibitor-tolerant master mix and modifying the primers with twisted intercalating nucleic acid molecules. Additionally, a modified protocol based on chaotropic lysis buffer and magnetic silica bead nucleic acid extraction was developed for complex water matrices. A validation of the modified extraction protocol on surface and drinking waters was performed. At least a 26-fold improvement was seen in the most complex surface water studied. The modified protocol resulted in average recoveries of 33, 13, 8, and 4% for mengovirus, norovirus GI, GII, and HAV, respectively. The modified protocol also improved the limit of detection for norovirus GI and HAV. RT-qPCR inhibition with C q shifts of 1.6, 2.8, and 3.5 for norovirus GI, GII, and HAV, respectively, obtained for the standard nucleic acid extraction were completely eliminated by the modified protocol. The standard nucleic acid extraction method worked well on drinking water with no RT-qPCR inhibition observed and average recoveries of 80, 124, 89, and 32% for mengovirus, norovirus GI, GII, and HAV, respectively.

Detection of PAX3-FKHR and PAX7-FKHR fusion transcripts in rhabdomyosarcoma by reverse transcriptase-polymerase chain reaction using paraffin-embedded tissue.

PubMed

Chen, B F; Chen, M L; Liang, D C; Huang, Y W; Liu, H C; Chen, S H

1999-02-01

Alveolar rhabdomyosarcoma (RMS) is associated with a characteristic chromosomal translocation t(2;13)(q35;q14). The genes involved in this translocation are paired box (PAX)3 on chromosome 2 and forkhead in RMS (FKHR) on chromosome 13. An occasional variant translocation t(1;13)(p36;q14) affecting PAX7 and FKHR on chromosomes 1 and 13, respectively, has also been described. Chromosomal translocations in RMS are detected using conventional cytogenetic analysis, fluorescence in situ hybridization (FISH) or reverse transcriptase-polymerase chain reaction (RT-PCR) on fresh or frozen tissue samples. We describe the results of RT-PCR analysis of PAX3-FKHR and PAX7-FKHR chimeric messages in formalin-fixed, paraffin-embedded tissue samples from 17 RMS cases. RNA was extracted from formalin-fixed, paraffin-embedded RMS tissue. Oligonucleotide primers corresponding to the regions of PAX3, PAX7 and FKHR were used for the detection of PAX3-FKHR and PAX7-FKHR chimeric messages. A seminested PCR of the PCR products was used to increase the sensitivity of detection. The amplified fragments were purified and directly sequenced to confirm the specificity of the methods. The PAX3-FKHR chimeric message was detected in all three cases of alveolar RMS but not in any of the 12 embryonal and two pleomorphic RMS cases. The PAX7-FKHR fusion transcript was detected in one case of embryonal RMS. The results indicate that the RT-PCR assay is a reliable method for the detection of the PAX3-FKHR fusion transcript of alveolar RMS in formalin-fixed, paraffin-embedded tissue. This simple method enables pathologists to identify chromosomal rearrangements in RMS as a diagnostic aid in cases where fresh or frozen tissue is not available.

Detection of Anaplasma marginale and A. phagocytophilum in Bovine Peripheral Blood Samples by Duplex Real-Time Reverse Transcriptase PCR Assay ▿

PubMed Central

Reinbold, James B.; Coetzee, Johann F.; Sirigireddy, Kamesh R.; Ganta, Roman R.

2010-01-01

Insufficient diagnostic sensitivity and specificity coupled with the potential for cross-reactivity among closely related Anaplasma species has made the accurate determination of infection status problematic. A method for the development of simplex and duplex real-time quantitative reverse transcriptase PCR (qRT-PCR) assays for the detection of A. marginale and A. phagocytophilum 16S rRNA in plasma-free bovine peripheral blood samples is described. The duplex assay was able to detect as few as 100 copies of 16S rRNA of both A. marginale and A. phagocytophilum in the same reaction. The ratio of 16S rRNA to 16S DNA copies for A. marginale was determined to be 117.9:1 (95% confidence interval [95% CI], 100.7:1, 135.2:1). Therefore, the detection limit is the minimum infective unit of one A. marginale bacterium. The duplex assay detected nonequivalent molar ratios as high as 100-fold. Additionally, the duplex assay and a competitive enzyme-linked immunosorbent assay (cELISA) were used to screen 237 samples collected from herds in which anaplasmosis was endemic. When the cELISA was evaluated by the results of the qRT-PCR, its sensitivity and specificity for the detection of A. marginale infection were found to be 65.2% (95% CI, 55.3%, 75.1%) and 97.3% (95% CI, 94.7%, 99.9%), respectively. A. phagocytophilum infection was not detected in the samples analyzed. One- and two-way receiver operator characteristic curves were constructed in order to recommend the optimum negative cutoff value for the cELISA. Percentages of inhibition of 20 and 15.3% were recommended for the one- and two-way curves, respectively. In conclusion, the duplex real-time qRT-PCR assay is a highly sensitive and specific diagnostic tool for the accurate and precise detection of A. marginale and A. phagocytophilum infections in cattle. PMID:20463162

Defining the Enterovirus Diversity Landscape of a Fecal Sample: A Methodological Challenge?

PubMed

Faleye, Temitope Oluwasegun Cephas; Adewumi, Moses Olubusuyi; Adeniji, Johnson Adekunle

2016-01-12

Enteroviruses are a group of over 250 naked icosahedral virus serotypes that have been associated with clinical conditions that range from intrauterine enterovirus transmission withfataloutcome through encephalitis and meningitis, to paralysis. Classically, enterovirus detection was done by assaying for the development of the classic enterovirus-specific cytopathic effect in cell culture. Subsequently, the isolates were historically identified by a neutralization assay. More recently, identification has been done by reverse transcriptase-polymerase chain reaction (RT-PCR). However, in recent times, there is a move towards direct detection and identification of enteroviruses from clinical samples using the cell culture-independent RT semi-nested PCR (RT-snPCR) assay. This RT-snPCR procedure amplifies the VP1 gene, which is then sequenced and used for identification. However, while cell culture-based strategies tend to show a preponderance of certain enterovirus species depending on the cell lines included in the isolation protocol, the RT-snPCR strategies tilt in a different direction. Consequently, it is becoming apparent that the diversity observed in certain enterovirus species, e.g., enterovirus species B(EV-B), might not be because they are the most evolutionarily successful. Rather, it might stem from cell line-specific bias accumulated over several years of use of the cell culture-dependent isolation protocols. Furthermore, it might also be a reflection of the impact of the relative genome concentration on the result of pan-enterovirus VP1 RT-snPCR screens used during the identification of cell culture isolates. This review highlights the impact of these two processes on the current diversity landscape of enteroviruses and the need to re-assess enterovirus detection and identification algorithms in a bid to better balance our understanding of the enterovirus diversity landscape.

Defining the Enterovirus Diversity Landscape of a Fecal Sample: A Methodological Challenge?

PubMed Central

Faleye, Temitope Oluwasegun Cephas; Adewumi, Moses Olubusuyi; Adeniji, Johnson Adekunle

2016-01-01

Enteroviruses are a group of over 250 naked icosahedral virus serotypes that have been associated with clinical conditions that range from intrauterine enterovirus transmission withfataloutcome through encephalitis and meningitis, to paralysis. Classically, enterovirus detection was done by assaying for the development of the classic enterovirus-specific cytopathic effect in cell culture. Subsequently, the isolates were historically identified by a neutralization assay. More recently, identification has been done by reverse transcriptase-polymerase chain reaction (RT-PCR). However, in recent times, there is a move towards direct detection and identification of enteroviruses from clinical samples using the cell culture-independent RT semi-nested PCR (RT-snPCR) assay. This RT-snPCR procedure amplifies the VP1 gene, which is then sequenced and used for identification. However, while cell culture-based strategies tend to show a preponderance of certain enterovirus species depending on the cell lines included in the isolation protocol, the RT-snPCR strategies tilt in a different direction. Consequently, it is becoming apparent that the diversity observed in certain enterovirus species, e.g., enterovirus species B(EV-B), might not be because they are the most evolutionarily successful. Rather, it might stem from cell line-specific bias accumulated over several years of use of the cell culture-dependent isolation protocols. Furthermore, it might also be a reflection of the impact of the relative genome concentration on the result of pan-enterovirus VP1 RT-snPCR screens used during the identification of cell culture isolates. This review highlights the impact of these two processes on the current diversity landscape of enteroviruses and the need to re-assess enterovirus detection and identification algorithms in a bid to better balance our understanding of the enterovirus diversity landscape. PMID:26771630

MZC Gel Inhibits SHIV-RT and HSV-2 in Macaque Vaginal Mucosa and SHIV-RT in Rectal Mucosa.

PubMed

Calenda, Giulia; Villegas, Guillermo; Barnable, Patrick; Litterst, Claudia; Levendosky, Keith; Gettie, Agegnehu; Cooney, Michael L; Blanchard, James; Fernández-Romero, José A; Zydowsky, Thomas M; Teleshova, Natalia

2017-03-01

The Population Council's microbicide gel MZC (also known as PC-1005) containing MIV-150 and zinc acetate dihydrate (ZA) in carrageenan (CG) has shown promise as a broad-spectrum microbicide against HIV, herpes simplex virus (HSV), and human papillomavirus. Previous data show antiviral activity against these viruses in cell-based assays, prevention of vaginal and rectal simian-human immunodeficiency virus reverse transcriptase (SHIV-RT) infection, and reduction of vaginal HSV shedding in rhesus macaques and also excellent antiviral activity against HSV and human papillomavirus in murine models. Recently, we demonstrated that MZC is safe and effective against SHIV-RT in macaque vaginal explants. Here we established models of ex vivo SHIV-RT/HSV-2 coinfection of vaginal mucosa and SHIV-RT infection of rectal mucosa in macaques (challenge of rectal mucosa with HSV-2 did not result in reproducible tissue infection), evaluated antiviral activity of MZC, and compared quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay readouts for monitoring SHIV-RT infection. MZC (at nontoxic dilutions) significantly inhibited SHIV-RT in vaginal and rectal mucosas and HSV-2 in vaginal mucosa when present during viral challenge. Analysis of SHIV-RT infection and MZC activity by 1-step simian immunodeficiency virus gag quantitative RT-PCR and p27 enzyme-linked immunosorbent assay demonstrated similar virus growth dynamics and MZC activity by both methods and higher sensitivity of quantitative RT-PCR. Our data provide more evidence that MZC is a promising dual compartment multipurpose prevention technology candidate.

Nipah virus in the fruit bat Pteropus vampyrus in Sumatera, Indonesia.

PubMed

Sendow, Indrawati; Ratnawati, Atik; Taylor, Trevor; Adjid, R M Abdul; Saepulloh, Muharam; Barr, Jennifer; Wong, Frank; Daniels, Peter; Field, Hume

2013-01-01

Nipah virus causes periodic livestock and human disease with high case fatality rate, and consequent major economic, social and psychological impacts. Fruit bats of the genus Pteropus are the natural reservoir. In this study, we used real time PCR to screen the saliva and urine of P. vampyrus from North Sumatera for Nipah virus genome. A conventional reverse transcriptase (RT-PCR) assay was used on provisionally positive samples to corroborate findings. This is the first report of Nipah virus detection in P. vampyrus in Sumatera, Indonesia.

Capsicum annum, a new host of watermelon mosaic virus.

PubMed

Hajizadeh, Mohammad; Mohammadi, Kazhal

2016-03-01

The occurrence of Watermelon mosaic virus (WMV) in sweet pepper (Capsicum annuum L.) in Kurdistan province, Iran was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and partial characterization of coat protein. To the best of our knowledge, this is the first report of WMV infecting C. annuum, adding a new host to list of more than 170 species infected by this virus.

Sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction detecting feline coronavirus mutations in effusion and serum/plasma of cats to diagnose feline infectious peritonitis.

PubMed

Felten, Sandra; Leutenegger, Christian M; Balzer, Hans-Joerg; Pantchev, Nikola; Matiasek, Kaspar; Wess, Gerhard; Egberink, Herman; Hartmann, Katrin

2017-08-02

Feline coronavirus (FCoV) exists as two pathotypes, and FCoV spike gene mutations are considered responsible for the pathotypic switch in feline infectious peritonitis (FIP) pathogenesis. The aim of this study was to evaluate sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction (RT-PCR) specifically designed to detect FCoV spike gene mutations at two nucleotide positions. It was hypothesized that this test would correctly discriminate feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV). The study included 63 cats with signs consistent with FIP. FIP was confirmed in 38 cats. Twenty-five control cats were definitively diagnosed with a disease other than FIP. Effusion and/or serum/plasma samples were examined by real-time RT-PCR targeting the two FCoV spike gene fusion peptide mutations M1058 L and S1060A using an allelic discrimination approach. Sensitivity, specificity, negative and positive predictive values including 95% confidence intervals (95% CI) were calculated. FIPV was detected in the effusion of 25/59 cats, one of them being a control cat with chronic kidney disease. A mixed population of FIPV/FECV was detected in the effusion of 2/59 cats; all of them had FIP. RT-PCR was negative or the pathotype could not be determined in 34/59 effusion samples. In effusion, sensitivity was 68.6% (95% CI 50.7-83.2), specificity was 95.8% (95% CI 78.9-99.9). No serum/plasma samples were positive for FIPV. Although specificity of the test in effusions was high, one false positive result occurred. The use of serum/plasma cannot be recommended due to a low viral load in blood.

Prostate stem cell antigen (PSCA) mRNA expression in peripheral blood in patients with benign prostatic hyperplasia and/or prostate cancer.

PubMed

Fawzy, Mohamed S; Mohamed, Randa H; Elfayoumi, Abdel-Rahman R

2015-03-01

The aim of this study was to determine whether detection of prostate stem cell antigen (PSCA) expression in BPH might be associated with the subsequent presence of Prostate cancer (PCa) and also to determine whether detection of PSCA expression has potential for prognosis in PCa. This study was comprised of 112 PCa patients, 111 BPH patients and 120 control subjects. We employed reverse-transcriptase polymerase chain reaction (RT-PCR) to detect PSCA mRNA-bearing cells in peripheral blood. PSCA mRNA was detected in the peripheral blood of 71.4% PCa patients and in 13.5% of patients with BPH by RT-PCR. PSCA was positive in 80% of high-grade diseases compared with 20% of low-grade diseases (P = 0.01). Whereas only 38.8% of prostate-confined diseases were PSCA positive, 61.2% of extraprostatic diseases were PSCA positive (P < 0.001). Patients with a lymphovascular invasion of tumor emboli tended to be PSCA positive (P = 0.02). BPH patients with RT-PCR PSCA positive were significantly more likely to develop prostate cancer (OR = 16, 95% CI = 8.1-31.6, P < 0.001). In conclusion, RT-PCR PSCA positivity is significantly associated with the Gleason score, LV tumor emboli and whether or not the tumor was organ confined. In this study, RT-PCR PSCA detection may be a promising tumor marker of diagnostic and metastasis detection for patients with prostate cancer. Also, it may be an important test for predicting BPH patients who are at high risk of subsequent cancer development.

Evaluation of Immunomagnetic Separation Method for the Recovery of Hepatitis A Virus and GI.1 and GII.4 Norovirus Strains Seeded on Oyster and Mussel.

PubMed

Ha, Ji-Hyoung; Choi, Changsun; Ha, Sang-Do

2014-12-01

Outbreaks of viral diseases are frequently associated with the consumption of minimally processed shellfish. Among the viruses in these outbreaks, hepatitis A virus (HAV) and human norovirus (NoV) have been increasingly reported as the most common food-borne pathogens. These viruses must be concentrated in tested samples in order to be detected. In this study, a method for the detection of NoV and HAV in shellfish using an immuno-magnetic separation (IMS) procedure combined with reverse transcriptase (RT)-PCR was developed. The IMS/RT-PCR method was applied to investigate the recovery rates of HAV, NoV GI.1, and GII.4 from oyster and mussel. Based on IMS/RT-PCR results, recovery rates for HAV from oyster and mussel test samples were 2.4 and 1.1%, respectively. The NoV GI.1 recovery rates from oyster and mussel samples were 4.9-9.2% (mean 6.9%) and 4.3-8.6% (mean 6.2%), respectively, and the NoV GII.4 recovery rates were 8.8 and 8.5%, respectively. These results verified that HAV, NoV GI.1, and GII.4 can be detected in all the test samples using the IMS/RT-PCR method, although the three inoculated viruses were recovered with low efficiency. In conclusion, the IMS/RT-PCR method can be used to efficiently and rapidly detect viruses such as HAV and NoV in shellfish such as oyster and mussel.

Performance characteristics of a reverse transcriptase-polymerase chain reaction assay for the detection of tumor-specific fusion transcripts from archival tissue.

PubMed

Fritsch, Michael K; Bridge, Julia A; Schuster, Amy E; Perlman, Elizabeth J; Argani, Pedram

2003-01-01

Pediatric small round cell tumors still pose tremendous diagnostic problems. In difficult cases, the ability to detect tumor-specific gene fusion transcripts for several of these neoplasms, including Ewing sarcoma/peripheral primitive neuroectodermal tumor (ES/PNET), synovial sarcoma (SS), alveolar rhabdomyosarcoma (ARMS), and desmoplastic small round cell tumor (DSRCT) using reverse transcriptase-polymerase chain reaction (RT-PCR), can be extremely helpful. Few studies to date, however, have systematically examined several different tumor types for the presence of multiple different fusion transcripts in order to determine the specificity and sensitivity of the RT-PCR method, and no study has addressed this issue for formalin-fixed material. The objectives of this study were to address the specificity, sensitivity, and practicality of such an assay applied strictly to formalin-fixed tissue blocks. Our results demonstrate that, for these tumors, the overall sensitivity for detecting each fusion transcript is similar to that reported in the literature for RT-PCR on fresh or formalin-fixed tissues. The specificity of the assay is very high, being essentially 100% for each primer pair when interpreting the results from visual inspection of agarose gels. However, when these same agarose gels were examined using Southern blotting, a small number of tumors also yielded reproducibly detectable weak signals for unexpected fusion products, in addition to a strong signal for the expected fusion product. Fluorescence in situ hybridization (FISH) studies in one such case indicated that a rearrangement that would account for the unexpected fusion was not present, while another case was equivocal. The overall specificity for each primer pair used in this assay ranged from 94 to 100%. Therefore, RT-PCR using formalin-fixed paraffin-embedded tissue sections can be used to detect chimeric transcripts as a reliable, highly sensitive, and highly specific diagnostic assay. However, we strongly suggest that the final interpretation of the results from this assay be viewed in light of the other features of the case, including clinical history, histology, and immunohistochemistry, by the diagnostic pathologist. Additional studies such as FISH may be useful in clarifying the nature of equivocal or unexpected results.

Diagnostic performance of influenza viruses and RSV rapid antigen detection tests in children in tertiary care.

PubMed

Moesker, F M; van Kampen, J J A; Aron, G; Schutten, M; van de Vijver, D A M C; Koopmans, M P G; Osterhaus, A D M E; Fraaij, P L A

2016-06-01

Rapid antigen detection tests (RADTs) are increasingly used to detect influenza viruses and respiratory syncytial virus (RSV). However, their sensitivity and specificity are a matter of debate, challenging their clinical usefulness. Comparing diagnostic performances of BinaxNow Influenza AB(®) (BNI) and BinaxNow RSV(®) (BNR), to those of real-time reverse transcriptase PCR (RT-PCR), virus isolation and direct immunofluorescence (D-IF) in paediatric patients. Between November 2005 and September 2013, 521 nasal washings from symptomatic children (age <5 years) attending our tertiary care centre were tested, with a combination of the respective assays using RT-PCR as gold standard. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of BNI were 69% (confidence interval [CI] [51-83]), 96% [94-97], 55% [39-70] and 98% [96-99] respectively. Of eleven false-negative samples, RT-PCR Ct-values were higher than all RT-PCR positive test results (27 vs 22, p=0.012). Of twenty false-positive samples, none were culture positive and two tested positive in D-IF. Sensitivity, specificity, PPV and NPV for BNR were 79% [73-85], 98% [96-99], 97% [93-99] and 88% [84-91]. Of the 42 false-negative samples the median Ct-value was higher than that of all RT-PCR positive samples (31 vs 23, p<0.0001). Five false-positive samples were detected. Three of these tested positive for RSV in virus isolation and D-IF. RADTs have a high specificity with BNR being superior to BNI. However, their relative low sensitivity limits their usefulness for clinical decision making in a tertiary care paediatric hospital. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Diagnostic Yield of Laboratory Methods and Value of Viral Genotyping during an Outbreak of Mumps in a Partially Vaccinated Population in British Columbia, Canada.

PubMed

Nunn, Alexandra; Masud, Shazia; Krajden, Mel; Naus, Monika; Jassem, Agatha N

2018-05-01

Mumps remains endemic in North America despite routine use of the measles, mumps, and rubella (MMR) vaccine. In 2016, an outbreak of mumps in British Columbia, Canada, provided an opportunity to determine the diagnostic utility of laboratory testing methods. Specimens from patients with clinical mumps were tested for infection using a commercial enzyme-linked immunosorbent assay (ELISA) for antibody detection and an in-house reverse transcriptase PCR (RT-PCR) targeting viral fusion and small hydrophobic (SH) genes. Viral genotyping was performed by SH gene sequencing. Laboratory data was linked with epidemiologic case data. Of the 139 confirmed cases, 94 (68%) had reported or documented history of MMR vaccination. Specimens were typically collected 1 day (for buccal and IgM tests) or 2 days (for urine tests) after symptom onset. Most confirmed cases (69%) were confirmed by buccal swab RT-PCR. Among cases tested by multiple methods, the percent positivity for buccal swab RT-PCR was 90% (96/107) compared to 43% (30/69) for both IgM ELISA and urine RT-PCR. Mumps IgM detection was higher in confirmed cases with no history of vaccination than in those with history (64% versus 34%, P = 0.02). The outbreak strain was identified as genotype G related to MuVi/Sheffield.GBR/1.05 but with conserved variations in five nucleotides within the SH gene that allowed linkage of geographically distinct cases. In conclusion, RT-PCR of buccal specimens had the highest diagnostic yield during a mumps outbreak in a partially vaccinated population. To optimize mumps diagnostic potential, clinicians should collect specimens depending on when the patient presents for care and their immunization history. © Crown copyright 2018.

Retrotransposon expression and incorporation of cloned human and mouse retroelements in human spermatozoa.

PubMed

Lazaros, Leandros; Kitsou, Chrysoula; Kostoulas, Charilaos; Bellou, Sofia; Hatzi, Elissavet; Ladias, Paris; Stefos, Theodoros; Markoula, Sofia; Galani, Vasiliki; Vartholomatos, Georgios; Tzavaras, Theodore; Georgiou, Ioannis

2017-03-01

To investigate the expression of long interspersed element (LINE) 1, human endogenous retrovirus (HERV) K10, and short interspersed element-VNTR-Alu element (SVA) retrotransposons in ejaculated human spermatozoa by means of reverse-transcription (RT) polymerase chain reaction (PCR) analysis as well as the potential incorporation of cloned human and mouse active retroelements in human sperm cell genome. Laboratory study. University research laboratories and academic hospital. Normozoospermic and oligozoospermic white men. RT-PCR analysis was performed to confirm the retrotransposon expression in human spermatozoa. Exogenous retroelements were tagged with a plasmid containing a green fluorescence (EGFP) retrotransposition cassette, and the de novo retrotransposition events were tested with the use of PCR, fluorescence-activated cell sorting analysis, and confocal microscopy. Retroelement expression in human spermatozoa, incorporation of cloned human and mouse active retroelements in human sperm genome, and de novo retrotransposition events in human spermatozoa. RT-PCR products of expressed human LINE-1, HERV-K10, and SVA retrotransposons were observed in ejaculated human sperm samples. The incubation of human spermatozoa with either retrotransposition-active human LINE-1 and HERV-K10 or mouse reverse transcriptase-deficient VL30 retrotransposons tagged with an EGFP-based retrotransposition cassette led to EGFP-positive spermatozo; 16.67% of the samples were positive for retrotransposition. The respective retrotransposition frequencies for the LINE-1, HERV-K10, and VL30 retrotransposons in the positive samples were 0.34 ± 0.13%, 0.37 ± 0.17%, and 0.30 ± 0.14% per sample of 10,000 spermatozoa. Our results show that: 1) LINE-1, HERV-K10, and SVA retrotransposons are transcriptionally expressed in human spermatozoa; 2) cloned active retroelements of human and mammalian origin can be incorporated in human sperm genome; 3) active reverse transcriptases exist in human spermatozoa; and 4) de novo retrotransposition events occur in human spermatozoa. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

A comparative kinetic RT/-PCR strategy for the quantitation of mRNAs in microdissected human renal biopsy specimens.

PubMed

Del Prete, D; Forino, M; Gambaro, G; D'Angelo, A; Baggio, B; Anglani, F

1998-01-01

Molecular biology techniques, to be applicable to a diagnostic renal biopsy specimen, should (1) be highly sensitive to be performed on a very small quantity of tissue; (2) be quantitative because they have to analyze genes normally expressed in the tissue and (3) allow the analysis of as large a number of genes as possible. Among different methods, only the reverse-transcriptase polymerase chain reaction (RT/-PCR) might comply with previous requisites, but the few RT/-PCR examples on renal biopsies in the literature do not allow starting RNA quantification and quality control; furthermore they have the drawback of analyzing only few genes. In an ongoing study to assess the expression of a number of genes in glomeruli and in tubulointerstitium of patients with different nephropathies, we developed a comparative RT/-PCR kinetic strategy based on the purification and quantification of total glomerular and tubulointerstitial RNA and on the use of an internal standard, the housekeeping gene G3PDH. We demonstrate that in microdissected diagnostic renal biopsies (1) glomerular and interstitial starting RNA can be quantified; (2) the G3PDH gene may be used both as an internal standard and as an indirect marker of RNA integrity; (3) as low as 28 ng of total RNA is sufficient to obtain PCR products of eight genes, and (4) it is worth to operate on microdissected biopsy specimens because of the different expression of genes in the two renal compartments.

Mutational analysis of the reverse transcriptase and ribonuclease H domains of the human foamy virus.

PubMed Central

Kögel, D; Aboud, M; Flügel, R M

1995-01-01

Human foamy or spuma virus (HFV) codes for a distinct set of pol gen products. To determine the minimal requirements for the HFV enzymatic activities, defined residues of the reverse transcriptase (RT) and ribo-nuclease H (RNase H) domain of the HFV pol gene were mutated by site-specific PCR mutagenesis. The mutant gene products were bacterially expressed, purified by Ni2+ chelate affinity chromatography and characterised by Western blotting. The enzymatic activities of the individual recombinant HFV pol mutant proteins were characterised by the situ RT, RNase H and RNase H assays. Two substitution mutants reached RT activity levels higher than that of the intact recombinant HFV RT-RH-His. When the catalytically essential D508 was substituted by A508, 5% of RNase H activity was retained while DNA polymerase activity increased 2-fold. A deletion of 11 amino acid residues in the hinge region completely abolished DNA polymerase while RNase H activity decreased 2-fold. A deletion mutant in the C-terminal RH domain showed no RNase H but retained RNase H activity indicating that the activities are genetically separable. The combined data reveal that the HFV DNA polymerase and RNase H activities are interdependent. Images PMID:7544460

Dengue Virus in Bats from Southeastern Mexico

PubMed Central

Sotomayor-Bonilla, Jesús; Chaves, Andrea; Rico-Chávez, Oscar; Rostal, Melinda K.; Ojeda-Flores, Rafael; Salas-Rojas, Mónica; Aguilar-Setien, Álvaro; Ibáñez-Bernal, Sergio; Barbachano-Guerrero, Arturo; Gutiérrez-Espeleta, Gustavo; Aguilar-Faisal, J. Leopoldo; Aguirre, A. Alonso; Daszak, Peter; Suzán, Gerardo

2014-01-01

To identify the relationship between landscape use and dengue virus (DENV) occurrence in bats, we investigated the presence of DENV from anthropogenically changed and unaltered landscapes in two Biosphere Reserves: Calakmul (Campeche) and Montes Azules (Chiapas) in southern Mexico. Spleen samples of 146 bats, belonging to 16 species, were tested for four DENV serotypes with standard reverse transcriptase polymerase chain reaction (RT-PCR) protocols. Six bats (4.1%) tested positive for DENV-2: four bats in Calakmul (two Glossophaga soricina, one Artibeus jamaicensis, and one A. lituratus) and two bats in Montes Azules (both A. lituratus). No effect of anthropogenic disturbance on the occurrence of DENV was detected; however, all three RT-PCR–positive bat species are considered abundant species in the Neotropics and well-adapted to disturbed habitats. To our knowledge, this study is the first study conducted in southeastern Mexico to identify DENV-2 in bats by a widely accepted RT-PCR protocol. The role that bats play on DENV's ecology remains undetermined. PMID:24752688

[Expression of cell adhesion molecules in acute leukemia cell].

PubMed

Ju, Xiaoping; Peng, Min; Xu, Xiaoping; Lu, Shuqing; Li, Yao; Ying, Kang; Xie, Yi; Mao, Yumin; Xia, Fang

2002-11-01

To investigate the role of cell adhesion molecule in the development and extramedullary infiltration (EI) of acute leukemia. The expressions of neural cell adhesion molecule (NCAM) gene, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1) genes in 25 acute leukemia patients bone marrow cells were detected by microarray and reverse transcriptase-polymerase chain reaction (RT-PCR). The expressions of NCAM, ICAM-1 and VCAM-1 gene were significantly higher in acute leukemia cells and leukemia cells with EI than in normal tissues and leukemia cells without EI, respectively, both by cDNA microarray and by RT-PCR. The cDNA microarray is a powerful technique in analysis of acute leukemia cells associated genes. High expressions of cell adhesion molecule genes might be correlated with leukemia pathogenesis and infiltration of acute leukemia cell.

Progress towards Rapid Detection of Measles Vaccine Strains: a Tool To Inform Public Health Interventions

PubMed Central

2016-01-01

ABSTRACT Rapid differentiation of vaccine from wild-type strains in suspect measles cases is a valuable epidemiological tool that informs the public health response to this highly infectious disease. Few public health laboratories sequence measles virus-positive specimens to determine genotype, and the vaccine-specific real-time reverse transcriptase PCR (rRT-PCR) assay described by F. Roy et al. (J. Clin. Microbiol. 55:735–743, 2017, https://doi.org/10.1128/JCM.01879-16) offers a rapid, easily adoptable method to identify measles vaccine strains in suspect cases. PMID:28003421

Progress towards Rapid Detection of Measles Vaccine Strains: a Tool To Inform Public Health Interventions.

PubMed

Hacker, Jill K

2017-03-01

Rapid differentiation of vaccine from wild-type strains in suspect measles cases is a valuable epidemiological tool that informs the public health response to this highly infectious disease. Few public health laboratories sequence measles virus-positive specimens to determine genotype, and the vaccine-specific real-time reverse transcriptase PCR (rRT-PCR) assay described by F. Roy et al. (J. Clin. Microbiol. 55:735-743, 2017, https://doi.org/10.1128/JCM.01879-16) offers a rapid, easily adoptable method to identify measles vaccine strains in suspect cases. Copyright © 2017 American Society for Microbiology.

Transcriptional expression of type-I interferon response genes and stability of housekeeping genes in the human endometrium and endometriosis.

PubMed

Vestergaard, Anna L; Knudsen, Ulla B; Munk, Torben; Rosbach, Hanne; Martensen, Pia M

2011-04-01

Endometriosis is a painful chronic female disease defined by the presence of endometrial tissue implants in ectopic (Ec) locations. The pathogenesis is much debated, and type-I interferons (IFNs) could be involved. The expression of genes of the type-I IFN response were profiled by a specific PCR array of RNA obtained from Ec and eutopic (Eu) endometrium collected from nine endometriosis patients and nine healthy control women. Transcriptional expression levels of selected IFN-regulated and housekeeping genes (HKGs) were investigated by real-time quantitative reverse transcriptase PCR (qRT-PCR). Stably expressed HKGs for valid normalization of transcriptional studies of endometrium and endometriosis have not yet been published. Here, seven HKGs were evaluated for stability using the GeNorm and NormFinder software. A normalization factor based on HMBS, TBP and YWHAZ expression was suitable for normalization of qRT-PCR studies of Eu versus Ec endometrium. In the endometrial cell lines HEC1A, HEC1B, Ishikawa and RL95-2, HMBS and HPRT1 were the most stably expressed. The IFN-specific PCR array indicated significantly different expression of the genes BST2, COL16A1, HOXB2 and ISG20 between the endometrial tissue types. However, by correctly normalized qRT-PCR, levels of BST2, COL16A1 and the highly type-I IFN-stimulated genes ISG12A and 6-16 displayed insignificant variations. Conversely, HOXB2 and ISG20 transcriptions were significantly reduced in endometriosis lesions compared with endometrium from endometriosis patients and healthy controls. In conclusion, appropriate HKGs for normalization of qRT-PCR studies of endometrium and endometriosis have been identified here. Abolished expression of ISG20 and HOX genes could be important in endometriosis.

Incidence of enteroviruses in Mamala Bay, Hawaii using cell culture and direct polymerase chain reaction methodologies.

PubMed

Reynolds, K A; Roll, K; Fujioka, R S; Gerba, C P; Pepper, I L

1998-06-01

The consequence of point and nonpoint pollution sources, discharged into marine waters, on public recreational beaches in Mamala Bay, Hawaii was evaluated using virus cell culture and direct reverse transcriptase-polymerase chain reaction (RT-PCR). Twelve sites, nine marine, two freshwater (one stream and one canal), and one sewage, were assessed either quarterly or monthly for 1 year to detect the presence of human enteric viruses. Water samples were concentrated from initial volumes of 400 L to final volumes of 30 mL using Filterite electronegative cartridge filters and a modified beef extract elution procedure. Cell culture was applied using the Buffalo Green Monkey kidney cell line to analyze samples for enteroviruses. Positive samples were also evaluated by RT-PCR, using enterovirus-specific primers. Levels of RT-PCR inhibition varied with each concentrated sample. Resin column purification increased PCR detection sensitivity by at least one order of magnitude in a variety of sewage outfall and recreational marine water samples but not in the freshwater canal samples. Using cell culture, viable enteroviruses were found in 50 and 17% of all outfall and canal samples, respectively. Samples were positive at beaches 8% of the time. These data illustrate the potential public health hazard associated with recreational waters. Using direct PCR, viruses were detected at the outfall but were not found in any beach or canal samples, in part, owing to substances that inhibit PCR. Therefore, conventional cell culture is the most effective means of detecting low levels of infectious enteroviruses in environmental waters, whereas direct RT-PCR is rendered less effective by inhibitory compounds and low equivalent reaction volumes.

Isolation and characterization of a FLOWERING LOCUS T homolog from pineapple (Ananas comosus (L.) Merr).

PubMed

Lv, LingLing; Duan, Jun; Xie, JiangHui; Wei, ChangBin; Liu, YuGe; Liu, ShengHui; Sun, GuangMing

2012-09-01

FLOWERING LOCUS T (FT)-like genes are crucial regulators of flowering in angiosperms. A homolog of FT, designated as AcFT (GenBank ID: HQ343233), was isolated from pineapple cultivar Comte de Paris by reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The cDNA sequence of AcFT is 915 bp in length and contains an ORF of 534 bp, which encodes a protein of 177 aa. Molecular weight was 19.9 kDa and isoelectric point was 6.96. The deduced protein sequence of AcFT was 84% and 82% identical to homologs encoded by CgFT in Cymbidium goeringii and OgFT in Oncidium Gower Ramsey respectively. Quantitative real-time PCR (qRT-PCR) analyses showed that the expression of AcFT was high in flesh and none in leaves. qRT-PCR analyses in different stages indicated that the expression of AcFT reached the highest level on 40 d after flower inducing, when the multiple fruit and floral organs were forming. The 35S::AcFT transgenic Arabidopsis plants flowered earlier and had more inflorescences or branches than wild type plants. Copyright © 2012 Elsevier B.V. All rights reserved.

Transfer and targeted overexpression of γ-tocopherol methyltransferase (γ-TMT) gene using seed-specific promoter improves tocopherol composition in Indian soybean cultivars.

PubMed

Arun, Muthukrishnan; Subramanyam, Kondeti; Theboral, Jeevaraj; Sivanandhan, Ganeshan; Rajesh, Manoharan; Kapil Dev, Gnanajothi; Jaganath, Balusamy; Manickavasagam, Markandan; Girija, Shanmugam; Ganapathi, Andy

2014-02-01

Soybean oil contains high levels of tocopherols which are an important source of vitamin E in human diet. The conversion of γ- to α-tocopherol catalyzed by γ-tocopherol methyltransferase (γ-TMT) is found to be the rate limiting factor in soybean which influences the tocopherol composition. Using Agrobacterium-mediated transformation, we overexpressed the γ-TMT gene of Perilla frutescens under the control of the seed-specific promoter vicillin in cultivar Pusa 16. Transgene integration and expression was confirmed in five independently transformed GUS positive soybean plants by polymerase chain reaction (PCR), Southern hybridization, and reverse transcriptase-PCR (RT-PCR). High-performance liquid chromatography (HPLC) analysis showed that overexpression of Pf-γ-TMT resulted in efficient conversion of γ-tocopherol to α-tocopherol and concomitant increase in seed α-tocopherol content in RT-PCR positive plants. The protocol was successfully applied to three more cultivars PK 416, Gujarat soybean 1, and VL soya 1 in which seeds of transformed plants showed elevated level of α-tocopherol than wild-type seeds.

Comparison of IHC, FISH and RT-PCR methods for detection of ALK rearrangements in 312 non-small cell lung cancer patients in Taiwan.

PubMed

Wu, Yi-Cheng; Chang, Il-Chi; Wang, Chi-Liang; Chen, Tai-Di; Chen, Ya-Ting; Liu, Hui-Ping; Chu, Yen; Chiu, Yu-Ting; Wu, Tzu-Hua; Chou, Li-Hui; Chen, Yi-Rong; Huang, Shiu-Feng

2013-01-01

Recently Echinoderm microtubule-associated protein-like 4- anaplastic lymphoma kinase (EML4-ALK) fusion gene has become an important biomarker for ALK tyrosine kinase inhibitor (crizotinib) treatment in NSCLC. However, the best detection method and the significance of EML4-ALK variant types remain uncertain. Reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescence in Situ hybridization (FISH) and Immunohistochemical (IHC) stain were performed on tumor tissues of 312 NSCLC patients for detection of ALK rearrangements. Mutation analyses for EGFR and KRAS genes were also performed. Thirteen of the 312 patients (4.17%) had ALK rearrangements detected by RT-PCR. If RT-PCR data was used as the gold standard, FISH tests had a low sensitivity (58.33%), but very good specificity (99.32%). IHC stain had better sensitivity (91.67%) than FISH, but lower specificity (79.52%), when the cut off was IHC2+. All of the 8 patients with high abundance of EML4-ALK positive cells in tumor tissues (assessed by the signal intensities of the RT-PCR product), were also have high expression of ALK protein (IHC3+), and positive for FISH, except one failed in FISH. Variants 3a+3b (4/5, 80%) of EML4-ALK fusion gene were more common to have high abundance of EML4-ALK positive cells in tumor tissues than variant 1 (1/3, 33.3%). Meta-analysis of the published data of 2273 NSCLC patients revealed that variant 3 (23/44, 52.3%) was the most common type in Chinese population, while variant 1 (28/37, 75.7%) was most common in Caucasian. Among the three detection methods, RT-PCR could detect not only the presence of EML4-ALK fusion gene and their variant types, but also the abundance of EML4-ALK positive cells in NSCLC tumor tissues. The latter two factors might affect the treatment response to anti-ALK inhibitor. Including RT-PCR as a diagnostic test for ALK inhibitor treatment in the prospective clinical trials is recommended.

Comparison of IHC, FISH and RT-PCR Methods for Detection of ALK Rearrangements in 312 Non-Small Cell Lung Cancer Patients in Taiwan

PubMed Central

Wang, Chi-Liang; Chen, Tai-Di; Chen, Ya-Ting; Liu, Hui-Ping; Chu, Yen; Chiu, Yu-Ting; Wu, Tzu-Hua; Chou, Li-Hui; Chen, Yi-Rong; Huang, Shiu-Feng

2013-01-01

Background Recently Echinoderm microtubule-associated protein-like 4- anaplastic lymphoma kinase (EML4-ALK) fusion gene has become an important biomarker for ALK tyrosine kinase inhibitor (crizotinib) treatment in NSCLC. However, the best detection method and the significance of EML4-ALK variant types remain uncertain. Methods Reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescence in Situ hybridization (FISH) and Immunohistochemical (IHC) stain were performed on tumor tissues of 312 NSCLC patients for detection of ALK rearrangements. Mutation analyses for EGFR and KRAS genes were also performed. Results Thirteen of the 312 patients (4.17%) had ALK rearrangements detected by RT-PCR. If RT-PCR data was used as the gold standard, FISH tests had a low sensitivity (58.33%), but very good specificity (99.32%). IHC stain had better sensitivity (91.67%) than FISH, but lower specificity (79.52%), when the cut off was IHC2+. All of the 8 patients with high abundance of EML4-ALK positive cells in tumor tissues (assessed by the signal intensities of the RT-PCR product), were also have high expression of ALK protein (IHC3+), and positive for FISH, except one failed in FISH. Variants 3a+3b (4/5, 80%) of EML4-ALK fusion gene were more common to have high abundance of EML4-ALK positive cells in tumor tissues than variant 1 (1/3, 33.3%). Meta-analysis of the published data of 2273 NSCLC patients revealed that variant 3 (23/44, 52.3%) was the most common type in Chinese population, while variant 1 (28/37, 75.7%) was most common in Caucasian. Conclusions Among the three detection methods, RT-PCR could detect not only the presence of EML4-ALK fusion gene and their variant types, but also the abundance of EML4-ALK positive cells in NSCLC tumor tissues. The latter two factors might affect the treatment response to anti-ALK inhibitor. Including RT-PCR as a diagnostic test for ALK inhibitor treatment in the prospective clinical trials is recommended. PMID:23951022

The sensitivity and specificity of Lassa virus IgM by ELISA as screening tool at early phase of Lassa fever infection

PubMed Central

Ibekwe, Titus S.; Nwegbu, Maxwell M.; Asogun, Daniel; Adomeh, Donatus I.; Okokhere, Peter O.

2012-01-01

Background: Early diagnosis, prompt treatment, and disease containment are vital measures in the management of Lassa fever (LF), a lethal and contagious arenaviral hemorrhagic disease prevalent in West Africa. Lassa Virus (LAV)-specific Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) test, the gold standard for diagnosis, is unavailable in most centers. Serologic detection of LAV IgM is a more accessible tool and this work was to investigate its adequacy as an early marker for LF. Patients and Methods: A prospective case–control study conducted July 2007-March 2011 in a tertiary referral health center in Nigeria. Blood samples for test and control were evaluated for Lassa specific antigens and IgM using RT-PCR (primers S36+ and LVS 339) and indirect ELISA (Lassa Nucleo-protein (NP)-Antigen) respectively. RT-PCR outcome was used as standard to test for the sensitivity and specificity of IgM. Results: Of the 37 confirmed cases of LF infection by RT-PCR, 21 (57%) were IgM positive. Amongst the 35 confirmed negative cases (control group), eight were IgM positive. The diagnostic sensitivity and specificity of the IgM assay were 57% and 77% respectively. The negative and positive predictive values of the IgM serological assay were 63% and 72%, respectively, while the efficiency of the test was 67%. Conclusion: The specificity and sensitivity of IgM as a screening tool for early detection of LF appear weak and, hence, the need for a reliable LF “rapid screening kit” since RT-PCR is unavailable in most centers. In the interim, “high clinical index of suspicion,” irrespective of IgM status, requires urgent referral to confirmatory centers. PMID:23661877

The sensitivity and specificity of Lassa virus IgM by ELISA as screening tool at early phase of Lassa fever infection.

PubMed

Ibekwe, Titus S; Nwegbu, Maxwell M; Asogun, Daniel; Adomeh, Donatus I; Okokhere, Peter O

2012-10-01

Early diagnosis, prompt treatment, and disease containment are vital measures in the management of Lassa fever (LF), a lethal and contagious arenaviral hemorrhagic disease prevalent in West Africa. Lassa Virus (LAV)-specific Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) test, the gold standard for diagnosis, is unavailable in most centers. Serologic detection of LAV IgM is a more accessible tool and this work was to investigate its adequacy as an early marker for LF. A prospective case-control study conducted July 2007-March 2011 in a tertiary referral health center in Nigeria. Blood samples for test and control were evaluated for Lassa specific antigens and IgM using RT-PCR (primers S36+ and LVS 339) and indirect ELISA (Lassa Nucleo-protein (NP)-Antigen) respectively. RT-PCR outcome was used as standard to test for the sensitivity and specificity of IgM. Of the 37 confirmed cases of LF infection by RT-PCR, 21 (57%) were IgM positive. Amongst the 35 confirmed negative cases (control group), eight were IgM positive. The diagnostic sensitivity and specificity of the IgM assay were 57% and 77% respectively. The negative and positive predictive values of the IgM serological assay were 63% and 72%, respectively, while the efficiency of the test was 67%. The specificity and sensitivity of IgM as a screening tool for early detection of LF appear weak and, hence, the need for a reliable LF "rapid screening kit" since RT-PCR is unavailable in most centers. In the interim, "high clinical index of suspicion," irrespective of IgM status, requires urgent referral to confirmatory centers.

Disappearance of Ph1 chromosome with intensive chemotherapy and detection of minimal residual disease by polymerase chain reaction in a patient with blast crisis of chronic myelogenous leukemia.

PubMed

Honda, H; Miyagawa, K; Endo, M; Takaku, F; Yazaki, Y; Hirai, H

1993-06-01

We diagnosed a patient with chronic myelogenous leukemia (CML) in chronic phase (CP) on the basis of clinical findings, Ph1 chromosome detected by cytogenetic analysis, and bcr-abl fusion mRNA detected by reverse transcriptase-dependent polymerase chain reaction (RT-PCR). One month after diagnosis, the patient developed extramedullary blast crisis in the lymph nodes, and then medullary blast crisis in the bone marrow, in which different surface markers were shown. Combination chemotherapy with BH-AC, VP16, and mitoxantrone was administered; this resulted in rapid disappearance of the lymphadenopathy, restoration of normal hematopoiesis, and no Ph1 chromosome being detected by cytogenetic analysis. RT-PCR performed to detect the residual Ph1 clone revealed that although the Ph1 clone was preferentially suppressed, it was still residual. The intensive chemotherapy regimen preferentially suppressed the Ph1-positive clone and led to both clinical and cytogenetic remission in this patient with BC of CML; we suggest that RT-PCR is a sensitive and useful method for detecting minimal residual disease during the clinical course of this disease.

Evaluation of NGS and RT-PCR Methods for ALK Rearrangement in European NSCLC Patients: Results from the European Thoracic Oncology Platform Lungscape Project.

PubMed

Letovanec, Igor; Finn, Stephen; Zygoura, Panagiota; Smyth, Paul; Soltermann, Alex; Bubendorf, Lukas; Speel, Ernst-Jan; Marchetti, Antonio; Nonaka, Daisuke; Monkhorst, Kim; Hager, Henrik; Martorell, Miguel; Sejda, Aleksandra; Cheney, Richard; Hernandez-Losa, Javier; Verbeken, Eric; Weder, Walter; Savic, Spasenija; Di Lorito, Alessia; Navarro, Atilio; Felip, Enriqueta; Warth, Arne; Baas, Paul; Meldgaard, Peter; Blackhall, Fiona; Dingemans, Anne-Marie; Dienemann, Hendrik; Dziadziuszko, Rafal; Vansteenkiste, Johan; O'Brien, Cathal; Geiger, Thomas; Sherlock, Jon; Schageman, Jeoffrey; Dafni, Urania; Kammler, Roswitha; Kerr, Keith; Thunnissen, Erik; Stahel, Rolf; Peters, Solange

2018-03-01

The reported prevalence of ALK receptor tyrosine kinase gene (ALK) rearrangement in NSCLC ranges from 2% to 7%. The primary standard diagnostic method is fluorescence in situ hybridization (FISH). Recently, immunohistochemistry (IHC) has also proved to be a reproducible and sensitive technique. Reverse-transcriptase polymerase chain reaction (RT-PCR) has also been advocated, and most recently, the advent of targeted next-generation sequencing (NGS) for ALK and other fusions has become possible. This study compares anaplastic lymphoma kinase (ALK) evaluation with all four techniques in resected NSCLC from the large European Thoracic Oncology Platform Lungscape cohort. A total of 96 cases from the European Thoracic Oncology Platform Lungscape iBiobank, with any ALK immunoreactivity were examined by FISH, central RT-PCR, and NGS. An H-score higher than 120 defines IHC positivity. RNA was extracted from the same formalin-fixed, paraffin-embedded tissues. For RT-PCR, primers covered the most frequent ALK translocations. For NGS, the Oncomine Solid Tumour Fusion Transcript Kit (Thermo Fisher Scientific, Waltham, MA) was used. The concordance was assessed using the Cohen κ coefficient (two-sided α ≤ 5%). NGS provided results for 77 of the 95 cases tested (81.1%), whereas RT-PCR provided results for 77 of 96 (80.2%). Concordance occurred in 55 cases of the 60 cases tested with all four methods (43 ALK negative and 12 ALK positive). Using ALK copositivity for IHC and FISH as the criterion standard, we derived a sensitivity for RT-PCR/NGS of 70.0%/85.0%, with a specificity of 87.1%/79.0%. When either RT-PCR or NGS was combined with IHC, the sensitivity remained the same, whereas the specificity increased to 88.7% and 83.9% respectively. NGS evaluation with the Oncomine Solid Tumour Fusion transcript kit and RT-PCR proved to have high sensitivity and specificity, advocating their use in routine practice. For maximal sensitivity and specificity, ALK status should be assessed by using two techniques and a third one in discordant cases. We therefore propose a customizable testing algorithm. These findings significantly influence existing testing paradigms and have clear clinical and economic impact. Copyright © 2017 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Mapping of RNA accessible sites by extension of random oligonucleotide libraries with reverse transcriptase.

PubMed Central

Allawi, H T; Dong, F; Ip, H S; Neri, B P; Lyamichev, V I

2001-01-01

A rapid and simple method for determining accessible sites in RNA that is independent of the length of target RNA and does not require RNA labeling is described. In this method, target RNA is allowed to hybridize with sequence-randomized libraries of DNA oligonucleotides linked to a common tag sequence at their 5'-end. Annealed oligonucleotides are extended with reverse transcriptase and the extended products are then amplified by using PCR with a primer corresponding to the tag sequence and a second primer specific to the target RNA sequence. We used the combination of both the lengths of the RT-PCR products and the location of the binding site of the RNA-specific primer to determine which regions of the RNA molecules were RNA extendible sites, that is, sites available for oligonucleotide binding and extension. We then employed this reverse transcription with the random oligonucleotide libraries (RT-ROL) method to determine the accessible sites on four mRNA targets, human activated ras (ha-ras), human intercellular adhesion molecule-1 (ICAM-1), rabbit beta-globin, and human interferon-gamma (IFN-gamma). Our results were concordant with those of other researchers who had used RNase H cleavage or hybridization with arrays of oligonucleotides to identify accessible sites on some of these targets. Further, we found good correlation between sites when we compared the location of extendible sites identified by RT-ROL with hybridization sites of effective antisense oligonucleotides on ICAM-1 mRNA in antisense inhibition studies. Finally, we discuss the relationship between RNA extendible sites and RNA accessibility. PMID:11233988

Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types.

PubMed

Kilian, A; Bowtell, D D; Abud, H E; Hime, G R; Venter, D J; Keese, P K; Duncan, E L; Reddel, R R; Jefferson, R A

1997-11-01

Telomerase is a multicomponent reverse transcriptase enzyme that adds DNA repeats to the ends of chromosomes using its RNA component as a template for synthesis. Telomerase activity is detected in the germline as well as the majority of tumors and immortal cell lines, and at low levels in several types of normal cells. We have cloned a human gene homologous to a protein from Saccharomyces cerevisiae and Euplotes aediculatus that has reverse transcriptase motifs and is thought to be the catalytic subunit of telomerase in those species. This gene is present in the human genome as a single copy sequence with a dominant transcript of approximately 4 kb in a human colon cancer cell line, LIM1215. The cDNA sequence was determined using clones from a LIM1215 cDNA library and by RT-PCR, cRACE and 3'RACE on mRNA from the same source. We show that the gene is expressed in several normal tissues, telomerase-positive post-crisis (immortal) cell lines and various tumors but is not expressed in the majority of normal tissues analyzed, pre-crisis (non-immortal) cells and telomerase-negative immortal (ALT) cell lines. Multiple products were identified by RT-PCR using primers within the reverse transcriptase domain. Sequencing of these products suggests that they arise by alternative splicing. Strikingly, various tumors, cell lines and even normal tissues (colonic crypt and testis) showed considerable differences in the splicing patterns. Alternative splicing of the telomerase catalytic subunit transcript may be important for the regulation of telomerase activity and may give rise to proteins with different biochemical functions.

Utility of dengue NS1 antigen rapid diagnostic test for use in difficult to reach areas and its comparison with dengue NS1 ELISA and qRT-PCR.

PubMed

Shukla, Mohan K; Singh, Neeru; Sharma, Ravendra K; Barde, Pradip V

2017-07-01

The objective of this study was to demonstrate the utility of dengue virus (DENV) non structural protein 1 (NS1) based rapid diagnostic test (RDT) for use in tribal and difficult to reach areas for early dengue (DEN) diagnosis in acute phase patients and evaluate its sensitivity and specificity against DENV NS1 enzyme linked immune sorbent assay (ELISA) and real time reverse transcriptase polymerase chain reaction (qRT-PCR). The DENV NS1 RDT was used for preliminary diagnosis during outbreaks in difficult to reach rural and tribal areas. The diagnosis was confirmed by DENV NS1 ELISA in the laboratory. The samples were also tested and serotyped by qRT-PCR. The results were evaluated using statistical tests. The DENV NS1 RDT showed 99.2% sensitivity and 96.0% specificity when analyzed using DENV NS1 ELISA as standard. The specificity and sensitivity of the RDT when compared with qRT-PCR was 93.6% and 91.1%, respectively. The serotype specific evaluation showed more than 90% sensitivity and specificity for DENV-1, 2, and 3. The RDT proved a good diagnostic tool in difficult to reach rural and tribal areas. Further evaluation studies with different commercially available RDTs in different field conditions are essential, that will help clinicians and patients for treatment and programme managers for timely intervention. © 2017 Wiley Periodicals, Inc.

Relationship Between Ebola Virus Real-Time Quantitative Polymerase Chain Reaction-Based Threshold Cycle Value and Virus Isolation From Human Plasma.

PubMed

Spengler, Jessica R; McElroy, Anita K; Harmon, Jessica R; Ströher, Ute; Nichol, Stuart T; Spiropoulou, Christina F

2015-10-01

We performed a longitudinal analysis of plasma samples obtained from 4 patients with Ebola virus (EBOV) disease (EVD) to determine the relationship between the real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)-based threshold cycle (Ct) value and the presence of infectious EBOV. EBOV was not isolated from plasma samples with a Ct value of >35.5 or >12 days after onset of symptoms. EBOV was not isolated from plasma samples in which anti-EBOV nucleoprotein immunoglobulin G was detected. These data demonstrate the utility of interpreting qRT-PCR results in the context of the course of EBOV infection and associated serological responses for patient-management decisions. Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Murine Leukemia Virus Reverse Transcriptase: Structural Comparison with HIV-1 Reverse Transcriptase

PubMed Central

Coté, Marie L.; Roth, Monica J.

2008-01-01

Recent X-ray crystal structure determinations of Moloney murine leukemia virus reverse transcriptase (MoMLV RT) have allowed for more accurate structure/function comparisons to HIV-1 RT than were formerly possible. Previous biochemical studies of MoMLV RT in conjunction with knowledge of sequence homologies to HIV-1 RT and overall fold similarities to RTs in general, provided a foundation upon which to build. In addition, numerous crystal structures of the MoMLV RT fingers/palm subdomain had also shed light on one of the critical functions of the enzyme, specifically polymerization. Now in the advent of new structural information, more intricate examination of MoMLV RT in its entirety can be realized, and thus the comparisons with HIV-1 RT may be more critically elucidated. Here, we will review the similarities and differences between MoMLV RT and HIV-1 RT via structural analysis, and propose working models for the MoMLV RT based upon that information. PMID:18294720

Internalization of Murine Norovirus 1 by Lactuca sativa during Irrigation ▿

PubMed Central

Wei, Jie; Jin, Yan; Sims, Tom; Kniel, Kalmia E.

2011-01-01

Romaine lettuce (Lactuca sativa) was grown hydroponically or in soil and challenged with murine norovirus 1 (MNV) under two conditions: one mimicking a severe one-time contamination event and another mimicking a lower level of contamination occurring over time. In each condition, lettuce was challenged with MNV delivered at the roots. In the first case, contamination occurred on day one with 5 × 108 reverse transcriptase quantitative PCR (RT-qPCR) U/ml MNV in nutrient buffer, and irrigation water was replaced with virus-free buffer every day for another 4 days. In the second case, contamination with 5 × 105 RT-qPCR U/ml MNV (freshly prepared) occurred every day for 5 days. Virus had a tendency to adsorb to soil particles, with a small portion suspended in nutrient buffer; e.g., ∼8 log RT-qPCR U/g MNV was detected in soil during 5 days of challenge with virus inoculums of 5 × 108 RT-qPCR U/ml at day one, but <6 log was found in nutrient buffer on days 3 and 5. For hydroponically grown lettuce, ∼3.4 log RT-qPCR U of viral RNA/50 mg of plant tissue was detected in some lettuce leaf samples after 5 days at high MNV inoculums, significantly higher than the internalized virus concentration (∼2.6 log) at low inoculums (P < 0.05). For lettuce grown in soil, approximately 2 log RT-qPCR U of viral RNA/50 mg of plant tissue was detected in lettuce with both high and low inoculums, showing no significant difference. For viral infectivity, infectious MNV was found in lettuce samples challenged with high virus inoculums grown hydroponically and in soil but not in lettuce grown with low virus inoculums. Lettuce grown hydroponically was further incubated in 99% and 70% relative humidities (RH) to evaluate plant transpiration relative to virus uptake. More lettuce samples were found positive for MNV at a significantly higher transpiration rate at 70% RH, indicating that transpiration might play an important role in virus internalization into L. sativa. PMID:21296944

Reverse transcription strand invasion based amplification (RT-SIBA): a method for rapid detection of influenza A and B.

PubMed

Eboigbodin, Kevin; Filén, Sanna; Ojalehto, Tuomas; Brummer, Mirko; Elf, Sonja; Pousi, Kirsi; Hoser, Mark

2016-06-01

Rapid and accurate diagnosis of influenza viruses plays an important role in infection control, as well as in preventing the misuse of antibiotics. Isothermal nucleic acid amplification methods offer significant advantages over the polymerase chain reaction (PCR), since they are more rapid and do not require the sophisticated instruments needed for thermal cycling. We previously described a novel isothermal nucleic acid amplification method, 'Strand Invasion Based Amplification' (SIBA®), with high analytical sensitivity and specificity, for the detection of DNA. In this study, we describe the development of a variant of the SIBA method, namely, reverse transcription SIBA (RT-SIBA), for the rapid detection of viral RNA targets. The RT-SIBA method includes a reverse transcriptase enzyme that allows one-step reverse transcription of RNA to complementary DNA (cDNA) and simultaneous amplification and detection of the cDNA by SIBA under isothermal reaction conditions. The RT-SIBA method was found to be more sensitive than PCR for the detection of influenza A and B and could detect 100 copies of influenza RNA within 15 min. The development of RT-SIBA will enable rapid and accurate diagnosis of viral RNA targets within point-of-care or central laboratory settings.

Molecular Analysis of Ammonia-Oxidizing Bacteria of the β Subdivision of the Class Proteobacteria in Compost and Composted Materials

PubMed Central

Kowalchuk, George A.; Naoumenko, Zinaida S.; Derikx, Piet J. L.; Felske, Andreas; Stephen, John R.; Arkhipchenko, Irina A.

1999-01-01

Although the practice of composting animal wastes for use as biofertilizers has increased in recent years, little is known about the microorganisms responsible for the nitrogen transformations which occur in compost and during the composting process. Ammonia is the principle available nitrogenous compound in composting material, and the conversion of this compound to nitrite in the environment by chemolithotrophic ammonia-oxidizing bacteria is an essential step in nitrogen cycling. Therefore, the distribution of ammonia-oxidizing members of the β subdivision of the class Proteobacteria in a variety of composting materials was assessed by amplifying 16S ribosomal DNA (rDNA) and 16S rRNA by PCR and reverse transcriptase PCR (RT-PCR), respectively. The PCR and RT-PCR products were separated by denaturing gradient gel electrophoresis (DGGE) and were identified by hybridization with a hierarchical set of oligonucleotide probes designed to detect ammonia oxidizer-like sequence clusters in the genera Nitrosospira and Nitrosomonas. Ammonia oxidizer-like 16S rDNA was detected in almost all of the materials tested, including industrial and experimental composts, manure, and commercial biofertilizers. A comparison of the DGGE and hybridization results after specific PCR and RT-PCR suggested that not all of the different ammonia oxidizer groups detected in compost are equally active. amoA, the gene encoding the active-site-containing subunit of ammonia monooxygenase, was also targeted by PCR, and template concentrations were estimated by competitive PCR. Detection of ammonia-oxidizing bacteria in the composts tested suggested that such materials may not be biologically inert with respect to nitrification and that the fate of nitrogen during composting and compost storage may be affected by the presence of these organisms. PMID:9925559

Nine year trends of dengue virus infection in Mumbai, Western India.

PubMed

Shastri, Jayanthi; Williamson, Manita; Vaidya, Nilima; Agrawal, Sachee; Shrivastav, Om

2017-01-01

Dengue virus (DENV) causes a wide range of diseases in humans, from acute febrile illness Dengue fever (DF) to life-threatening Dengue hemorrhagic fever (DHF) or Dengue shock syndrome (DSS). Factors believed to be responsible for spread of Dengue virus infection include explosive population growth, unplanned urban overpopulation with inadequate public health systems, poor standing water and vector control, climate changes and increased international recreational, business, military travel to endemic areas. All of these factors must be addressed to control the spread of Dengue and other mosquito-borne infections. The detection of Dengue virus RNA by reverse transcriptase PCR (RT-PCR) in human serum or plasma samples is highly indicative of acute Dengue fever. Moreover, the method is able to identify the Dengue virus serotype by demonstrating defined sequence homologies in the viral genomic RNA. During the nine year period of this study analysis, 6767 strongly suspected cases were tested by RT-PCR. 1685 (24.9%) were Dengue PCR positive and confirmed as Dengue cases. Observations on the seasonality were based on the nine year's data as the intensity of sampling was at its maximum during monsoon season. Dengue typing was done on 100 positive samples after storage of Dengue RNA at - 80°C. Dengue serotypes were detected in 69 samples of which Dengue 2 was most predominant. 576 samples were processed for NS1 antigen and PCR simultaneously. 19/576 were positive (3.3 %) for NS1 as well as by PCR. 23/576 samples were negative for NS1 antigen, but were positive by RT-PCR. The remaining 534 samples which were negative for NS1 antigen were also negative by Dengue RT-PCR. In this study we sought to standardize rapid, sensitive, and specific fluorogenic probe-based RT-PCR assay to screen and serotype a representative range of Dengue viruses that are found in and around Mumbai. Qualitative Dengue virus TaqMan assays could have tremendous utility for the epidemiological investigation of Dengue illness and especially for the study of the viremic response with candidate live-attenuated dengue virus vaccines.

A rapid single-tube protocol for HAV detection by nested real-time PCR.

PubMed

Hu, Yuan; Arsov, Ivica

2014-09-01

Infections by food-borne viruses such as hepatitis A virus (HAV) and norovirus are significant public health concerns worldwide. Since food-borne viruses are rarely confirmed through direct isolation from contaminated samples, highly sensitive molecular techniques remain the methods of choice for the detection of viral genetic material. Our group has previously developed a specific nested real-time PCR (NRT-PCR) assay for HAV detection that improved overall sensitivity. Furthermore in this study, we have developed a single-tube NRT-PCR approach for HAV detection in food samples that reduces the likelihood of cross contamination between tubes during sample manipulation. HAV RNA was isolated from HAV-spiked food samples and HAV-infected cell cultures. All reactions following HAV RNA isolation, including conventional reverse transcriptase PCR, nested-PCR, and RT-PCR were performed in a single tube. Our results demonstrated that all the samples tested positive by RT-PCR and nested-PCR were also positive by a single-tube NRT-PCR. The detection limits observed for HAV-infected cell cultures and HAV-spiked green onions were 0.1 and 1 PFU, respectively. This novel method retained the specificity and robustness of the original NRT-PCR method, while greatly reducing sample manipulation, turnaround time, and the risk of carry-over contamination. Single-tube NRT-PCR thus represents a promising new tool that can potentially facilitate the detection of HAV in foods thereby improving food safety and public health.

Specific, sensitive, and rapid assay for human immunodeficiency virus type 1 pol mutations associated with resistance to zidovudine and didanosine.

PubMed Central

Frenkel, L M; Wagner, L E; Atwood, S M; Cummins, T J; Dewhurst, S

1995-01-01

The effectiveness of antiretroviral therapy may be limited by the development of human immunodeficiency virus type 1 (HIV-1) resistance. Monitoring for resistance will perhaps allow changes in therapy prior to deterioration in the patient's clinical or immunologic status. Our objective was to develop a rapid, specific, and sensitive genotypic assay for HIV-1 resistance to zidovudine (ZDV) and didanosine (ddI) which is simple to perform. In our assay the DNA of HIV-1 pol was amplified by PCR using two sets of nested oligonucleotide primers. Mutations of reverse transcriptase (RT) encoding amino acids (aa) 74 and 41, 70, and 215 which have been associated with HIV-1 resistance to ddI and ZDV, respectively, were detected with a ligase detection reaction (LDR) and indicated colorimetrically. The RT genotypes of 35 patient specimens (140 codons) blindly assessed for these mutations were in agreement by PCR-LDR and by dideoxynucleotide sequencing. To evaluate the limits of the assay, other specimens with mutations close to the ligation site were evaluated by PCR-LDR. The assay was sensitive and specific for all specimens except when mutations occurred within 2 bases on either side of the ligation site. In summary, this PCR-LDR assay specifically, sensitively, and rapidly detected pol mutations (RT aa 74, 41, 70, and 215) associated with HIV-1 resistance to ddI and ZDV. PMID:7714190

[Difference of three standard curves of real-time reverse-transcriptase PCR in viable Vibrio parahaemolyticus quantification].

PubMed

Jin, Mengtong; Sun, Wenshuo; Li, Qin; Sun, Xiaohong; Pan, Yingjie; Zhao, Yong

2014-04-04

We evaluated the difference of three standard curves in quantifying viable Vibrio parahaemolyticus in samples by real-time reverse-transcriptase PCR (Real-time RT-PCR). The standard curve A was established by 10-fold diluted cDNA. The cDNA was reverse transcripted after RNA synthesized in vitro. The standard curve B and C were established by 10-fold diluted cDNA. The cDNA was synthesized after RNA isolated from Vibrio parahaemolyticus in pure cultures (10(8) CFU/mL) and shrimp samples (10(6) CFU/g) (Standard curve A and C were proposed for the first time). Three standard curves were performed to quantitatively detect V. parahaemolyticus in six samples, respectively (Two pure cultured V. parahaemolyticus samples, two artificially contaminated cooked Litopenaeus vannamei samples and two artificially contaminated Litopenaeus vannamei samples). Then we evaluated the quantitative results of standard curve and the plate counting results and then analysed the differences. The three standard curves all show a strong linear relationship between the fractional cycle number and V. parahaemolyticus concentration (R2 > 0.99); The quantitative results of Real-time PCR were significantly (p < 0.05) lower than the results of plate counting. The relative errors compared with the results of plate counting ranked standard curve A (30.0%) > standard curve C (18.8%) > standard curve B (6.9%); The average differences between standard curve A and standard curve B and C were - 2.25 Lg CFU/mL and - 0.75 Lg CFU/mL, respectively, and the mean relative errors were 48.2% and 15.9%, respectively; The average difference between standard curve B and C was among (1.47 -1.53) Lg CFU/mL and the average relative errors were among 19.0% - 23.8%. Standard curve B could be applied to Real-time RT-PCR when quantify the number of viable microorganisms in samples.

Monitoring virologic responses to antiretroviral therapy in HIV-infected adults in Kenya: evaluation of a low-cost viral load assay.

PubMed

Sivapalasingam, Sumathi; Wangechi, Beatrice; Marshed, Fatuma; Laverty, Maura; Essajee, Shaffiq; Holzman, Robert S; Valentine, Fred

2009-08-28

A key advantage of monitoring HIV viral load (VL) in persons receiving antiretroviral therapy (ART) is the ability to detect virologic failure before clinical deterioration or resistance occurs. Detection of virologic failure will help clarify the need for enhanced adherence counseling or a change to second- line therapy. Low-cost, locally performable alternates to expensive VL assays are needed where resources are limited. We monitored the response to 48-week ART in 100 treatment-naïve Kenyan adults using a low-cost VL measurement, the Cavidi reverse transcriptase (RT) assay and gold-standard assays, Roche RNA PCR and Bayer Versant HIV-1 RNA (bDNA) assays. In Altman-Bland plots, the mean difference in viral loads between the three assays was small (<0.5 log(10) copies/mL). However, the limits of agreement between the methods exceeded the biologically relevant change of 0.5 log copies/ml. Therefore, the RT assay cannot be used interchangeably with the other assays to monitor individual patients. The RT assay was 100% sensitive in detecting viral loads of > or =400 copies/ml compared to gold-standard assays. After 24 weeks of treatment, viral load measured by the RT assay was undetectable in 95% of 65 patients with undetectable RNA PCR VL (<400 copies/ml), 90% of 67 patients with undetectable bDNA VL, and 96% of 57 patients with undetectable VL in both RNA PCR and bDNA assays. The negative predictive value of the RT assay was 100% compared to either assay; the positive predictive value was 86% compared to RNA PCR and 70% compared to bDNA. The RT assay compared well with gold standard assays. Our study highlights the importance of not interchanging viral load assays when monitoring an individual patient. Furthermore, the RT assay may be limited by low positive predictive values when used in populations with low prevalence of virologic failure.

Monitoring Virologic Responses to Antiretroviral Therapy in HIV-Infected Adults in Kenya: Evaluation of a Low-Cost Viral Load Assay

PubMed Central

Sivapalasingam, Sumathi; Wangechi, Beatrice; Marshed, Fatuma; Laverty, Maura; Essajee, Shaffiq; Holzman, Robert S.; Valentine, Fred

2009-01-01

Background A key advantage of monitoring HIV viral load (VL) in persons receiving antiretroviral therapy (ART) is the ability to detect virologic failure before clinical deterioration or resistance occurs. Detection of virologic failure will help clarify the need for enhanced adherence counseling or a change to second- line therapy. Low-cost, locally performable alternates to expensive VL assays are needed where resources are limited. Methodology/Principal Findings We monitored the response to 48-week ART in 100 treatment-naïve Kenyan adults using a low-cost VL measurement, the Cavidi reverse transcriptase (RT) assay and gold-standard assays, Roche RNA PCR and Bayer Versant HIV-1 RNA (bDNA) assays. In Altman-Bland plots, the mean difference in viral loads between the three assays was small (<0.5 log10 copies/mL). However, the limits of agreement between the methods exceeded the biologically relevant change of 0.5 log copies/ml. Therefore, the RT assay cannot be used interchangeably with the other assays to monitor individual patients. The RT assay was 100% sensitive in detecting viral loads of ≥400 copies/ml compared to gold-standard assays. After 24 weeks of treatment, viral load measured by the RT assay was undetectable in 95% of 65 patients with undetectable RNA PCR VL (<400 copies/ml), 90% of 67 patients with undetectable bDNA VL, and 96% of 57 patients with undetectable VL in both RNA PCR and bDNA assays. The negative predictive value of the RT assay was 100% compared to either assay; the positive predictive value was 86% compared to RNA PCR and 70% compared to bDNA. Conclusion The RT assay compared well with gold standard assays. Our study highlights the importance of not interchanging viral load assays when monitoring an individual patient. Furthermore, the RT assay may be limited by low positive predictive values when used in populations with low prevalence of virologic failure. PMID:19714253

Use of molecular and milk production information for the cost-effective diagnosis of bovine viral diarrhoea infection in New Zealand dairy cattle.

PubMed

Hill, F I; Reichel, M P; Tisdall, D J

2010-04-21

An increase in veterinary and farmer interest in bovine viral diarrhoea (BVD) in New Zealand over recent years led to requests for cost-effective identification of BVD virus (BVDV) infected herds and individuals. This study was undertaken to determine if the use of real-time reverse transcriptase polymerase chain reaction (RT-PCR) technology and dairy cow production data could identify persistently infected (PI) animals in milking herds. Milk samples were collected from the vats of dairy herds and tested for the presence of BVDV by RT-PCR till four herds were found containing PI animals. Individual serum samples were then collected from every cow in the herd and tested by both RT-PCR and antigen capture enzyme-linked immunosorbent assays (ACE) to identify the PI animals. Individual animal testing found 1/223, 1/130, 2/800 and 1/275 PI's respectively in the four herds. Based on these results a maximum pool size of 400 cows contributing to the bulk tank milk was selected. After removal of the PI from the herds, further bulk milk samples were shown to be BVDV negative by RT-PCR. All the PI animals identified by this method were found in the lowest producing 10-20% of herd. This approach of targeted testing of dairy herds using PCR technology, in conjunction with animal production information, markedly reduced the cost of diagnostic testing for BVDV in dairy herds in New Zealand. Questionnaire follow-up on 81 BVDV-positive herds (15% of those tested) indicated the stratification approach identified milking PIs successfully over 90% of the time and reduced the number of individual tests to 12% of the milking herd. Copyright 2009 Elsevier B.V. All rights reserved.

Preservation of pathological tissue specimens by freeze-drying for immunohistochemical staining and various molecular biological analyses.

PubMed

Matsuo, S; Sugiyama, T; Okuyama, T; Yoshikawa, K; Honda, K; Takahashi, R; Maeda, S

1999-05-01

Conditions of preserving DNA, RNA and protein in pathological specimens are of great importance as degradation of such macromolecules would critically affect results of molecular biological analysis. The feasibility of freeze-drying as a means of preserving pathological tissue samples for molecular analysis has previously been shown. In the present study, further tests on long-term storage conditions and analyses of freeze-dried samples by polymerase chain reaction (PCR), reverse transcriptase (RT)-PCR, western blotting and immunohistochemistry are reported. Rat chromosomal DNA of freeze-dried samples stored for 4 years showed slight degradation while RNA degradation was more prominently seen at an earlier stage of storage. However, these 4 year DNA and RNA samples were still able to serve as a template for some PCR and RT-PCR analyses, respectively. Overexpression of c-erbB-2 and p53 protein was demonstrated by western blotting and immunohistochemical staining using freeze-dried human breast cancer tissues. Although macromolecules in freeze-dried samples degrade to some extent during the preservation period, they should still be of value for certain molecular biological analyses and morphological examination; hence, providing more convenient and inexpensive ways of pathological tissue storage.

RHEB expression in fibroadenomas of the breast.

PubMed

Eom, Minseob; Han, Airi; Yi, Sang Yeop; Shin, John Junghun; Cui, Ying; Park, Kwang Hwa

2008-04-01

Although fibroadenoma is one of the most common types of benign breast tumor, genes specific to the tumor have not been identified. Microarrays were used to identify differentially expressed genes between fibroadenoma and infiltrating ductal carcinoma. The comparative expression of one of the identified genes, RAS homolog enriched in the brain (RHEB), was further explored using reverse transcriptase-polymerase chain reaction (RT-PCR). Microarray analysis was performed on tissue samples from five patients with fibroadenoma. In the fibroadenoma samples, the genes HDAC1, ROS1, TNFRSF10A, WASP2, TYRP1, WEE1, and RHEB were expressed at levels more than twofold higher than in the normal tissues. RT-PCR for RHEB indicated increased expression of RHEB in fibroadenoma compared to breast cancer. When studied with real-time PCR, the average RHEB/beta-actin ratio in fibroadenoma samples was 1.99, 2.46-fold greater than the average RHEB/beta-actin ratio in breast carcinoma of 0.81 (P < 0.01). Immunohistochemistry and PCR followed by microdissection shows increased expression of RHEB in epithelial cells compared to the stromal cells of fibroadenoma. Therefore, RHEB could be used cytopathologically to distinguish fibroadenoma from malignant breast carcinomas as a secondary diagnostic tool.

Selection of reference genes for RT-qPCR analysis in a predatory biological control agent, Coleomegilla maculata (Coleoptera: Coccinellidae).

PubMed

Yang, Chunxiao; Pan, Huipeng; Noland, Jeffrey Edward; Zhang, Deyong; Zhang, Zhanhong; Liu, Yong; Zhou, Xuguo

2015-12-10

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for quantifying gene expression across various biological processes, of which requires a set of suited reference genes to normalize the expression data. Coleomegilla maculata (Coleoptera: Coccinellidae), is one of the most extensively used biological control agents in the field to manage arthropod pest species. In this study, expression profiles of 16 housekeeping genes selected from C. maculata were cloned and investigated. The performance of these candidates as endogenous controls under specific experimental conditions was evaluated by dedicated algorithms, including geNorm, Normfinder, BestKeeper, and ΔCt method. In addition, RefFinder, a comprehensive platform integrating all the above-mentioned algorithms, ranked the overall stability of these candidate genes. As a result, various sets of suitable reference genes were recommended specifically for experiments involving different tissues, developmental stages, sex, and C. maculate larvae treated with dietary double stranded RNA. This study represents the critical first step to establish a standardized RT-qPCR protocol for the functional genomics research in a ladybeetle C. maculate. Furthermore, it lays the foundation for conducting ecological risk assessment of RNAi-based gene silencing biotechnologies on non-target organisms; in this case, a key predatory biological control agent.

MAEWEST expression in flower development of two petunia species.

PubMed

Segatto, Ana Lúcia A; Turchetto-Zolet, Andreia Carina; Aizza, Lilian Cristina B; Monte-Bello, Carolina C; Dornelas, Marcelo C; Margis, Rogerio; Freitas, Loreta B

2013-07-03

Changes in flower morphology may influence the frequency and specificity of animal visitors. In Petunia (Solanaceae), adaptation to different pollinators is one of the factors leading to species diversification within the genus. This study provides evidence that differential expression patterns of MAWEWEST (MAW) homologs in different Petunia species may be associated with adaptive changes in floral morphology. The Petunia × hybrida MAW gene belongs to the WOX (WUSCHEL-related homeobox) transcription factor family and has been identified as a controller of petal fusion during corolla formation. We analyzed the expression patterns of P. inflata and P. axillaris MAW orthologs (PiMAW and PaMAW, respectively) by reverse transcriptase polymerase chain reaction (RT-PCR), reverse transcription-quantitative PCR (qRT-PCR) and in situ hybridization in different tissues and different developmental stages of flowers in both species. The spatial expression patterns of PiMAW and PaMAW were similar in P. inflata and P. axillaris. Nevertheless, PaMAW expression level in P. axillaris was higher during the late bud development stage as compared to PiMAW in P. inflata. This work represents an expansion of petunia developmental research to wild accessions.

Persistence of Viral Reservoirs in Multiple Tissues after Antiretroviral Therapy Suppression in a Macaque RT-SHIV Model

PubMed Central

Franks, Tamera; Kiser, Rebecca; Coalter, Vicky; Smedley, Jeremy; Piatak, Michael; Mellors, John W.; Lifson, Jeffrey D.; Ambrose, Zandrea

2013-01-01

Although antiretroviral therapy (ART) can suppress HIV-1 replication sufficiently to eliminate measurable plasma viremia, infected cells remain and ensure viral recrudescence after discontinuation of ART. We used a macaque model of HIV-1/AIDS to evaluate the location of infected cells during ART. Twelve macaques were infected with RT-SHIVmne, a SIV containing HIV-1 reverse transcriptase, conferring sensitivity to non-nucleoside reverse transcriptase inhibitors (NNRTIs). Ten to fourteen weeks post-infection, 6 animals were treated with 3 or 4 antiretroviral drugs for 17-20 weeks; 6 control animals remained untreated. Viral DNA (vDNA) and RNA (vRNA) were measured in peripheral blood mononuclear cells (PBMC) and at necropsy in multiple tissues by quantitative PCR and RT-PCR. The majority of virally infected cells were located in lymphoid tissues with variable levels in the gastrointestinal tract of both treated and untreated animals. Tissue viral DNA levels correlated with week 1 plasma viremia, suggesting that tissues that harbor proviral DNA are established within the first week of infection. PBMC vDNA levels did not correlate with plasma viremia or tissue levels of vDNA. vRNA levels were high in lymphoid and gastrointestinal tissues of the untreated animals; animals on ART had little vRNA expressed in tissues and virus could not be cultured from lymph node resting CD4+ cells after 17-20 weeks on ART, indicating little or no ongoing viral replication. Strategies for eradication of HIV-1 will need to target residual virus in ART suppressed individuals, which may not be accurately reflected by frequencies of infected cells in blood. PMID:24367650

The development of a real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay using TaqMan technology for the pan detection of bluetongue virus (BTV).

PubMed

Mulholland, Catherine; McMenamy, Michael J; Hoffmann, Bernd; Earley, Bernadette; Markey, Bryan; Cassidy, Joseph; Allan, Gordon; Welsh, Michael D; McKillen, John

2017-07-01

Bluetongue virus (BTV) is an infectious, non-contagious viral disease of domestic and wild ruminants that is transmitted by adult females of certain Culicoides species. Since 2006, several serotypes including BTV-1, 2, 4, 6, 8, 9 and 16, have spread from the Mediterranean basin into Northern Europe for the first time. BTV-8 in particular, caused a major epidemic in northern Europe. As a result, it is evident that most European countries are at risk of BTV infection. The objective of this study was to develop and validate a real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assay based on TaqMan technology for the detection of representative strains of all BTV serotypes. Primers and probes were based on genome segment 10 of the virus, the NS3 gene. The assay was tested for sensitivity, and specificity. The analytical sensitivity of the rRT-PCR assay was 200 copies of RNA per reaction. The assay did not amplify the closely related orbivirus epizootic hemorrhagic disease virus (EHDV) but successfully detected all BTV reference strains including clinical samples from animals experimentally infected with BTV-8. This real time RT-PCR assay offers a sensitive, specific and rapid alternative assay for the pan detection of BTV that could be used as part of a panel of diagnostic assays for the detection of all serotypes of BTV. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Human epidermal growth factor receptor 2 assessment in a case-control study: comparison of fluorescence in situ hybridization and quantitative reverse transcription polymerase chain reaction performed by central laboratories.

PubMed

Baehner, Frederick L; Achacoso, Ninah; Maddala, Tara; Shak, Steve; Quesenberry, Charles P; Goldstein, Lynn C; Gown, Allen M; Habel, Laurel A

2010-10-01

The optimal method to assess human epidermal growth factor receptor 2 (HER2) status remains highly controversial. Before reporting patient HER2 results, American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines mandate that laboratories demonstrate ≥ 95% concordance to another approved laboratory or methodology. Here, we compare central laboratory HER2 assessed by fluorescence in situ hybridization (FISH) and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) using Oncotype DX in lymph node-negative, chemotherapy-untreated patients from a large Kaiser Permanente case-control study. Breast cancer specimens from the Kaiser-Genomic Health study were examined. Central FISH assessment of HER2 amplification and polysomy 17 was conducted by PhenoPath Laboratories (ratios > 2.2, 1.8 to 2.2, and < 1.8 define HER2 positive, HER2 equivocal, and HER2 negative, respectively). HER2 expression by RT-PCR was conducted using Oncotype DX by Genomic Health (normalized expression units ≥ 11.5, 10.7 to < 11.5, and < 10.7 define HER2 positive, HER2 equivocal, and HER2 negative, respectively). Concordance analyses followed ASCO/CAP guidelines. HER2 concordance by central FISH and central RT-PCR was 97% (95% CI, 96% to 99%). Twelve percent (67 of 568 patients) and 11% (60 of 568 patients) of patients were HER2 positive by RT-PCR and FISH, respectively. HER2-positive patients had increased odds of dying from breast cancer compared with HER2-negative patients. Polysomy 17 was demonstrated in 12.5% of all patients and 33% of FISH-positive patients. Nineteen of 20 FISH-positive patients with polysomy 17 were also RT-PCR HER2 positive. Although not statistically significantly different, HER2-positive/polysomy 17 patients tended to have the worst prognosis, followed by HER2-positive/eusomic, HER2-negative/polysomy 17, and HER2-negative/eusomic patients. There is a high degree of concordance between central FISH and quantitative RT-PCR using Oncotype DX for HER2 status, and the assay warrants additional study in a trastuzumab-treated population.

Failure to detect Xenotropic murine leukaemia virus-related virus in Chinese patients with chronic fatigue syndrome

PubMed Central

2010-01-01

Background Recent controversy has surrounded the question of whether xenotropic murine leukaemia virus-related virus (XMRV) contributes to the pathogenesis of chronic fatigue syndrome (CFS). To investigate the question in a Chinese population, 65 CFS patients and 85 blood donor controls were enrolled and multiplex real-time PCR or reverse transcriptase PCR (RT-PCR) was developed to analyze the XMRV infection status of the study participants. The assay was standardized by constructing plasmid DNAs and armored RNAs as XMRV standards and competitive internal controls (CICs), respectively. Results The sensitivities of the multiplex real-time PCR and RT-PCR assays were 20 copies/reaction and 10 IU/ml, respectively, with 100% specificity. The within-run precision coefficient of variation (CV) ranged from 1.76% to 2.80% and 1.70% to 2.59%, while the between-run CV ranged from 1.07% to 2.56% and 1.06% to 2.74%. XMRV was not detected in the 65 CFS patients and 65 normal individuals out of 85 controls. Conclusions This study failed to show XMRV in peripheral blood mononuclear cells (PBMCs) and plasma of Chinese patients with CFS. The absence of XMRV nucleic acids does not support an association between XMRV infection and the development of CFS in Chinese. PMID:20836869

Validation of Reference Genes for Real-Time Quantitative PCR (qPCR) Analysis of Avibacterium paragallinarum.

PubMed

Wen, Shuxiang; Chen, Xiaoling; Xu, Fuzhou; Sun, Huiling

2016-01-01

Real-time quantitative reverse transcription PCR (qRT-PCR) offers a robust method for measurement of gene expression levels. Selection of reliable reference gene(s) for gene expression study is conducive to reduce variations derived from different amounts of RNA and cDNA, the efficiency of the reverse transcriptase or polymerase enzymes. Until now reference genes identified for other members of the family Pasteurellaceae have not been validated for Avibacterium paragallinarum. The aim of this study was to validate nine reference genes of serovars A, B, and C strains of A. paragallinarum in different growth phase by qRT-PCR. Three of the most widely used statistical algorithms, geNorm, NormFinder and ΔCT method were used to evaluate the expression stability of reference genes. Data analyzed by overall rankings showed that in exponential and stationary phase of serovar A, the most stable reference genes were gyrA and atpD respectively; in exponential and stationary phase of serovar B, the most stable reference genes were atpD and recN respectively; in exponential and stationary phase of serovar C, the most stable reference genes were rpoB and recN respectively. This study provides recommendations for stable endogenous control genes for use in further studies involving measurement of gene expression levels.

Nevirapine resistance mutation at codon 181 of the HIV-1 reverse transcriptase confers stavudine resistance by increasing nucleotide substrate discrimination and phosphorolytic activity.

PubMed

Blanca, Giuseppina; Baldanti, Fausto; Paolucci, Stefania; Skoblov, Alexander Yu; Victorova, Lyubov; Hübscher, Ulrich; Gerna, Giuseppe; Spadari, Silvio; Maga, Giovanni

2003-05-02

Recombinant HIV-1 reverse transcriptase (RT) carrying non-nucleoside inhibitors (NNRTIs) resistance mutation at codon 181 showed reduced incorporation and high efficiency of phosphorolytic removal of stavudine, a nucleoside RT inhibitor. These results reveal a new mechanism for cross-resistance between different classes of HIV-1 RT inhibitors.

Successful Validation of Sample Processing and Quantitative Real-Time PCR Capabilities on the International Space Station

NASA Technical Reports Server (NTRS)

Parra, Macarena; Jung, Jimmy; Almeida, Eduardo; Boone, Travis; Schonfeld, Julie; Tran, Luan

2016-01-01

The WetLab-2 system was developed by NASA Ames Research Center to offer new capabilities to researchers. The system can lyse cells and extract RNA (Ribonucleic Acid) on-orbit from different sample types ranging from microbial cultures to animal tissues. The purified RNA can then either be stabilized for return to Earth or can be used to conduct on-orbit quantitative Reverse Transcriptase PCR (Polymerase Chain Reaction) (qRT-PCR) analysis without the need for sample return. The qRT-PCR results can be downlinked to the ground a few hours after the completion of the run. The validation flight of the WetLab-2 system launched on SpaceX-8 on April 8, 2016. On orbit operations started on April 15th with system setup and was followed by three quantitative PCR runs using an E. coli genomic DNA template pre-loaded at three different concentrations. These runs were designed to discern if quantitative PCR functions correctly in microgravity and if the data is comparable to that from the ground control runs. The flight data showed no significant differences compared to the ground data though there was more variability in the values, this was likely due to the numerous small bubbles observed. The capability of the system to process samples and purify RNA was then validated using frozen samples prepared on the ground. The flight data for both E. coli and mouse liver clearly shows that RNA was successfully purified by our system. The E. coli qRT-PCR run showed successful singleplex, duplex and triplex capability. Data showed high variability in the resulting Cts (Cycle Thresholds [for the PCR]) likely due to bubble formation and insufficient mixing during the procedure run. The mouse liver qRT-PCR run had successful singleplex and duplex reactions and the variability was slightly better as the mixing operation was improved. The ability to purify and stabilize RNA and to conduct qRT-PCR on-orbit is an important step towards utilizing the ISS as a National Laboratory facility. The ability to get on-orbit data will provide investigators with the opportunity to adjust experimental parameters in real time without the need for sample return and re-flight. The WetLab-2 Project is supported by the Research Integration Office in the ISS Program.

A general method to eliminate laboratory induced recombinants during massive, parallel sequencing of cDNA library.

PubMed

Waugh, Caryll; Cromer, Deborah; Grimm, Andrew; Chopra, Abha; Mallal, Simon; Davenport, Miles; Mak, Johnson

2015-04-09

Massive, parallel sequencing is a potent tool for dissecting the regulation of biological processes by revealing the dynamics of the cellular RNA profile under different conditions. Similarly, massive, parallel sequencing can be used to reveal the complexity of viral quasispecies that are often found in the RNA virus infected host. However, the production of cDNA libraries for next-generation sequencing (NGS) necessitates the reverse transcription of RNA into cDNA and the amplification of the cDNA template using PCR, which may introduce artefact in the form of phantom nucleic acids species that can bias the composition and interpretation of original RNA profiles. Using HIV as a model we have characterised the major sources of error during the conversion of viral RNA to cDNA, namely excess RNA template and the RNaseH activity of the polymerase enzyme, reverse transcriptase. In addition we have analysed the effect of PCR cycle on detection of recombinants and assessed the contribution of transfection of highly similar plasmid DNA to the formation of recombinant species during the production of our control viruses. We have identified RNA template concentrations, RNaseH activity of reverse transcriptase, and PCR conditions as key parameters that must be carefully optimised to minimise chimeric artefacts. Using our optimised RT-PCR conditions, in combination with our modified PCR amplification procedure, we have developed a reliable technique for accurate determination of RNA species using NGS technology.

Absence of Measles Virus Detection from Stapes of Patients with Otosclerosis.

PubMed

Flores-García, María de Lourdes; Colín-Castro, Claudia Adriana; Hernández-Palestina, Mario Sabas; Sánchez-Larios, Roberto; Franco-Cendejas, Rafael

2018-01-01

Objective To determine molecularly the presence of measles virus genetic material in the stapes of patients with otosclerosis. Study Design A cross-sectional study. Setting A tertiary referral hospital. Subjects and Methods Genetic material was extracted from the stapes of patients with otosclerosis (n = 93) during the period from March 2011 to April 2012. The presence of viral measles sequences was evaluated by the real-time reverse transcriptase polymerase chain reaction (RT-PCR). The expression of the CD46 gene was determined. Results Ninety-three patients were included in the study. No sample was positive for any of 3 measles virus genes (H, N, and F). Measles virus RNA was not detected in any sample by real-time RT-PCR. CD46 levels were positive in 3.3% (n = 3) and negative in 96.7% (n = 90). Conclusion This study does not support the theory of measles virus as the cause of otosclerosis. It is necessary to do more research about other causal theories to clarify its etiology and prevention.

Identification of genotypes of Influenza A virus in Malaysia

PubMed Central

MM, Rahman; KK, Wong; I, Isahak; ZZ, Rashid; H, Alfizah

2014-01-01

Objective: Influenza is considered as an emerging disease until today. The present study was undertaken to determine the prevalent genotypes of Influenza A virus in Malaysia. Methods: Influenza A virus was identified from respiratory specimens by real-time reverse transcriptase polymerase chain reaction (rRT-PCR). Phylogenetic analysis of the identified isolates was performed and genotypes were detected. Results: A total number of 505 throat swabs and nasopharyngeal aspirates were examined by rRT-PCR at Universiti Kebangsaan Malaysia Medical Centre (UKMMC) in which 65(12.87%) were positive for influenza A. The identified isolates were successfully genotyped by phylogenetic analysis. The identified influenza A genotypes were: H1N1 (42), H3N2 (20) and H5N1 (3). Conclusion: The findings indicated that 3 genotypes were circulating in Malaysia during 2011 in which H1N1 was the predominant. Results added new genotype (H5N1) identification record in Malaysia that may be added in data base of WHO and CDC. PMID:25225528

Identification of genotypes of Influenza A virus in Malaysia.

PubMed

Mm, Rahman; Kk, Wong; I, Isahak; Zz, Rashid; H, Alfizah

2014-09-01

Influenza is considered as an emerging disease until today. The present study was undertaken to determine the prevalent genotypes of Influenza A virus in Malaysia. Influenza A virus was identified from respiratory specimens by real-time reverse transcriptase polymerase chain reaction (rRT-PCR). Phylogenetic analysis of the identified isolates was performed and genotypes were detected. A total number of 505 throat swabs and nasopharyngeal aspirates were examined by rRT-PCR at Universiti Kebangsaan Malaysia Medical Centre (UKMMC) in which 65(12.87%) were positive for influenza A. The identified isolates were successfully genotyped by phylogenetic analysis. The identified influenza A genotypes were: H1N1 (42), H3N2 (20) and H5N1 (3). The findings indicated that 3 genotypes were circulating in Malaysia during 2011 in which H1N1 was the predominant. RESULTS added new genotype (H5N1) identification record in Malaysia that may be added in data base of WHO and CDC.

Expression of chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) in bladder transitional cell carcinoma.

PubMed

Ham, Won Sik; Lee, Joo Hyoung; Yu, Ho Song; Choi, Young Deuk

2008-10-01

An analysis of differentially expressed genes (DEGs) between bladder transitional cell carcinoma (TCC) and the surrounding urothelium to help identify what lies behind the mechanism of multifocal tumor development has not yet been performed. We sought to find a new DEG related to the development of bladder TCC. Thirty-nine bladder TCC tissues paired with normal-appearing urothelium tissues obtained from the same patient were used as subjects. Initially, we compared the messenger RNA (mRNA) profiles between normal-appearing urothelium and TCC tissue of 1 patient by using annealing control primer (ACP)-based GeneFishing polymerase chain reaction (PCR) and selective amplification of family members (SAFM) PCR to identify potential DEGs. To validate the results of the ACP data, reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on those of all 39 patients. Among the several DEGs discovered in the ACP data, 1 DEG was chosen as the candidate for the RT-PCR, that is present or markedly upregulated in normal-appearing urothelial tissue compared with TCC tissue. Gene sequence searching revealed that this DEG is chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI). Downregulation of COUP-TFI mRNA expression in TCC tissue compared to normal-appearing urothelium tissue of the same patient, irrespective of tumor stage and grade, was confirmed by RT-PCR in 39 patients. Our results suggest that the loss of COUP-TFI may play a role in the transition from normal epithelium to TCC. Further characterization of the COUP-TFI gene is expected to give us informations about bladder TCC tumorigenesis.

A candidate gene for autoimmune myasthenia gravis

PubMed Central

Landouré, Guida; Knight, Melanie A.; Stanescu, Horia; Taye, Addis A.; Shi, Yijun; Diallo, Oumarou; Johnson, Janel O.; Hernandez, Dena; Traynor, Bryan J.; Biesecker, Leslie G.; Elkahloun, Abdel; Rinaldi, Carlo; Vincent, Angela; Willcox, Nick; Kleta, Robert; Fischbeck, Kenneth H.

2012-01-01

Objective: We sought to identify a causative mutation in a previously reported kindred with parental consanguinity and 5 of 10 siblings with adult-onset autoimmune myasthenia gravis. Methods: We performed genome-wide homozygosity mapping, and sequenced all known genes in the one region of extended homozygosity. Quantitative and allele-specific reverse transcriptase PCR (RT-PCR) were performed on a candidate gene to determine the RNA expression level in affected siblings and controls and the relative abundance of the wild-type and mutant alleles in a heterozygote. Results: A region of shared homozygosity at chromosome 13q13.3–13q14.11 was found in 4 affected siblings and 1 unaffected sibling. A homozygous single nucleotide variant was found in the 3′-untranslated region of the ecto-NADH oxidase 1 gene (ENOX1). No other variants likely to be pathogenic were found in genes in this region or elsewhere. The ENOX1 sequence variant was not found in 764 controls. Quantitative RT-PCR showed that expression of ENOX1 decreased to about 20% of normal levels in lymphoblastoid cells from individuals homozygous for the variant and to about 50% in 2 unaffected heterozygotes. Allele-specific RT-PCR showed a 55%–60% reduction in the level of the variant transcript in heterozygous cells due to reduced mRNA stability. Conclusion: These results indicate that this sequence variant in ENOX1 may contribute to the familial autoimmune myasthenia in these patients. PMID:22744667

Comprehensive Multiplex One-Step Real-Time TaqMan qRT-PCR Assays for Detection and Quantification of Hemorrhagic Fever Viruses

PubMed Central

Li, Jiandong; Qu, Jing; He, Chengcheng; Zhang, Shuo; Li, Chuan; Zhang, Quanfu; Liang, Mifang; Li, Dexin

2014-01-01

Background Viral hemorrhagic fevers (VHFs) are a group of animal and human illnesses that are mostly caused by several distinct families of viruses including bunyaviruses, flaviviruses, filoviruses and arenaviruses. Although specific signs and symptoms vary by the type of VHF, initial signs and symptoms are very similar. Therefore rapid immunologic and molecular tools for differential diagnosis of hemorrhagic fever viruses (HFVs) are important for effective case management and control of the spread of VHFs. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay is one of the reliable and desirable methods for specific detection and quantification of virus load. Multiplex PCR assay has the potential to produce considerable savings in time and resources in the laboratory detection. Results Primers/probe sets were designed based on appropriate specific genes for each of 28 HFVs which nearly covered all the HFVs, and identified with good specificity and sensitivity using monoplex assays. Seven groups of multiplex one-step real-time qRT-PCR assays in a universal experimental system were then developed by combining all primers/probe sets into 4-plex reactions and evaluated with serial dilutions of synthesized viral RNAs. For all the multiplex assays, no cross-reactivity with other HFVs was observed, and the limits of detection were mainly between 45 and 150 copies/PCR. The reproducibility was satisfactory, since the coefficient of variation of Ct values were all less than 5% in each dilution of synthesized viral RNAs for both intra-assays and inter-assays. Evaluation of the method with available clinical serum samples collected from HFRS patients, SFTS patients and Dengue fever patients showed high sensitivity and specificity of the related multiplex assays on the clinical specimens. Conclusions Overall, the comprehensive multiplex one-step real-time qRT-PCR assays were established in this study, and proved to be specific, sensitive, stable and easy to serve as a useful tool for rapid detection of HFVs. PMID:24752452

Dynamic Changes of Smooth Muscle and Endothelial Markers in the Early Healing Process of Dacron Vascular Grafts in the Dog, Using RT-PCR.

PubMed

Ishida; Wu; Shi; Fujita; Sauvage; Hammond; Wijelath

2000-03-01

Previous studies of neointima formation on Dacron vascular grafts mainly focused on the late stages using immunohistochemistry staining for von Willebrand factor (vWF) and smooth muscle (SM) alpha-actin. However, it is impossible to use immunohistochemistry to study the early events of neointima formation, because graft samples lack sufficient cellular material. Therefore, we used reverse transcriptase-polymerase chain reaction (RT-PCR) to demonstrate dynamic changes of SM and endothelial markers during the early stages of neointima formation. Preclotted Dacron grafts were implanted in the descending thoracic aorta of 14 mongrel dogs. Specimens were retrieved at 1-4 weeks. Total RNAs were extracted from mid-portion of graft flow surfaces, and RT-PCR for vWF, SM myosin heavy chain (MHC), and SM alpha-actin were performed and expressed as a ratio to the ribosome s17 signal. SM MHC and vWF mRNA expression was low at 1-2 weeks but elevated at 3-4 weeks (P < 0.05). However, SM alpha-actin mRNA levels were expressed consistently throughout the study period. At 3-4 weeks, vWF mRNA expression was inversely correlated to thrombus formation on the graft flow surface. Increased expressions of SM MHC and vWF mRNA corresponded to the formation of neointima and an endothelial layer at the later stages. However, SM alpha-actin mRNA expression did not vary during the healing process. The application of RT-PCR should permit further studies of gene regulation in the early vascular graft healing process in vivo. This model can also be used to study the molecular events that are involved in SM cell differentiation.

Molecular epidemiology of human metapneumovirus in Ireland.

PubMed

Carr, Michael J; Waters, Allison; Fenwick, Fiona; Toms, Geoffrey L; Hall, William W; O'Kelly, Edwin

2008-03-01

Human metapneumovirus (hMPV) is a cause of respiratory illness ranging from wheezing to bronchiolitis and pneumonia in children. A quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay was developed for the detection of all four main genetic lineages of hMPV and employed to validate an indirect immunofluorescence (IF) assay to detect hMPV positive specimens. The IF assay detected 24 positives from a screen of 625 randomly selected pediatric respiratory specimens collected (3.8% prevalence). From this cohort of 625 specimens, 229 were also tested by real-time RT-PCR assay. This included the 24 IF positive specimens and 205 randomly selected specimens from both study periods. In addition to confirming all the IF positives, the real-time assay detected an additional six hMPV positive specimens giving rise to a combined prevalence of 4.8%. Phylogenetic analysis showed that hMPV subtypes A2b and B2 to be the most prevalent genotypes circulating in our population and surprisingly no hMPV subgroups A1 or B1 were detected during this study period. Based on this phylogenetic analysis, we propose the existence of sub-clusters of hMPV genotype B2 present in our population which we term subtypes B2a and B2b. The mean log 10 copies/ml of quantitative RT-PCR determinations from these 30 hMPV positive respiratory specimens was 6.35 (range = 4.44-8.15). Statistical analysis of quantitative RT-PCR determinations of viral load from these 30 respiratory specimens suggests that hMPV genotype B specimens have a higher viral load than hMPV genotype A isolates (P < 0.03).

Evaluation of reference genes in mouse preimplantation embryos for gene expression studies using real-time quantitative RT-PCR (RT-qPCR).

PubMed

Jeong, Jae-Kyo; Kang, Min-Hee; Gurunathan, Sangiliyandi; Cho, Ssang-Goo; Park, Chankyu; Seo, Han Geuk; Kim, Jin-Hoi

2014-09-25

Real-time quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) is the most sensitive, and valuable technique for rare mRNA detection. However, the expression profiles of reference genes under different experimental conditions, such as different mouse strains, developmental stage, and culture conditions have been poorly studied. mRNA stability of the actb, gapdh, sdha, ablim, ywhaz, sptbn, h2afz, tgfb1, 18 s and wrnip genes was analyzed. Using the NormFinder program, the most stable genes are as follows: h2afz for the B6D2F-1 and C57BL/6 strains; sptbn for ICR; h2afz for KOSOM and CZB cultures of B6D2F-1 and C57BL/6 strain-derived embryos; wrnip for M16 culture of B6D2F-1 and C57BL/6 strain-derived embryos; ywhaz, tgfb1, 18 s, 18 s, ywhaz, and h2afz for zygote, 2-cell, 4-cell, 8-cell, molular, and blastocyst embryonic stages cultured in KSOM medium, respectively; h2afz, wrnip, wrnip, h2afz, ywhaz, and ablim for zygote, 2-cell, 4-cell, 8-cell, molular, and blastocyst stage embryos cultured in CZB medium, respectively; 18 s, h2afz, h2afz, actb, h2afz, and wrnip for zygote, 2-cell, 4-cell, 8-cell, molular, and blastocyst stage embryos cultured in M16 medium, respectively. These results demonstrated that candidate reference genes for normalization of target gene expression using RT-qPCR should be selected according to mouse strains, developmental stage, and culture conditions.

The relationship between quantitative human epidermal growth factor receptor 2 gene expression by the 21-gene reverse transcriptase polymerase chain reaction assay and adjuvant trastuzumab benefit in Alliance N9831.

PubMed

Perez, Edith A; Baehner, Frederick L; Butler, Steven M; Thompson, E Aubrey; Dueck, Amylou C; Jamshidian, Farid; Cherbavaz, Diana; Yoshizawa, Carl; Shak, Steven; Kaufman, Peter A; Davidson, Nancy E; Gralow, Julie; Asmann, Yan W; Ballman, Karla V

2015-10-01

The N9831 trial demonstrated the efficacy of adjuvant trastuzumab for patients with human epidermal growth factor receptor 2 (HER2) locally positive tumors by protein or gene analysis. We used the 21-gene assay to examine the association of quantitative HER2 messenger RNA (mRNA) gene expression and benefit from trastuzumab. N9831 tested the addition of trastuzumab to chemotherapy in stage I-III HER2-positive breast cancer. For two of the arms of the trial, doxorubicin and cyclophosphamide followed by paclitaxel (AC-T) and doxorubicin and cyclophosphamide followed by paclitaxel and trastuzumab concurrent chemotherapy-trastuzumab (AC-TH), recurrence score (RS) and HER2 mRNA expression were determined by the 21-gene assay (Oncotype DX®) (negative <10.7, equivocal 10.7 to <11.5, and positive ≥11.5 log2 expression units). Cox regression was used to assess the association of HER2 expression with trastuzumab benefit in preventing distant recurrence. Median follow-up was 7.4 years. Of 1,940 total patients, 901 had consent and sufficient tissue. HER2 by reverse transcriptase polymerase chain reaction (RT-PCR) was negative in 130 (14 %), equivocal in 85 (9 %), and positive in 686 (76 %) patients. Concordance between HER2 assessments was 95 % for RT-PCR versus central immunohistochemistry (IHC) (>10 % positive cells = positive), 91 % for RT-PCR versus central fluorescence in situ hybridization (FISH) (≥2.0 = positive) and 94 % for central IHC versus central FISH. In the primary analysis, the association of HER2 expression by 21-gene assay with trastuzumab benefit was marginally nonsignificant (nonlinear p = 0.057). In hormone receptor-positive patients (local IHC) the association was significant (p = 0.002). The association was nonlinear with the greatest estimated benefit at lower and higher HER2 expression levels. Concordance among HER2 assessments by central IHC, FISH, and RT-PCR were similar and high. Association of HER2 mRNA expression with trastuzumab benefit as measured by time to distant recurrence was nonsignificant. A consistent benefit of trastuzumab irrespective of mHER2 levels was observed in patients with either IHC-positive or FISH-positive tumors. Trend for benefit was observed also for the small groups of patients with negative results by any or all of the central assays. Clinicaltrials.gov NCT00005970 . Registered 5 July 2000.

Determination of internal controls for quantitative real time RT-PCR analysis of the effect of Edwardsiella tarda infection on gene expression in turbot (Scophthalmus maximus).

PubMed

Dang, Wei; Sun, Li

2011-02-01

In recent years, quantitative real time reverse transcriptase-PCR (qRT-PCR) has been used frequently in the study of gene expression in turbot (Scophthalmus maximus) in relation to bacterial infection. However, no investigations on appropriate qRT-PCR reference genes have been documented. In this report, we determined the potential of eight housekeeping genes, i.e. β-actin (ACTB), ribosomal protein L17 (RPL17), α-tubulin (TUBA), elongation factor-1-α(EF1A), β-2-Microglobulin (B2M), RNA polymerase II subunit D (RPSD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18S ribosomal RNA (18S rRNA), as internal standards for qRT-PCR analysis of gene expression in turbot as a function of bacterial infection. For this purpose, the expression of the eight housekeeping genes in seven turbot tissues was determined by qRT-PCR before and after bacterial challenge, and the data were analyzed with the geNorm and NormFinder algorisms. The results showed that the expression of all the examined genes exhibited tissue-dependent variations both before and after bacterial challenge. Before bacterial challenge, geNorm and NormFinder identified RPSD as the gene that showed least tissue specific expression. At 12 h post-bacterial infection, geNorm ranked ACTB/GAPDH, 18S rRNA/ACTB, ACTB/GAPDH, 18S rRNA/ACTB, RPL17/TUBA, RPSD/GAPDH, and RPSD/B2M, respectively, as the most stably expressed genes in liver, spleen, kidney, gill, heart, muscle, and brain. Comparable ranking orders were produced by NormFinder. Similar results were obtained at 24 h post-bacterial infection. Taken together, these results indicate that RPSD is the most stable gene across tissue types under normal physiological conditions and that, during bacterial infection, ACTB might be used as an internal standard for the normalization of gene expression in immune relevant organs; however, no single gene or single pair of genes in the examined set of housekeeping genes can serve as a universal reference across all tissue types under the condition of bacterial infection. Copyright © 2011 Elsevier Ltd. All rights reserved.

Serologic evaluation, efficacy, and safety of a commerical modified-live canine distemper vaccine in domestic ferrets.

PubMed

Wimsatt, J; Jay, M T; Innes, K E; Jessen, M; Collins, J K

2001-05-01

To determine efficacy and safety of a commercial modified-live canine distemper virus (CDV) vaccine used for prophylaxis in domestic ferrets. Sixteen 16-week-old neutered male ferrets. Equal groups of ferrets were inoculated subcutaneously at 16 and 20 weeks of age with saline (0.9% NaCl) solution or a vaccine derived from the Onderstepoort CDV strain and attenuated in a primate cell line. Live virulent CDV was administered to all ferrets intranasally and orally 3 weeks after the second inoculation. Clinical signs and body weights were monitored regularly during the study. Blood samples for serologic examination were drawn prior to each inoculation, before challenge exposure, and 10, 15, and 21 days after exposure. Blood samples for reverse transcriptase polymerase chain reaction (RT-PCR) were obtained 5 days after the first vaccination, and 5, 10, 15, and 21 days after challenge exposure. After challenge exposure, control ferrets had significantly more clinical signs and weight loss, compared with vaccinates. All vaccinated ferrets survived, whereas all control ferrets died. The RT-PCR assay was successful in detecting CDV in blood and fresh or formalin-fixed tissues from infected ferrets. Findings suggest that the vaccine when given SC to domestic ferrets as directed is safe and protective against challenge exposure with virulent CDV. The RT-PCR assay may simplify detection of CDV in fresh and fixed tissues.

Synthesis, structure-activity relationship and molecular docking of cyclohexenone based analogous as potent non-nucleoside reverse-transcriptase inhibitors

NASA Astrophysics Data System (ADS)

Nazar, Muhammad Faizan; Abdullah, Muhammad Imran; Badshah, Amir; Mahmood, Asif; Rana, Usman Ali; Khan, Salah Ud-Din

2015-04-01

The chalcones core in compounds is advantageously chosen effective synthons, which offer exciting perspectives in biological and pharmacological research. The present study reports the successful development of eight new cyclohexenone based anti-reverse transcriptase analogous using rational drug design synthesis principles. These new cyclohexenone derivatives (CDs) were synthesized by following a convenient route of Robinson annulation, and the molecular structure of these CDs were later confirmed by various analytical techniques such as 1H NMR, 13C NMR, FT-IR, UV-Vis spectroscopy and mass spectrometry. All the synthesized compounds were screened theoretically and experimentally against reverse transcriptase (RT) and found potentially active reverse transcriptase (RT) inhibitors. Of the compounds studied, the compound 2FC4 showed high interaction with RT at non-nucleoside binding site, contributing high free binding energy (ΔG -8.01 Kcal) and IC50 (0.207 μg/ml), respectively. Further results revealed that the compounds bearing more halogen groups, with additional hydrophobic character, offered superior anti-reverse transcriptase activity as compared to rest of compounds. It is anticipate that the present study would be very useful for the selection of potential reverse transcriptase inhibitors featuring inclusive pharmacological profiles.

Validation of housekeeping genes as an internal control for gene expression studies in Giardia lamblia using quantitative real-time PCR.

PubMed

Marcial-Quino, Jaime; Fierro, Francisco; De la Mora-De la Mora, Ignacio; Enríquez-Flores, Sergio; Gómez-Manzo, Saúl; Vanoye-Carlo, America; Garcia-Torres, Itzhel; Sierra-Palacios, Edgar; Reyes-Vivas, Horacio

2016-04-25

The analysis of transcript levels of specific genes is important for understanding transcriptional regulation and for the characterization of gene function. Real-time quantitative reverse transcriptase PCR (RT-qPCR) has become a powerful tool to quantify gene expression. The objective of this study was to identify reliable housekeeping genes in Giardia lamblia. Twelve genes were selected for this purpose, and their expression was analyzed in the wild type WB strain and in two strains with resistance to nitazoxanide (NTZ) and metronidazole (MTZ), respectively. RefFinder software analysis showed that the expression of the genes is different in the three strains. The integrated data from the four analyses showed that the NADH oxidase (NADH) and aldolase (ALD) genes were the most steadily expressed genes, whereas the glyceraldehyde-3-phosphate dehydrogenase gene was the most unstable. Additionally, the relative expression of seven genes were quantified in the NTZ- and MTZ-resistant strains by RT-qPCR, using the aldolase gene as the internal control, and the results showed a consistent differential pattern of expression in both strains. The housekeeping genes found in this work will facilitate the analysis of mRNA expression levels of other genes of interest in G. lamblia. Copyright © 2016 Elsevier B.V. All rights reserved.

A two-step lyssavirus real-time polymerase chain reaction using degenerate primers with superior sensitivity to the fluorescent antigen test.

PubMed

Suin, Vanessa; Nazé, Florence; Francart, Aurélie; Lamoral, Sophie; De Craeye, Stéphane; Kalai, Michael; Van Gucht, Steven

2014-01-01

A generic two-step lyssavirus real-time reverse transcriptase polymerase chain reaction (qRT-PCR), based on a nested PCR strategy, was validated for the detection of different lyssavirus species. Primers with 17 to 30% of degenerate bases were used in both consecutive steps. The assay could accurately detect RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV. In silico sequence alignment showed a functional match with the remaining lyssavirus species. The diagnostic specificity was 100% and the sensitivity proved to be superior to that of the fluorescent antigen test. The limit of detection was ≤ 1 50% tissue culture infectious dose. The related vesicular stomatitis virus was not recognized, confirming the selectivity for lyssaviruses. The assay was applied to follow the evolution of rabies virus infection in the brain of mice from 0 to 10 days after intranasal inoculation. The obtained RNA curve corresponded well with the curves obtained by a one-step monospecific RABV-qRT-PCR, the fluorescent antigen test, and virus titration. Despite the presence of degenerate bases, the assay proved to be highly sensitive, specific, and reproducible.

A Two-Step Lyssavirus Real-Time Polymerase Chain Reaction Using Degenerate Primers with Superior Sensitivity to the Fluorescent Antigen Test

PubMed Central

Nazé, Florence; Francart, Aurélie; Lamoral, Sophie; De Craeye, Stéphane; Kalai, Michael

2014-01-01

A generic two-step lyssavirus real-time reverse transcriptase polymerase chain reaction (qRT-PCR), based on a nested PCR strategy, was validated for the detection of different lyssavirus species. Primers with 17 to 30% of degenerate bases were used in both consecutive steps. The assay could accurately detect RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV. In silico sequence alignment showed a functional match with the remaining lyssavirus species. The diagnostic specificity was 100% and the sensitivity proved to be superior to that of the fluorescent antigen test. The limit of detection was ≤1 50% tissue culture infectious dose. The related vesicular stomatitis virus was not recognized, confirming the selectivity for lyssaviruses. The assay was applied to follow the evolution of rabies virus infection in the brain of mice from 0 to 10 days after intranasal inoculation. The obtained RNA curve corresponded well with the curves obtained by a one-step monospecific RABV-qRT-PCR, the fluorescent antigen test, and virus titration. Despite the presence of degenerate bases, the assay proved to be highly sensitive, specific, and reproducible. PMID:24822188

Coinfection and vertical transmission of Brucella and Morbillivirus in a neonatal sperm whale (Physeter macrocephalus) in Hawaii, USA.

PubMed

West, Kristi L; Levine, Gregg; Jacob, Jessica; Jensen, Brenda; Sanchez, Susan; Colegrove, Kathleen; Rotstein, David

2015-01-01

The viral genus Morbillivirus and the bacterial genus Brucella have emerged as important groups of pathogens that are known to affect cetacean health on a global scale, but neither pathogen has previously been reported from endangered sperm whales (Physeter macrocephalus). A female neonate sperm whale stranded alive and died near Laie on the island of Oahu, Hawaii, US, in May of 2011. Congestion of the cerebrum and enlarged lymph nodes were noted on the gross necropsy. Microscopic findings included lymphoid depletion, chronic meningitis, and pneumonia, suggesting an in utero infection. Cerebrum, lung, umbilicus, and select lymph nodes (tracheobronchial and mediastinal) were positive for Brucella by PCR. Brucella sp. was also cultured from the cerebrum and from mediastinal and tracheobronchial lymph nodes. Twelve different tissues were screened for Morbillivirus by reverse-transcriptase (RT)-PCR and select tissues by immunohistochemistry, but only the tracheobronchial lymph node and spleen were positive by RT-PCR. Pathologic findings observed were likely a result of Brucella, but Morbillivirus may have played a key role in immune suppression of the mother and calf. The in utero infection in this individual strongly supports vertical transmission of both pathogens.

MAEWEST Expression in Flower Development of Two Petunia Species

PubMed Central

Segatto, Ana Lúcia A.; Turchetto-Zolet, Andreia Carina; Aizza, Lilian Cristina B.; Monte-Bello, Carolina C.; Dornelas, Marcelo C.; Margis, Rogerio; Freitas, Loreta B.

2013-01-01

Changes in flower morphology may influence the frequency and specificity of animal visitors. In Petunia (Solanaceae), adaptation to different pollinators is one of the factors leading to species diversification within the genus. This study provides evidence that differential expression patterns of MAWEWEST (MAW) homologs in different Petunia species may be associated with adaptive changes in floral morphology. The Petunia × hybrida MAW gene belongs to the WOX (WUSCHEL-related homeobox) transcription factor family and has been identified as a controller of petal fusion during corolla formation. We analyzed the expression patterns of P. inflata and P. axillaris MAW orthologs (PiMAW and PaMAW, respectively) by reverse transcriptase polymerase chain reaction (RT-PCR), reverse transcription–quantitative PCR (qRT-PCR) and in situ hybridization in different tissues and different developmental stages of flowers in both species. The spatial expression patterns of PiMAW and PaMAW were similar in P. inflata and P. axillaris. Nevertheless, PaMAW expression level in P. axillaris was higher during the late bud development stage as compared to PiMAW in P. inflata. This work represents an expansion of petunia developmental research to wild accessions. PMID:23823801

AtDMC1, the Arabidopsis homologue of the yeast DMC1 gene: characterization, transposon-induced allelic variation and meiosis-associated expression.

PubMed

Klimyuk, V I; Jones, J D

1997-01-01

Based on homologies between the yeast DMC1 and the lily LIM15 meiosis-specific genes, degenerate PCR primers were designed that amplified the Arabidopsis DMC1 gene (AtDMC1). AtDMC1 genomic DNA (8 kb) was sequenced, and the transcript was characterized by reverse transcriptase-polymerase chain reaction (RT-PCR) and by 5' and 3' RACE (rapid amplification of cDNA ends). The AtDMC1 gene contains 15 exons and 14 introns. RNA in situ hybridization analysis showed that expression of the AtDMC1 is restricted to pollen mother cells in anthers and to megaspore mother cells in ovules. The AtDMC1 promoter was fused to the GUS reporter gene, and conferred meiosis-associated expression in both male and female floral lineages. Comparison of AtDMC1 isolated from Landsberg erecta ecotype to its Columbia allele ArLIM15, revealed the presence of a 1874 bp transposon-like element within the promoter region of ArLIM15. RT-PCR analysis showed that the expression levels of AtDMC1 and ArLIM15 are similar. Possible uses for the AtDMC1 promoter are discussed.

Development of a reverse transcription polymerase chain reaction method for yellow fever virus detection.

PubMed

Méndez, María C; Domingo, Cristina; Tenorio, Antonio; Pardo, Lissethe C; Rey, Gloria J; Méndez, Jairo A

2013-09-01

Yellow fever is considered a re-emerging disease and is endemic in tropical regions of Africa and South America. At present, there are no standardized or commercialized kits available for yellow fever virus detection. Therefore, diagnosis must be made by time-consuming routine techniques, and sometimes, the virus or its proteins are not detected. Furthermore, co-circulation with other flaviviruses, including dengue virus, increases the difficulty of diagnosis. To develop a specific reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR-based assay to improve the detection and diagnosis of yellow fever virus using both serum and fresh tissue samples. RT-PCR primers were designed to amplify a short fragment of all yellow fever virus genotypes reported. A second set of primers was used in a nested PCR to increase sensitivity. Thirty-three clinical samples were tested with the standardized reaction. The expected amplicon was obtained in 25 out of 33 samples analyzed using this approach, and 2 more samples tested positive after a subsequent nested PCR approach. This improved technique not only ensures the specific detection of a wide range of yellow fever virus genotypes but also may increase the sensitivity of detection by introducing a second round of amplification, allowing a rapid differential diagnosis between dengue and yellow fever infection, which is required for effective surveillance and opportune epidemiologic measures.

An immortalized goat mammary epithelial cell line induced with human telomerase reverse transcriptase (hTERT) gene transfer.

PubMed

He, Y L; Wu, Y H; He, X N; Liu, F J; He, X Y; Zhang, Y

2009-06-01

Although mammary epithelial cell lines can provide a rapid and reliable indicator of gene expression efficiency of transgenic animals, their short lifespan greatly limits this application. To provide stable and long lifespan cells, goat mammary epithelial cells (GMECs) were transduced with pLNCX2-hTERT by retrovirus-mediated gene transfer. Transduced GMECs were evaluated by reverse transcriptase polymerase chain reaction (RT-PCR), proliferation assays, karyotype analysis, telomerase activity assay, western blotting, soft agar assay, and injection into nude mice. Non-transduced GMECs were used as a control. The hTERT-GMECs had higher telomerase activity and extended proliferative lifespan compared to non-transfected GMECs; even after Passage 50, hTERT-GMECs had a near diploid complement of chromosomes. Furthermore, they did not gain the anchorage-independent growth property and were not associated with a malignant phenotype in vitro or in vivo.

Guillain-Barré Syndrome Associated with Zika Virus Infection in Colombia.

PubMed

Parra, Beatriz; Lizarazo, Jairo; Jiménez-Arango, Jorge A; Zea-Vera, Andrés F; González-Manrique, Guillermo; Vargas, José; Angarita, Jorge A; Zuñiga, Gonzalo; Lopez-Gonzalez, Reydmar; Beltran, Cindy L; Rizcala, Karen H; Morales, Maria T; Pacheco, Oscar; Ospina, Martha L; Kumar, Anupama; Cornblath, David R; Muñoz, Laura S; Osorio, Lyda; Barreras, Paula; Pardo, Carlos A

2016-10-20

Zika virus (ZIKV) infection has been linked to the Guillain-Barré syndrome. From November 2015 through March 2016, clusters of cases of the Guillain-Barré syndrome were observed during the outbreak of ZIKV infection in Colombia. We characterized the clinical features of cases of Guillain-Barré syndrome in the context of this ZIKV infection outbreak and investigated their relationship with ZIKV infection. A total of 68 patients with the Guillain-Barré syndrome at six Colombian hospitals were evaluated clinically, and virologic studies were completed for 42 of the patients. We performed reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assays for ZIKV in blood, cerebrospinal fluid, and urine, as well as antiflavivirus antibody assays. A total of 66 patients (97%) had symptoms compatible with ZIKV infection before the onset of the Guillain-Barré syndrome. The median period between the onset of symptoms of ZIKV infection and symptoms of the Guillain-Barré syndrome was 7 days (interquartile range, 3 to 10). Among the 68 patients with the Guillain-Barré syndrome, 50% were found to have bilateral facial paralysis on examination. Among 46 patients in whom nerve-conduction studies and electromyography were performed, the results in 36 patients (78%) were consistent with the acute inflammatory demyelinating polyneuropathy subtype of the Guillain-Barré syndrome. Among the 42 patients who had samples tested for ZIKV by RT-PCR, the results were positive in 17 patients (40%). Most of the positive RT-PCR results were in urine samples (in 16 of the 17 patients with positive RT-PCR results), although 3 samples of cerebrospinal fluid were also positive. In 18 of 42 patients (43%) with the Guillain-Barré syndrome who underwent laboratory testing, the presence of ZIKV infection was supported by clinical and immunologic findings. In 20 of these 42 patients (48%), the Guillain-Barré syndrome had a parainfectious onset. All patients tested were negative for dengue virus infection as assessed by RT-PCR. The evidence of ZIKV infection documented by RT-PCR among patients with the Guillain-Barré syndrome during the outbreak of ZIKV infection in Colombia lends support to the role of the infection in the development of the Guillain-Barré syndrome. (Funded by the Bart McLean Fund for Neuroimmunology Research and others.).

Identification of suitable reference genes for gene expression normalization in qRT-PCR analysis in watermelon.

PubMed

Kong, Qiusheng; Yuan, Jingxian; Gao, Lingyun; Zhao, Shuang; Jiang, Wei; Huang, Yuan; Bie, Zhilong

2014-01-01

Watermelon is one of the major Cucurbitaceae crops and the recent availability of genome sequence greatly facilitates the fundamental researches on it. Quantitative real-time reverse transcriptase PCR (qRT-PCR) is the preferred method for gene expression analyses, and using validated reference genes for normalization is crucial to ensure the accuracy of this method. However, a systematic validation of reference genes has not been conducted on watermelon. In this study, transcripts of 15 candidate reference genes were quantified in watermelon using qRT-PCR, and the stability of these genes was compared using geNorm and NormFinder. geNorm identified ClTUA and ClACT, ClEF1α and ClACT, and ClCAC and ClTUA as the best pairs of reference genes in watermelon organs and tissues under normal growth conditions, abiotic stress, and biotic stress, respectively. NormFinder identified ClYLS8, ClUBCP, and ClCAC as the best single reference genes under the above experimental conditions, respectively. ClYLS8 and ClPP2A were identified as the best reference genes across all samples. Two to nine reference genes were required for more reliable normalization depending on the experimental conditions. The widely used watermelon reference gene 18SrRNA was less stable than the other reference genes under the experimental conditions. Catalase family genes were identified in watermelon genome, and used to validate the reliability of the identified reference genes. ClCAT1and ClCAT2 were induced and upregulated in the first 24 h, whereas ClCAT3 was downregulated in the leaves under low temperature stress. However, the expression levels of these genes were significantly overestimated and misinterpreted when 18SrRNA was used as a reference gene. These results provide a good starting point for reference gene selection in qRT-PCR analyses involving watermelon.

Comparison of Assays for Sensitive and Reproducible Detection of Cell Culture-Infectious Cryptosporidium parvum and Cryptosporidium hominis in Drinking Water

PubMed Central

Di Giovanni, George D.; Rochelle, Paul A.

2012-01-01

This study compared the three most commonly used assays for detecting Cryptosporidium sp. infections in cell culture: immunofluorescent antibody and microscopy assay (IFA), PCR targeting Cryptosporidium sp.-specific DNA, and reverse transcriptase PCR (RT-PCR) targeting Cryptosporidium sp.-specific mRNA. Monolayers of HCT-8 cells, grown in 8-well chamber slides or 96-well plates, were inoculated with a variety of viable and inactivated oocysts to assess assay performance. All assays detected infection with low doses of flow cytometry-enumerated Cryptosporidium parvum oocysts, including infection with one oocyst and three oocysts. All methods also detected infection with Cryptosporidium hominis. The RT-PCR assay, IFA, and PCR assay detected infection in 23%, 25%, and 51% of monolayers inoculated with three C. parvum oocysts and 10%, 9%, and 16% of monolayers inoculated with one oocyst, respectively. The PCR assay was the most sensitive, but it had the highest frequency of false positives with mock-infected cells and inactivated oocysts. IFA was the only infection detection assay that did not produce false positives with mock-infected monolayers. IFA was also the only assay that detected infections in all experiments with spiked oocysts recovered from Envirochek capsules following filtration of 1,000 liters of treated water. Consequently, cell culture with IFA detection is the most appropriate method for routine and sensitive detection of infectious Cryptosporidium parvum and Cryptosporidium hominis in drinking water. PMID:22038611

Kaposi's Sarcoma-Associated Herpesvirus Can Productively Infect Primary Human Keratinocytes and Alter Their Growth Properties

PubMed Central

Cerimele, Francesca; Curreli, Francesca; Ely, Scott; Friedman-Kien, Alvin E.; Cesarman, Ethel; Flore, Ornella

2001-01-01

Previous studies have shown the presence of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) DNA in endothelial cells, in keratinocytes in the basal layer of the epidermis overlying plaque-stage nodular lesions of cutaneous Kaposi's sarcoma (KS), and in the epithelial cells of eccrine glands within KS lesions. We infected primary cell cultures of human keratinocytes with KSHV/HHV8. At 6 days post infection, transcription of viral genes was detected by reverse transcriptase PCR (RT-PCR), and protein expression was documented by an immunofluorescence assay with an anti-LANA monoclonal antibody. To determine whether the viral lytic cycle was inducible by chemical treatment, KSHV/HHV8-infected keratinocytes were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) and RT-PCR was performed to confirm the transcription of lytic genes such as open reading frame 26, (which encodes a capsid protein). Finally, to assess infectious viral production, other primary human cells (human umbilical vein endothelial cells), were infected with concentrated supernatant of KSHV-infected, TPA-induced keratinocytes and the presence of viral transcripts was confirmed by RT-PCR. The uninfected keratinocytes senesced 3 to 5 weeks after mock infection, while the KSHV/HHV8-infected keratinocytes continued to proliferate and to date are still in culture. However, 8 weeks after infection, viral genomes were no longer detectable by nested PCR. Although the previously KSHV/HHV8-infected keratinocytes still expressed epithelial markers, they acquired new characteristics such as contact inhibition loss, telomerase activity, anchorage-independent growth, and changes in cytokine production. These results show that KSHV/HHV8, like other herpesviruses, can infect and replicate in epithelial cells in vitro and suggest that in vivo these cells may play a significant role in the establishment of KSHV/HHV8 infection and viral transmission. PMID:11160746

SYBR green-based real-time reverse transcription-PCR for typing and subtyping of all hemagglutinin and neuraminidase genes of avian influenza viruses and comparison to standard serological subtyping tests.

PubMed

Tsukamoto, Kenji; Panei, Carlos Javier; Javier, Panei Carlos; Shishido, Makiko; Noguchi, Daigo; Pearce, John; Kang, Hyun-Mi; Jeong, Ok Mi; Lee, Youn-Jeong; Nakanishi, Koji; Ashizawa, Takayoshi

2012-01-01

Continuing outbreaks of H5N1 highly pathogenic (HP) avian influenza virus (AIV) infections of wild birds and poultry worldwide emphasize the need for global surveillance of wild birds. To support the future surveillance activities, we developed a SYBR green-based, real-time reverse transcriptase PCR (rRT-PCR) for detecting nucleoprotein (NP) genes and subtyping 16 hemagglutinin (HA) and 9 neuraminidase (NA) genes simultaneously. Primers were improved by focusing on Eurasian or North American lineage genes; the number of mixed-base positions per primer was set to five or fewer, and the concentration of each primer set was optimized empirically. Also, 30 cycles of amplification of 1:10 dilutions of cDNAs from cultured viruses effectively reduced minor cross- or nonspecific reactions. Under these conditions, 346 HA and 345 NA genes of 349 AIVs were detected, with average sensitivities of NP, HA, and NA genes of 10(1.5), 10(2.3), and 10(3.1) 50% egg infective doses, respectively. Utility of rRT-PCR for subtyping AIVs was compared with that of current standard serological tests by using 104 recent migratory duck virus isolates. As a result, all HA genes and 99% of the NA genes were genetically subtyped, while only 45% of HA genes and 74% of NA genes were serologically subtyped. Additionally, direct subtyping of AIVs in fecal samples was possible by 40 cycles of amplification: approximately 70% of HA and NA genes of NP gene-positive samples were successfully subtyped. This validation study indicates that rRT-PCR with optimized primers and reaction conditions is a powerful tool for subtyping varied AIVs in clinical and cultured samples.

Lack of detection of a putative retrovirus associated with haemic neoplasia in the soft shell clam Mya arenaria.

PubMed

AboElkhair, M; Iwamoto, T; Clark, K F; McKenna, P; Siah, A; Greenwood, S J; Berthe, F C J; Casey, J W; Cepica, A

2012-01-01

Haemic neoplasia (HN) is a leukemia-like disease that affects at least 20 species of marine bivalves including soft shell clam, Mya arenaria. Since the disease was discovered in 1969, the etiology remains unknown. A retroviral etiology has been suggested based on the detection of reverse transcriptase activity and electron microscopic observation of retroviral-like particles using negative staining. To date, however no virus isolate and no retroviral sequence from HN has been obtained. Moreover, transmission of the disease by cell-free filtrate from affected clams has not been reproduced. In the current study, we reinvestigated the association of HN with a putative retrovirus. Sucrose gradient centrifugation followed by assessment of reverse transcriptase activity, electrophoretic analysis of protein and RNA, and electron microscopic examinations of fractions corresponding to retroviral density were employed. Detection of retroviral pol sequences using degenerate RT-PCR approaches was also attempted. Our results showed visible bands at the expected density of retrovirus in HN-positive and HN-negative clam tissues and both with reverse transcriptase activity. Electron microscopy, RNA analysis, protein analysis, and PCR systems targeting the pol gene of retroviruses did not however provide clear evidence supporting presence of a retrovirus. We point out that the retrovirus etiology of HN of Mya arenaria proposed some 25 years ago should be reconsidered in the absence of a virus isolate or virus sequences. Copyright © 2011 Elsevier Inc. All rights reserved.

An outbreak of West Nile Virus infection in the region of Monastir, Tunisia, 2003

PubMed Central

Riabi, Samira; Gaaloul, Imed; Mastouri, Maha; Hassine, Mohsen; Aouni, Mahjoub

2014-01-01

Background A West Nile (WN) fever epidemic occurred in the region of Monastir, Tunisia, between August and October 2003. Aim of the study We attempt to describe the epidemiology, clinical presentation, and outcome of patients with confirmed West Nile virus (WNV) infection. Methods Three groups of specimens were prepared. One was made up of serum only (n  =  43), the other of cerebrospinal fluid (CSF) only (n  =  30), and the third group was made up of both (n  =  40). These specimens were obtained from 113 patients. A serological diagnosis and evidence of WNV genome by nested reverse-transcriptase polymerase chain reaction (nRT-PCR) and TaqMan reverse transcription-polymerase chain reaction (RT-PCR) were carried out. Results Thirty-eight cases (33.6%) were serologically positive. Results of nRT-PCR showed a total of 10 positive cases of WNV (8.8%) detected in group 1 (n  =  1/43), group 2 (n  =  5/30), and group 3 (n  =  4/40) whereas the PCR TaqMan showed 18 positive samples (15.9%) found in group 1 (n  =  3/43), group 2 (n  =  9/30), and group 3 (n  =  6/40). All TaqMan PCR positive cases were nRT-PCR positive. In addition, four serologically probable cases were confirmed by TaqMan PCR. The attempts to isolate WNV by cell culture were unsuccessful. Considering the results of TaqMan assay and the serological diagnosis, WNV infection was confirmed in a total of 42 patients. The main clinical presentations were meningoencephalitis (40%), febrile disease (95%), and meningitis (36%). Eight patients (19%) died. The highest case-fatality rates occurred among patients aged ≧55 years. The phylogenetic analysis revealed that isolates of WNV were closely related to the Tunisian strain 1997 (PAH001) and the Israeli one (Is-98). Conclusions West Nile virus is a reemerging global pathogen that remains an important public health challenge in the next decade. PMID:24766339

Molecular detection and serotyping of infectious bronchitis virus from FTA filter paper.

PubMed

Moscoso, Hugo; Raybon, Erine O; Thayer, Stephan G; Hofacre, Charles L

2005-03-01

We investigated the feasibility of using Flinders Technology Associates (FTA) filter cards for the storage of allantoic fluid containing an infectious bronchitis virus (IBV), such as Arkansas-DPI, Connecticut, and Massachusetts, and for their identification by reverse transcriptase (RT)-polymerase chain reaction (PCR) and characterization by restriction fragment length polymorphism (RFLP) or nucleotide sequencing. FTA paper is a cotton-based cellulose membrane containing lyophilized chemicals that lyses many types of bacteria and viruses. IBV was inactivated upon contact with the FTA, as shown by the inability of the virus to be propagated in embryonating chicken eggs. RT-PCR of the S1 gene showed that viral RNA in allantoic fluid remained stable after storage on FTA filter cards and that the stability was time and temperature sensitive for the large (1700 base pair [bp]) but not the small (383 bp) PCR products. Analysis of the amplified products showed that molecular characterization is feasible in allantoic fluid stored on FTA under nonfavorable environmental conditions (41 C) for at least 15 days. The use of FTA cards for the collection, transport, and storage of IBV-containing samples is safe, inexpensive, and adequate for molecular diagnosis. We propose that specimens coming from overseas on FTA cards would be first analyzed by RT-PCR with primers yielding a 1700-bp product followed by RFLP of the positive cases. Negative cases would be analyzed with primers yielding a 383-bp product (to exdude detrimental effect of the storage conditions) followed by nucleotide sequencing of the positive cases.

Mycophenolate mofetil prevents the delayed T cell response after pilocarpine-induced status epilepticus in mice

PubMed Central

Engelmann, Robby; Sellmann, Tina; Köhling, Rüdiger; Müller-Hilke, Brigitte

2017-01-01

Growing clinical and laboratory evidence corroborates a role for the immune system in the pathophysiology of epilepsy. In order to delineate the immune response following pilocarpine-induced status epilepticus (SE) in the mouse, we monitored the kinetics of leukocyte presence in the hippocampus over the period of four weeks. SE was induced following a ramping protocol of pilocarpine injection into 4–5 weeks old C57BL/6 mice. Brains were removed at days 1–4, 14 or 28 after SE, and the hippocampi were analyzed via flow cytometry, via quantitative reverse transcriptase PCR (qRT-PCR) and via immunohistochemistry. Epileptogenesis was confirmed by Timm staining of mossy fiber sprouting in the inner molecular layer of the dentate gyrus. The flow cytometry data revealed a biphasic immune response following pilocarpine-induced SE with a transient increase in activated CD11b+ and F4/80+ macrophages within the first four days replaced by an increase in CD3+ T-lymphocytes around day 28. This delayed T cell response was confirmed via qRT-PCR and via immunohistochemistry. In addition, qRT-PCR data could show that the delayed T cell response was associated with an increased CD8/CD4 ratio indicating a cytotoxic T cell response after SE. Intriguingly, early intervention with mycophenolate mofetil administration on days 0–3 after SE prevented this delayed T cell response. These results show an orchestrated immunological sequela and provide evidence that the delayed T cell response is sensitive to early immunomodulatory intervention. PMID:29182639

Biodegradation of trichloroethylene (TCE) by methanotrophic community.

PubMed

Shukla, Awadhesh K; Vishwakarma, Pranjali; Upadhyay, S N; Tripathi, Anil K; Prasana, H C; Dubey, Suresh K

2009-05-01

Laboratory incubation experiments were carried out to assess the potential of methanotrophic culture for degrading TCE. Measurements of the growth rate and TCE degradation showed that the methanotrophs not only grew in presence of TCE but also degraded TCE. The rate of TCE degradation was found to be 0.19 ppm h(-1). The reverse transcriptase-PCR test was conducted to quantify expression of pmoA and mmoX genes. RT-PCR revealed expression of pmoA gene only. This observation provides evidence that the pmoA gene was functionally active for pMMO enzyme during the study. The diversity of the methanotrophs involved in TCE degradation was assessed by PCR amplification, cloning, restriction fragment length polymorphism and phylogenetic analysis of pmoA genes. Results suggested the occurrence of nine different phylotypes belonging to Type II methanotrophs in the enriched cultures. Out of the nine, five clustered with, genera Methylocystis and rest got clustered in to a separate group.

Use of a novel FISH assay on paraffin-embedded tissues as an adjunct to diagnosis of alveolar rhabdomyosarcoma.

PubMed

Nishio, Jun; Althof, Pamela A; Bailey, Jacqueline M; Zhou, Ming; Neff, James R; Barr, Frederic G; Parham, David M; Teot, Lisa; Qualman, Stephen J; Bridge, Julia A

2006-06-01

A valuable diagnostic adjunct and important prognostic parameter in alveolar rhabdomyosarcoma (ARMS) is the identification of translocations t(2;13)(q35;q14) and t(1;13)(p36;q14), and the associated PAX3-FKHR and PAX7-FKHR fusion transcripts, respectively. Most RMS fusion gene type studies have been based on reverse transcriptase-polymerase chain reaction (RT-PCR) detection of the fusion transcript, a technique limited by RNA quality and failure of devised primer sets to detect unusual variants. As an alternative approach, we developed a fluorescence in situ hybridization (FISH) assay that can: (1) distinguish between the two most common ARMS-associated fusion genes; (2) identify potential unusual variant translocations; (3) assess histologic components in mixed alveolar/embryonal RMS; and (4) be performed on paraffinized tissue. FISH analyses of 75 specimens (40 ARMS, 16 ERMS, 8 mixed ARMS/ERMS, and 11 non-RMS tumors) using selected cosmid clone, bacterial, P1-derived, and yeast artificial chromosome probe sets were successful in all but two cases. Among specimens with informative results for both FISH and RT-PCR or standard karyotyping, PAX/FKHR classification results were concordant in 94.6% (53/56). The three discordant cases included one exhibiting a t(2;13) by FISH that was subsequently confirmed by repeat RT-PCR, a second showing a rearrangement of the PAX3 locus only (consistent with the presence of a PAX3 variant translocation), and a third revealing a t(2;13) by FISH that lacked this translocation cytogenetically. Both alveolar and embryonal components of the mixed ARMS/ERMS subtype were negative for PAX3, PAX7, and FKHR rearrangements, a surprising finding confirmed by RT-PCR and/or conventional karyotyping. These data demonstrate that FISH with newly designed probe sets is a reliable and highly specific method of detecting t(1;13) and t(2;13) in routinely processed tissue and may be useful in differentiating ARMS from other small round cell tumors. The findings also suggest that FISH may be a more sensitive assay than RT-PCR in some settings, capable of revealing variant translocations.

The Reverse Transcription Inhibitor Abacavir Shows Anticancer Activity in Prostate Cancer Cell Lines

PubMed Central

Molinari, Agnese; Parisi, Chiara; Bozzuto, Giuseppina; Toccacieli, Laura; Formisano, Giuseppe; De Orsi, Daniela; Paradisi, Silvia; Grober, OlÌ Maria Victoria; Ravo, Maria; Weisz, Alessandro; Arcieri, Romano; Vella, Stefano; Gaudi, Simona

2010-01-01

Background Transposable Elements (TEs) comprise nearly 45% of the entire genome and are part of sophisticated regulatory network systems that control developmental processes in normal and pathological conditions. The retroviral/retrotransposon gene machinery consists mainly of Long Interspersed Nuclear Elements (LINEs-1) and Human Endogenous Retroviruses (HERVs) that code for their own endogenous reverse transcriptase (RT). Interestingly, RT is typically expressed at high levels in cancer cells. Recent studies report that RT inhibition by non-nucleoside reverse transcriptase inhibitors (NNRTIs) induces growth arrest and cell differentiation in vitro and antagonizes growth of human tumors in animal model. In the present study we analyze the anticancer activity of Abacavir (ABC), a nucleoside reverse transcription inhibitor (NRTI), on PC3 and LNCaP prostate cancer cell lines. Principal Findings ABC significantly reduces cell growth, migration and invasion processes, considerably slows S phase progression, induces senescence and cell death in prostate cancer cells. Consistent with these observations, microarray analysis on PC3 cells shows that ABC induces specific and dose-dependent changes in gene expression, involving multiple cellular pathways. Notably, by quantitative Real-Time PCR we found that LINE-1 ORF1 and ORF2 mRNA levels were significantly up-regulated by ABC treatment. Conclusions Our results demonstrate the potential of ABC as anticancer agent able to induce antiproliferative activity and trigger senescence in prostate cancer cells. Noteworthy, we show that ABC elicits up-regulation of LINE-1 expression, suggesting the involvement of these elements in the observed cellular modifications. PMID:21151977

Applicability of integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) for the simultaneous detection of the four human enteric enterovirus species in disinfection studies

EPA Science Inventory

A newly developed integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) method and its applicability in UV disinfection studies is described. This method utilizes a singular cell culture system coupled with four RTqPCR assays to detect infectious serotypes t...

Spore-forming organisms in platelet concentrates: a challenge in transfusion bacterial safety.

PubMed

Störmer, M; Vollmer, T; Kleesiek, K; Dreier, J

2008-12-01

Bacterial detection and pathogen reduction are widely used methods of minimizing the risk of transfusion-transmitted bacterial infection. But, bacterial spores are highly resistant to chemical and physical agents. In this study, we assessed the bacterial proliferation of spore-forming organisms seeded into platelet concentrates (PCs) to demonstrate that spores can enter the vegetative state in PCs during storage. In the in vitro study, PCs were inoculated with 1-10 spores mL(-1)of Bacillus cereus (n = 1), Bacillus subtilis (n = 2) and Clostridium sporogenes (n = 2). Sampling was performed during 6-day aerobic storage at 22 degrees C. The presence of bacteria was assessed by plating culture, automated culture and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Spores of the C. sporogenes do not enter the vegetative phase under PC storage conditions, whereas B. subtilis and B. cereus showed growth in the PC and could be detected using RT-PCR and automated culture. Depending on the species and inoculums, bacterial spores may enter the vegetative phase during PC storage and can be detected by bacterial detection methods.

The cDNA-derived amino acid sequence of hemoglobin II from Lucina pectinata.

PubMed

Torres-Mercado, Elineth; Renta, Jessicca Y; Rodríguez, Yolanda; López-Garriga, Juan; Cadilla, Carmen L

2003-11-01

Hemoglobin II from the clam Lucina pectinata is an oxygen-reactive protein with a unique structural organization in the heme pocket involving residues Gln65 (E7), Tyr30 (B10), Phe44 (CD1), and Phe69 (E11). We employed the reverse transcriptase-polymerase chain reaction (RT-PCR) and methods to synthesize various cDNA(HbII). An initial 300-bp cDNA clone was amplified from total RNA by RT-PCR using degenerate oligonucleotides. Gene-specific primers derived from the HbII-partial cDNA sequence were used to obtain the 5' and 3' ends of the cDNA by RACE. The length of the HbII cDNA, estimated from overlapping clones, was approximately 2114 bases. Northern blot analysis revealed that the mRNA size of HbII agrees with the estimated size using cDNA data. The coding region of the full-length HbII cDNA codes for 151 amino acids. The calculated molecular weight of HbII, including the heme group and acetylated N-terminal residue, is 17,654.07 Da.

Evaluation of Flinders Technology Associates cards for storage and molecular detection of avian metapneumoviruses.

PubMed

Awad, Faez; Baylis, Matthew; Jones, Richard C; Ganapathy, Kannan

2014-01-01

The feasibility of using Flinders Technology Associates (FTA) cards for the molecular detection of avian metapneumovirus (aMPV) by reverse transcriptase-polymerase chain reaction (RT-PCR) was investigated. Findings showed that no virus isolation was possible from aMPV-inoculated FTA cards, confirming viral inactivation upon contact with the cards. The detection limits of aMPV from the FTA card and tracheal organ culture medium were 10(1.5) median ciliostatic doses/ml and 10(0.75) median ciliostatic doses/ml respectively. It was possible to perform molecular characterization of both subtypes A and B aMPV using inoculated FTA cards stored for up to 60 days at 4 to 6°C. Tissues of the turbinate, trachea and lung of aMPV-infected chicks sampled either by direct impression smears or by inoculation of the tissue homogenate supernatants onto the FTA cards were positive by RT-PCR. However, the latter yielded more detections. FTA cards are suitable for collecting and transporting aMPV-positive samples, providing a reliable and hazard-free source of RNA for molecular characterization.

Involvement of a banana MADS-box transcription factor gene in ethylene-induced fruit ripening.

PubMed

Liu, Juhua; Xu, Biyu; Hu, Lifang; Li, Meiying; Su, Wei; Wu, Jing; Yang, Jinghao; Jin, Zhiqiang

2009-01-01

To investigate the regulation of MADS-box genes in banana (Musa acuminata L. AAA group cv. Brazilian) fruit development and postharvest ripening, we isolated from banana fruit a MADS-box gene designated MuMADS1. Amino acid alignment indicated MuMADS1 belongs to the AGAMOUS subfamily, and phylogenetic analysis indicates that this gene is most similar to class D MADS-box genes. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that MuMADS1 is expressed in the stamen and pistil of male and female flowers and in the rhizome, the vegetative reproductive organ of the banana plant. In preharvest banana fruit, MuMADS1 is likely expressed throughout banana fruit development. In postharvest banana ripening, MuMADS1 is associated with ethylene biosynthesis. Expression patterns of MuMADS1 during postharvest ripening as determined by real-time RT-PCR suggest that differential expression of MuMADS1 may not only be induced by ethylene biosynthesis associated with postharvest banana ripening, but also may be induced by exogenous ethylene.

Comprehensive evaluation of candidate reference genes for gene expression studies in Lysiphlebia japonica (Hymenoptera: Aphidiidae) using RT-qPCR.

PubMed

Gao, Xue-Ke; Zhang, Shuai; Luo, Jun-Yu; Wang, Chun-Yi; Lü, Li-Min; Zhang, Li-Juan; Zhu, Xiang-Zhen; Wang, Li; Lu, Hui; Cui, Jin-Jie

2017-12-30

Lysiphlebia japonica (Ashmead) is a predominant parasitoid of cotton-melon aphids in the fields of northern China with a proven ability to effectively control cotton aphid populations in early summer. For accurate normalization of gene expression in L. japonica using quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR), reference genes with stable gene expression patterns are essential. However, no appropriate reference genes is L. japonica have been investigated to date. In the present study, 12 selected housekeeping genes from L. japonica were cloned. We evaluated the stability of these genes under various experimental treatments by RT-qPCR using four independent (geNorm, NormFinder, BestKeeper and Delta Ct) and one comparative (RefFinder) algorithm. We identified genes showing the most stable levels of expression: DIMT, 18S rRNA, and RPL13 during different stages; AK, RPL13, and TBP among sexes; EF1A, PPI, and RPL27 in different tissues, and EF1A, RPL13, and PPI in adults fed on different diets. Moreover, the expression profile of a target gene (odorant receptor 1, OR1) studied during the developmental stages confirms the reliability of the chosen selected reference genes. This study provides for the first time a comprehensive list of suitable reference genes for gene expression studies in L. japonica and will benefit subsequent genomics and functional genomics research on this natural enemy. Copyright © 2017. Published by Elsevier B.V.

A putative peroxidase cDNA from turnip and analysis of the encoded protein sequence.

PubMed

Romero-Gómez, S; Duarte-Vázquez, M A; García-Almendárez, B E; Mayorga-Martínez, L; Cervantes-Avilés, O; Regalado, C

2008-12-01

A putative peroxidase cDNA was isolated from turnip roots (Brassica napus L. var. purple top white globe) by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Total RNA extracted from mature turnip roots was used as a template for RT-PCR, using a degenerated primer designed to amplify the highly conserved distal motif of plant peroxidases. The resulting partial sequence was used to design the rest of the specific primers for 5' and 3' RACE. Two cDNA fragments were purified, sequenced, and aligned with the partial sequence from RT-PCR, and a complete overlapping sequence was obtained and labeled as BbPA (Genbank Accession No. AY423440, named as podC). The full length cDNA is 1167bp long and contains a 1077bp open reading frame (ORF) encoding a 358 deduced amino acid peroxidase polypeptide. The putative peroxidase (BnPA) showed a calculated Mr of 34kDa, and isoelectric point (pI) of 4.5, with no significant identity with other reported turnip peroxidases. Sequence alignment showed that only three peroxidases have a significant identity with BnPA namely AtP29a (84%), and AtPA2 (81%) from Arabidopsis thaliana, and HRPA2 (82%) from horseradish (Armoracia rusticana). Work is in progress to clone this gene into an adequate host to study the specific role and possible biotechnological applications of this alternative peroxidase source.

The role of environmental contamination with small round structured viruses in a hospital outbreak investigated by reverse-transcriptase polymerase chain reaction assay.

PubMed

Green, J; Wright, P A; Gallimore, C I; Mitchell, O; Morgan-Capner, P; Brown, D W

1998-05-01

In May 1994 an outbreak of vomiting and diarrhoea occurred in a 28-bed long-stay ward for the mentally infirm. The predominant symptoms were vomiting, diarrhoea, malaise and abdominal pain lasting for approximately 12 h in most cases. The attack rate was 62% (13/21) for patients and 46% (16/35) for staff members. Infection control measures were implemented (containment of infectious individuals, hand hygiene among staff and environmental decontamination) and the ward was closed to admissions. Affected staff were excluded from contact with patients and their food until asymptomatic for 72 h. The outbreak lasted for 17 days. Faecal samples from nine symptomatic persons were negative for bacterial enteric pathogens, Giardia, Cryptosporidium and group A rotavirus. Electron microscopy of 12 faecal samples and one sample of vomitus revealed small round structured virus (SRSV) particles in one faecal sample. A further 30 faecal samples and seven vomitus samples were tested by reverse transcription polymerase chain reaction (RT-PCR) for SRSV of which 12 (40%) and 1 (14%) were positive respectively. Twenty-eight throat swabs from symptomatic and asymptomatic patients were collected, three (9.5%) of which were positive for SRSV by RT-PCR. Thirty-six environmental swabs were collected on the affected ward, and 11 (30%) were positive by RT-PCR. Positive swabs were from lockers, curtains and commodes and confined to the immediate environment of symptomatic patients. The distribution of contamination supports the rationale of cohorting sick patients.

Mumps outbreak and laboratory diagnosis.

PubMed

Maillet, Mylène; Bouvat, Eric; Robert, Nicole; Baccard-Longère, Monique; Morel-Baccard, Christine; Morand, Patrice; Vabret, Astrid; Stahl, Jean-Paul

2015-01-01

Several mumps outbreaks have been reported in Europe and in the United States among highly vaccinated populations. Biological diagnosis is classically based on the detection of mumps-specific IgM, but the ability of serological tests to confirm mumps infection seems to be limited among vaccinated patients. We aim to report a mumps outbreak in an engineering school in Grenoble, France, from February to June 2013 and results of the biological testing. WHO definitions were used to define cases. Mumps--specific IgM and IgG were assessed by a commercially available EIA. Mumps RNA detection by real time reverse transcriptase polymerase chain reaction tests (RT-PCR) and mumps genotyping were performed by the French National Reference Centre for Paramyxoviridae. Sixty two mumps patient-cases were identified using WHO case definitions, 20 being biologically explored, of which 17 were confirmed by biological tests. Vaccination status was documented for 27 patients/62: 4 (14.8%) patients had received one dose of MMR vaccine, and 23 (85.2) two doses of MMR vaccine. Among the biologically explored patients, 83% had a positive RT PCR at the first sampling whereas only 45% had positive or equivocal IgM. All the genotyped strains were genotype G. Mumps laboratory diagnosis in a highly vaccinated population is challenging. Serological tests among vaccinated patients should be interpreted cautiously and confirmed by RT-PCR tests at the beginning of a mumps outbreak. Copyright © 2014 Elsevier B.V. All rights reserved.

Field-based evaluation of a male-specific (F+) RNA coliphage concentration method.

PubMed

Chandler, J C; Pérez-Méndez, A; Paar, J; Doolittle, M M; Bisha, B; Goodridge, L D

2017-01-01

Fecal contamination of water poses a significant risk to public health due to the potential presence of pathogens, including enteric viruses. Therefore, sensitive, reliable and easy to use methods for the concentration, detection and quantification of microorganisms associated with the safety and quality of water are needed. In this study, we performed a field evaluation of an anion exchange resin-based method to concentrate male-specific (F+) RNA coliphages (FRNA), fecal indicator organisms, from diverse environmental waters that were suspected to be contaminated with feces. In this system, FRNA coliphages are adsorbed to anion exchange resin and direct nucleic acid isolation is performed, yielding a sample amenable to real-time reverse transcriptase (RT)-PCR detection. Matrix-dependent inhibition of this method was evaluated using known quantities of spiked FRNA coliphages belonging to four genogroups (GI, GII, GII and GIV). RT-PCR-based detection was successful in 97%, 72%, 85% and 98% of the samples spiked (10 6 pfu/l) with GI, GII, GIII and GIV, respectively. Differential FRNA coliphage genogroup detection was linked to inhibitors that altered RT-PCR assay efficiency. No association between inhibition and the physicochemical properties of the water samples was apparent. Additionally, the anion exchange resin method facilitated detection of naturally present FRNA coliphages in 40 of 65 environmental water samples (61.5%), demonstrating the viability of this system to concentrate FRNA coliphages from water. Copyright © 2016 Elsevier B.V. All rights reserved.

Novel Method for Simultaneous Quantification of Phenotypic Resistance to Maturation, Protease, Reverse Transcriptase, and Integrase HIV Inhibitors Based on 3′Gag(p2/p7/p1/p6)/PR/RT/INT-Recombinant Viruses: a Useful Tool in the Multitarget Era of Antiretroviral Therapy▿†

PubMed Central

Weber, Jan; Vazquez, Ana C.; Winner, Dane; Rose, Justine D.; Wylie, Doug; Rhea, Ariel M.; Henry, Kenneth; Pappas, Jennifer; Wright, Alison; Mohamed, Nizar; Gibson, Richard; Rodriguez, Benigno; Soriano, Vicente; King, Kevin; Arts, Eric J.; Olivo, Paul D.; Quiñones-Mateu, Miguel E.

2011-01-01

Twenty-six antiretroviral drugs (ARVs), targeting five different steps in the life cycle of the human immunodeficiency virus type 1 (HIV-1), have been approved for the treatment of HIV-1 infection. Accordingly, HIV-1 phenotypic assays based on common cloning technology currently employ three, or possibly four, different recombinant viruses. Here, we describe a system to assess HIV-1 resistance to all drugs targeting the three viral enzymes as well as viral assembly using a single patient-derived, chimeric virus. Patient-derived p2-INT (gag-p2/NCp7/p1/p6/pol-PR/RT/IN) products were PCR amplified as a single fragment (3,428 bp) or two overlapping fragments (1,657 bp and 2,002 bp) and then recombined into a vector containing a near-full-length HIV-1 genome with the Saccharomyces cerevisiae uracil biosynthesis gene (URA3) replacing the 3,428 bp p2-INT segment (Dudley et al., Biotechniques 46:458–467, 2009). P2-INT-recombinant viruses were employed in drug susceptibility assays to test the activity of protease (PI), nucleoside/nucleotide reverse transcriptase (NRTI), nonnucleoside reverse transcriptase (NNRTI), and integrase strand-transfer (INSTI) inhibitors. Using a single standardized test (ViralARTS HIV), this new technology permits the rapid and automated quantification of phenotypic resistance for all known and candidate antiretroviral drugs targeting all viral enzymes (PR, RT, including polymerase and RNase H activities, and IN), some of the current and potential assembly inhibitors, and any drug targeting Pol or Gag precursor cleavage sites (relevant for PI and maturation inhibitors) This novel assay may be instrumental (i) in the development and clinical assessment of novel ARV drugs and (ii) to monitor patients failing prior complex treatment regimens. PMID:21628544

High Degree of Interlaboratory Reproducibility of Human Immunodeficiency Virus Type 1 Protease and Reverse Transcriptase Sequencing of Plasma Samples from Heavily Treated Patients

PubMed Central

Shafer, Robert W.; Hertogs, Kurt; Zolopa, Andrew R.; Warford, Ann; Bloor, Stuart; Betts, Bradley J.; Merigan, Thomas C.; Harrigan, Richard; Larder, Brendon A.

2001-01-01

We assessed the reproducibility of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and protease sequencing using cryopreserved plasma aliquots obtained from 46 heavily treated HIV-1-infected individuals in two laboratories using dideoxynucleotide sequencing. The rates of complete sequence concordance between the two laboratories were 99.1% for the protease sequence and 99.0% for the RT sequence. Approximately 90% of the discordances were partial, defined as one laboratory detecting a mixture and the second laboratory detecting only one of the mixture's components. Only 0.1% of the nucleotides were completely discordant between the two laboratories, and these were significantly more likely to occur in plasma samples with lower plasma HIV-1 RNA levels. Nucleotide mixtures were detected at approximately 1% of the nucleotide positions, and in every case in which one laboratory detected a mixture, the second laboratory either detected the same mixture or detected one of the mixture's components. The high rate of concordance in detecting mixtures and the fact that most discordances between the two laboratories were partial suggest that most discordances were caused by variation in sampling of the HIV-1 quasispecies by PCR rather than by technical errors in the sequencing process itself. PMID:11283081

Identification and characterization of jute LTR retrotransposons:

PubMed Central

Ahmed, Salim; Shafiuddin, MD; Azam, Muhammad Shafiul; Islam, Md. Shahidul; Ghosh, Ajit

2011-01-01

Long Terminal Repeat (LTR) retrotransposons constitute a significant part of eukaryotic genomes and play an important role in genome evolution especially in plants. Jute is an important fiber crop with a large genome of 1,250 Mbps. This genome is still mostly unexplored. In this study we aimed at identifying and characterizing the LTR retrotransposons of jute with a view to understanding the jute genome better. In this study, the Reverse Transcriptase domain of Ty1-copia and Ty3-gypsy LTR retrotransposons of jute were amplified by degenerate primers and their expressions were examined by reverse transcription PCR. Copy numbers of reverse transcriptase (RT) genes of Ty1-copia and Ty3-gypsy elements were determined by dot blot analysis. Sequence analysis revealed higher heterogeneity among Ty1-copia retrotransposons than Ty3-gypsy and clustered each of them in three groups. Copy number of RT genes in Ty1-copia was found to be higher than that of Ty3-gypsy elements from dot blot hybridization. Cumulatively Ty1-copia and Ty3-gypsy may constitute around 19% of the jute genome where two groups of Ty1-copia were found to be transcriptionally active. Since the LTR retrotransposons constitute a large portion of jute genome, these findings imply the importance of these elements in the evolution of jute genome. PMID:22016842

Minority Human Immunodeficiency Virus Type 1 Variants in Antiretroviral-Naive Persons with Reverse Transcriptase Codon 215 Revertant Mutations▿ †

PubMed Central

Mitsuya, Yumi; Varghese, Vici; Wang, Chunlin; Liu, Tommy F.; Holmes, Susan P.; Jayakumar, Prerana; Gharizadeh, Baback; Ronaghi, Mostafa; Klein, Daniel; Fessel, W. Jeffrey; Shafer, Robert W.

2008-01-01

T215 revertant mutations such as T215C/D/E/S that evolve from the nucleoside reverse transcriptase (RT) inhibitor mutations T215Y/F have been found in about 3% of human immunodeficiency virus type 1 (HIV-1) isolates from newly diagnosed HIV-1-infected persons. We used a newly developed sequencing method—ultradeep pyrosequencing (UDPS; 454 Life Sciences)—to determine the frequency with which T215Y/F or other RT inhibitor resistance mutations could be detected as minority variants in samples from untreated persons that contain T215 revertants (“revertant” samples) compared with samples from untreated persons that lack such revertants (“control” samples). Among the 22 revertant and 29 control samples, UDPS detected a mean of 3.8 and 4.8 additional RT amino acid mutations, respectively. In 6 of 22 (27%) revertant samples and in 4 of 29 control samples (14%; P = 0.4), UDPS detected one or more RT inhibitor resistance mutations. T215Y or T215F was not detected in any of the revertant or control samples; however, 4 of 22 revertant samples had one or more T215 revertants that were detected by UDPS but not by direct PCR sequencing. The failure to detect viruses with T215Y/F in the 22 revertant samples in this study may result from the overwhelming replacement of transmitted T215Y variants by the more fit T215 revertants or from the primary transmission of a T215 revertant in a subset of persons with T215 revertants. PMID:18715933

Identification and characterization of avian retroviruses in chicken embryo-derived yellow fever vaccines: investigation of transmission to vaccine recipients.

PubMed

Hussain, Althaf I; Johnson, Jeffrey A; Da Silva Freire, Marcos; Heneine, Walid

2003-01-01

All currently licensed yellow fever (YF) vaccines are propagated in chicken embryos. Recent studies of chick cell-derived measles and mumps vaccines show evidence of two types of retrovirus particles, the endogenous avian retrovirus (EAV) and the endogenous avian leukosis virus (ALV-E), which originate from the chicken embryonic fibroblast substrates. In this study, we investigated substrate-derived avian retrovirus contamination in YF vaccines currently produced by three manufacturers (YF-vax [Connaught Laboratories], Stamaril [Aventis], and YF-FIOCRUZ [FIOCRUZ-Bio-Manguinhos]). Testing for reverse transcriptase (RT) activity was not possible because of assay inhibition. However, Western blot analysis of virus pellets with anti-ALV RT antiserum detected three distinct RT proteins in all vaccines, indicating that more than one source is responsible for the RTs present in the vaccines. PCR analysis of both chicken substrate DNA and particle-associated RNA from the YF vaccines showed no evidence of the long terminal repeat sequences of exogenous ALV subgroups A to D in any of the vaccines. In contrast, both ALV-E and EAV particle-associated RNA were detected at equivalent titers in each vaccine by RT-PCR. Quantitative real-time RT-PCR revealed 61,600, 348,000, and 1,665,000 ALV-E RNA copies per dose of Stamaril, YF-FIOCRUZ, and YF-vax vaccines, respectively. ev locus-specific PCR testing of the vaccine-associated chicken substrate DNA was positive both for the nondefective ev-12 locus in two vaccines and for the defective ev-1 locus in all three vaccines. Both intact and ev-1 pol sequences were also identified in the particle-associated RNA. To investigate the risks of transmission, serum samples from 43 YF vaccine recipients were studied. None of the samples were seropositive by an ALV-E-based Western blot assay or had detectable EAV or ALV-E RNA sequences by RT-PCR. YF vaccines produced by the three manufacturers all have particles containing EAV genomes and various levels of defective or nondefective ALV-E sequences. The absence of evidence of infection with ALV-E or EAV in 43 YF vaccine recipients suggests low risks for transmission of these viruses, further supporting the safety of these vaccines.

Chemical crosslinking of the subunits of HIV-1 reverse transcriptase.

PubMed Central

Debyser, Z.; De Clercq, E.

1996-01-01

The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) is composed of two subunits of 66 and 51 kDa in a 1 to 1 ratio. Because dimerization is a prerequisite for enzymatic activity, interference with the dimerization process could constitute an alternative antiviral strategy for RT inhibition. Here we describe an in vitro assay for the study of the dimerization state of HIV-1 reverse transcriptase based on chemical crosslinking of the subunits with dimethylsuberimidate. Crosslinking results in the formation of covalent bonds between the subunits, so that the crosslinked species can be resolved by denaturing gel electrophoresis. Crosslinked RT species with molecular weight greater than that of the dimeric form accumulate during a 1-15-min time course. Initial evidence suggests that those high molecular weight species represent trimers and tetramers and may be the result of intramolecular crosslinking of the subunits of a higher-order RT oligomer. A peptide that corresponds to part of the tryptophan repeat motif in the connection domain of HIV-1 RT inhibits crosslink formation as well as enzymatic activity. The crosslinking assay thus allows the investigation of the effect of inhibitors on the dimerization of HIV-1 RT. PMID:8745406

Blood-Stage Parasitaemia and Age Determine Plasmodium falciparum and P. vivax Gametocytaemia in Papua New Guinea.

PubMed

Koepfli, Cristian; Robinson, Leanne J; Rarau, Patricia; Salib, Mary; Sambale, Naomi; Wampfler, Rahel; Betuela, Inoni; Nuitragool, Wang; Barry, Alyssa E; Siba, Peter; Felger, Ingrid; Mueller, Ivo

2015-01-01

A better understanding of human-to-mosquito transmission is crucial to control malaria. In order to assess factors associated with gametocyte carriage, 2083 samples were collected in a cross-sectional survey in Papua New Guinea. Plasmodium species were detected by light microscopy and qPCR and gametocytes by detection of pfs25 and pvs25 mRNA transcripts by reverse-transcriptase PCR (qRT-PCR). The parasite prevalence by PCR was 18.5% for Plasmodium falciparum and 13.0% for P. vivax. 52.5% of all infections were submicroscopic. Gametocytes were detected in 60% of P. falciparum-positive and 51% of P. vivax-positive samples. Each 10-fold increase in parasite density led to a 1.8-fold and 3.3-fold increase in the odds of carrying P. falciparum and P. vivax gametocytes. Thus the proportion of gametocyte positive and gametocyte densities was highest in young children carrying high asexual parasite densities and in symptomatic individuals. Dilution series of gametocytes allowed absolute quantification of gametocyte densities by qRT-PCR and showed that pvs25 expression is 10-20 fold lower than pfs25 expression. Between 2006 and 2010 parasite prevalence in the study site has decreased by half. 90% of the remaining infections were asymptomatic and likely constitute an important reservoir of transmission. However, mean gametocyte densities were low (approx. 1-2 gametocyte/μL) and it remains to be determined to what extent low-density gametocyte positive individuals are infective to mosquitos.

RT-LAMP assay: an alternative approach for profiling of bovine heat shock protein 70 gene in PBMC cultured model.

PubMed

Sengar, Gyanendra Singh; Deb, Rajib; Raja, T V; Singh, Umesh; Kant, Rajiv; Bhanuprakash, V; Alyethodi, R R; Kumar, Sushil; Verma, Preetam; Chakraborty, Soumendu; Alex, Rani; Singh, Rani

2017-07-01

The purpose of this study is to develop a novel Reverse Transcriptase Loop-mediated isothermal amplification (RT-LAMP) based assay for in vitro profiling of heat shock protein 70 (Hsp70) in bovine peripheral blood mononuclear cell (PBMC) culture model utilizing the absorbance level of magnesium pyrophosphate-a by-product of LAMP reaction. A set of bovine Hsp70 specific RT-LAMP primers were designed to detect the differential absorbance level of magnesium pyrophosphate by-product which signifies the degree of Hsp70 amplification from cDNA of thermally induced cultured cells at different recovery periods. The study revealed significant (P < 0.05) correlation between absorbance level and the fold change of Hsp70 transcripts at different kinetic intervals of heat stress recovery in bovine PBMC cell culture models. RT-LAMP based absorbance assay can be used as an indicator to measure the degree of bovine Hsp70 transcripts produced during thermal stress and can be used as an alternative to the traditional Real time PCR assay. Developed RT-LAMP assay can be used as a cost-effective method for profiling of bovine HSP70 gene.

Unconventional plasticity of HIV-1 reverse transcriptase: how inhibitors could open a connection "gate" between allosteric and catalytic sites.

PubMed

Bellucci, Luca; Angeli, Lucilla; Tafi, Andrea; Radi, Marco; Botta, Maurizio

2013-12-23

Targeted molecular dynamics (TMD) simulations allowed for identifying the chemical/structural features of the nucleotide-competitive HIV-1 inhibitor DAVP-1, which is responsible for the disruption of the T-shape motif between Try183 and Trp229 of the reverse transcriptase (RT). DAVP-1 promoted the opening of a connection "gate" between allosteric and catalytic sites of HIV-1 RT, thus explaining its peculiar mechanism of action and providing useful insights to develop novel nucleotide-competitive RT inhibitors.

Laboratory Evaluation of Commercially Available Platforms to Detect West Nile and Zika Viruses From Honey Cards.

PubMed

Burkhalter, Kristen L; Wiggins, Keenan; Burkett-Cadena, Nathan; Alto, Barry W

2018-05-04

Commercially available assays utilizing antigen or nucleic acid detection chemistries provide options for mosquito control districts to screen their mosquito populations for arboviruses and make timely operational decisions regarding vector control. These assays may be utilized even more advantageously when combined with honey-soaked nucleic acid preservation substrate ('honey card') testing by reducing or replacing the time- and labor-intensive efforts of identifying and processing mosquito pools. We tested artificially inoculated honey cards and cards fed upon individually by West Nile virus (WNV) and Zika virus (ZIKV)-infected mosquitoes with three assays to compare detection rates and the limit of detection for each platform with respect to virus detection of a single infected mosquito and quantify the time interval of virus preservation on the cards. Assays evaluated included CDC protocols for real-time reverse transcriptase polymerase chain reaction (RT-PCR) for WNV and ZIKV, Pro-Lab Diagnostics ProAmpRT WNV loop-mediated amplification (LAMP) and ZIKV LAMP assays, and the Rapid Analyte Measurement Platform (RAMP) WNV assay. Real-time RT-PCR was the most sensitive assay and the most robust to viral RNA degradation over time. To maximize the detection of virus, honey cards should be left in the traps ≤1 d if using LAMP assays and ≤3 d if using real-time RT-PCR to detect viruses from field samples. The WNV RAMP assay, although effective for pool screening, lacks sensitivity required for honey card surveillance. Future studies may determine the minimum number of infectious mosquitoes required to feed on a honey card that would be reliably detected by the LAMP or RAMP assays.

Outbreak of H3N2 influenza at a US military base in Djibouti during the H1N1 pandemic of 2009.

PubMed

Cosby, Michael T; Pimentel, Guillermo; Nevin, Remington L; Fouad Ahmed, Salwa; Klena, John D; Amir, Ehab; Younan, Mary; Browning, Robert; Sebeny, Peter J

2013-01-01

Influenza pandemics have significant operational impact on deployed military personnel working in areas throughout the world. The US Department of Defense global influenza-like illness (ILI) surveillance network serves an important role in establishing baseline trends and can be leveraged to respond to outbreaks of respiratory illness. We identified and characterized an operationally unique outbreak of H3N2 influenza at Camp Lemonnier, Djibouti occurring simultaneously with the H1N1 pandemic of 2009 [A(H1N1)pdm09]. Enhanced surveillance for ILI was conducted at Camp Lemonnier in response to local reports of a possible outbreak during the A(H1N1)pdm09 pandemic. Samples were collected from consenting patients presenting with ILI (utilizing a modified case definition) and who completed a case report form. Samples were cultured and analyzed using standard real-time reverse transcriptase PCR (rt-RT-PCR) methodology and sequenced genetic material was phylogenetically compared to other published strains. rt-RT-PCR and DNA sequencing revealed that 25 (78%) of the 32 clinical samples collected were seasonal H3N2 and only 2 (6%) were A(H1N1)pdm09 influenza. The highest incidence of H3N2 occurred during the month of May and 80% of these were active duty military personnel. Phylogenetic analysis revealed that sequenced H3N2 strains were genetically similar to 2009 strains from the United States of America, Australia, and South east Asia. This outbreak highlights challenges in the investigation of influenza among deployed military populations and corroborates the public health importance of maintaining surveillance systems for ILI that can be enhanced locally when needed.

Outbreak of H3N2 Influenza at a US Military Base in Djibouti during the H1N1 Pandemic of 2009

PubMed Central

Cosby, Michael T.; Pimentel, Guillermo; Nevin, Remington L.; Fouad Ahmed, Salwa; Klena, John D.; Amir, Ehab; Younan, Mary; Browning, Robert; Sebeny, Peter J.

2013-01-01

Background Influenza pandemics have significant operational impact on deployed military personnel working in areas throughout the world. The US Department of Defense global influenza-like illness (ILI) surveillance network serves an important role in establishing baseline trends and can be leveraged to respond to outbreaks of respiratory illness. Objective We identified and characterized an operationally unique outbreak of H3N2 influenza at Camp Lemonnier, Djibouti occurring simultaneously with the H1N1 pandemic of 2009 [A(H1N1)pdm09]. Methods Enhanced surveillance for ILI was conducted at Camp Lemonnier in response to local reports of a possible outbreak during the A(H1N1)pdm09 pandemic. Samples were collected from consenting patients presenting with ILI (utilizing a modified case definition) and who completed a case report form. Samples were cultured and analyzed using standard real-time reverse transcriptase PCR (rt-RT-PCR) methodology and sequenced genetic material was phylogenetically compared to other published strains. Results rt-RT-PCR and DNA sequencing revealed that 25 (78%) of the 32 clinical samples collected were seasonal H3N2 and only 2 (6%) were A(H1N1)pdm09 influenza. The highest incidence of H3N2 occurred during the month of May and 80% of these were active duty military personnel. Phylogenetic analysis revealed that sequenced H3N2 strains were genetically similar to 2009 strains from the United States of America, Australia, and South east Asia. Conclusions This outbreak highlights challenges in the investigation of influenza among deployed military populations and corroborates the public health importance of maintaining surveillance systems for ILI that can be enhanced locally when needed. PMID:24339995

On-Orbit Quantitative Real-Time Gene Expression Analysis Using the Wetlab-2 System

NASA Technical Reports Server (NTRS)

Parra, Macarena; Jung, Jimmy; Almeida, Eduardo; Boone, Travis; Tran, Luan; Schonfeld, Julie

2015-01-01

NASA Ames Research Center's WetLab-2 Project enables on-orbit quantitative Reverse Transcriptase PCR (qRT-PCR) analysis without the need for sample return. The WetLab-2 system is capable of processing sample types ranging from microbial cultures to animal tissues dissected on-orbit. The project developed a RNA preparation module that can lyse cells and extract RNA of sufficient quality and quantity for use as templates in qRT-PCR reactions. Our protocol has the advantage of using non-toxic chemicals and does not require alcohols or other organics. The resulting RNA is dispensed into reaction tubes that contain all lyophilized reagents needed to perform qRT-PCR reactions. System operations require simple and limited crew actions including syringe pushes, valve turns and pipette dispenses. The project selected the Cepheid SmartCycler (TradeMark), a Commercial-Off-The-Shelf (COTS) qRT-PCR unit, because of its advantages including rugged modular design, low power consumption, rapid thermal ramp times and four-color multiplex detection. Single tube multiplex assays can be used to normalize for RNA concentration and integrity, and to study multiple genes of interest in each module. The WetLab-2 system can downlink data from the ISS to the ground after a completed run and uplink new thermal cycling programs. The ability to conduct qRT-PCR and generate results on-orbit is an important step towards utilizing the ISS as a National Laboratory facility. Specifically, the ability to get on-orbit data will provide investigators with the opportunity to adjust experimental parameters in real time without the need for sample return and re-flight. On orbit gene expression analysis can also eliminate the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples and provide on-orbit gene expression benchmarking prior to sample return. Finally, the system can also be used for analysis of air, surface, water, and clinical samples to monitor environmental pathogens and crew health. The validation flight of the WetLab-2 system using E. coli bacteria and mouse liver launched on SpaceX-7 in June 2015 and will remain on the ISS National Laboratory.

[Typing and subtyping avian influenza virus using DNA microarrays].

PubMed

Yang, Zhongping; Wang, Xiurong; Tian, Lina; Wang, Yu; Chen, Hualan

2008-07-01

Outbreaks of highly pathogenic avian influenza (HPAI) virus has caused great economic loss to the poultry industry and resulted in human deaths in Thailand and Vietnam since 2004. Rapid typing and subtyping of viruses, especially HPAI from clinical specimens, are desirable for taking prompt control measures to prevent spreading of the disease. We described a simultaneous approach using microarray to detect and subtype avian influenza virus (AIV). We designed primers of probe genes and used reverse transcriptase PCR to prepare cDNAs of AIV M gene, H5, H7, H9 subtypes haemagglutinin genes and N1, N2 subtypes neuraminidase genes. They were cloned, sequenced, reamplified and spotted to form a glass-bound microarrays. We labeled samples using Cy3-dUTP by RT-PCR, hybridized and scanned the microarrays to typing and subtyping AIV. The hybridization pattern agreed perfectly with the known grid location of each probe, no cross hybridization could be detected. Examinating of HA subtypes 1 through 15, 30 infected samples and 21 field samples revealed the DNA microarray assay was more sensitive and specific than RT-PCR test and chicken embryo inoculation. It can simultaneously detect and differentiate the main epidemic AIV. The results show that DNA microarray technology is a useful diagnostic method.

Investigation of PAX3/7-FKHR fusion genes and IGF2 gene expression in rhabdomyosarcoma tumors.

PubMed

de Souza, Robson Ramos; Oliveira, Indhira Dias; Caran, Eliana Maria Monteiro; Alves, Maria Teresa de Seixas; Abib, Simone; Toledo, Silvia Regina Caminada

2012-12-01

The purpose of our study was to investigate the prevalence of the PAX3/7-FKHR fusion genes and quantify the IGF2 gene expression in rhabdomyosarcoma (RMS) samples. Soft tissue sarcomas account 5% of childhood cancers and 50% of them are RMS. Morphological evaluation of pediatric RMS has defined two histological subtypes, embryonal (ERMS) and alveolar (ARMS). Chromosomal analyses have demonstrated two translocations associated with ARMS, resulting in the PAX3/7-FKHR rearrangements. Reverse transcriptase-polymerase chain reaction (RT-PCR) is extremely useful in the diagnosis of ARMS positive for these rearrangements. Additionally, several studies have shown a significant involvement of IGF pathway in the pathogenesis of RMS. The presence of PAX3/7-FKHR gene fusions was studied in 25 RMS samples from patients attending the IOP-GRAACC/UNIFESP and three RMS cell lines by RT-PCR. IGF2 gene expression was quantified by qPCR and related with clinic pathological parameters. Of the 25 samples, nine (36%) were ARMS and 16 (64%) were ERMS. PAX3/7-FKHR gene fusions expression was detected in 56% of ARMS tumor samples. IGF2 overexpression was observed in 80% of samples and could indicate an important role of this pathway in RMS biology. Copyright © 2012 Elsevier Ltd. All rights reserved.

Molecular cloning of allelopathy related genes and their relation to HHO in Eupatorium adenophorum.

PubMed

Guo, Huiming; Pei, Xixiang; Wan, Fanghao; Cheng, Hongmei

2011-10-01

In this study, conserved sequence regions of HMGR, DXR, and CHS (encoding 3-hydroxy-3-methylglutaryl-CoA reductase, 1-deoxyxylulose-5-phosphate reductoisomerase and chalcone synthase, respectively) were amplified by reverse transcriptase (RT)-PCR from Eupatorium adenophorum. Quantitative real-time PCR showed that the expression of CHS was related to the level of HHO, an allelochemical isolated from E. adenophorum. Semi-quantitative RT-PCR showed that there was no significant difference in expression of genes among three different tissues, except for CHS. Southern blotting indicated that at least three CHS genes are present in the E. adenophorum genome. A full-length cDNA from CHS genes (named EaCHS1, GenBank ID: FJ913888) was cloned. The 1,455 bp cDNA contained an open reading frame (1,206 bp) encoding a protein of 401 amino acids. Preliminary bioinformatics analysis of EaCHS1 revealed that EaCHS1 was a member of CHS family, the subcellular localization predicted that EaCHS1 was a cytoplasmic protein. To the best of our knowledge, this is the first report of conserved sequences of these genes and of a full-length EaCHS1 gene in E. adenophorum. The results indicated that CHS gene is related to allelopathy of E. adenophorum.

Probing the molecular mechanism of action of the HIV-1 reverse transcriptase inhibitor 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) using pre-steady-state kinetics.

PubMed

Muftuoglu, Yagmur; Sohl, Christal D; Mislak, Andrea C; Mitsuya, Hiroaki; Sarafianos, Stefan G; Anderson, Karen S

2014-06-01

The novel antiretroviral 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a potent nucleoside HIV-1 reverse transcriptase (RT) inhibitor (NRTI). Unlike other FDA-approved NRTIs, EFdA contains a 3'-hydroxyl. Pre-steady-state kinetics showed RT preferred incorporating EFdA-TP over native dATP. Moreover, RT slowly inserted nucleotides past an EFdA-terminated primer, resulting in delayed chain termination with unaffected fidelity. This is distinct from KP1212, another 3'-hydroxyl-containing RT inhibitor considered to promote viral lethal mutagenesis. New mechanistic features of RT inhibition by EFdA are revealed. Copyright © 2014 Elsevier B.V. All rights reserved.

Probing the molecular mechanism of action of the HIV-1 reverse transcriptase inhibitor 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) using pre-steady-state kinetics

PubMed Central

Muftuoglu, Yagmur; Sohl, Christal D.; Mislak, Andrea C.; Mitsuya, Hiroaki; Sarafianos, Stefan G.; Anderson, Karen S.

2014-01-01

The novel antiretroviral 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is a potent nucleoside HIV-1 reverse transcriptase (RT) inhibitor (NRTI). Unlike other FDA-approved NRTIs, EFdA contains a 3′-hydroxyl. Pre-steady-state kinetics showed RT preferred incorporating EFdA-TP over native dATP. Moreover, RT slowly inserted nucleotides past an EFdA-terminated primer, resulting in delayed chain termination with unaffected fidelity. This is distinct from KP1212, another 3′-hydroxyl-containing RT inhibitor considered to promote viral lethal mutagenesis. New mechanistic features of RT inhibition by EFdA are revealed. PMID:24632447

Assessment Effects of Resveratrol on Human Telomerase Reverse Transcriptase Messenger Ribonucleic Acid Transcript in Human Glioblastoma.

PubMed

Mirzazadeh, Azin; Kheirollahi, Majid; Farashahi, Ehsan; Sadeghian-Nodoushan, Fatemeh; Sheikhha, Mohammad Hasan; Aflatoonian, Behrouz

2017-01-01

Glioblastoma (GBM) is the most common and aggressive brain tumor, which has a poor prognosis despite the advent of different therapeutic strategies. There are numerous molecular biomarkers to contribute diagnosis, prognosis, and prediction of response to the current therapy in GBM. One of the most important markers that are potentially valuable is immortalization-specific or immortalization-associated marker named "hTERT messenger ribonucleic acid (mRNA)" the key subunit of telomerase enzyme, which is expressed in more than 85% of cancer cells, in spite of the majority of normal somatic cells. In this study, we investigated the effects of resveratrol (RSV) on this mRNA marker level, leading to cancer progression. U-87MG cell line was obtained from Pasteur Institute of Iran and treated with various concentrations of 0-160 μg/mL of RSV and at different time points (24, 48, and 72 h). To evaluate viability of U-87MG cells, standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed. Real-time polymerase chain reaction (RT-PCR) was used for comparative and quantitative assessment of human telomerase reverse transcriptase (hTERT) mRNA copy number versus control-untreated group. The results of our investigation suggested that RSV effectively inhibited cell growth and caused cell death in dose-dependent ( P < 0.05) and not in time-dependent manner ( P > 0.05), in vitro . Interestingly, quantitative RT-PCR analysis demonstrated that at half inhibition concentration, RSV dramatically decreased mRNA expression of hTERT, the catalytic subunit of telomerase enzyme, which leads to prevention of cell division and tumor progression. With regard to downregulation of this immortalization-associated marker, RSV may potentially be used as a therapeutic agent against GBM.

Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing.

PubMed

Mohr, Sabine; Ghanem, Eman; Smith, Whitney; Sheeter, Dennis; Qin, Yidan; King, Olga; Polioudakis, Damon; Iyer, Vishwanath R; Hunicke-Smith, Scott; Swamy, Sajani; Kuersten, Scott; Lambowitz, Alan M

2013-07-01

Mobile group II introns encode reverse transcriptases (RTs) that function in intron mobility ("retrohoming") by a process that requires reverse transcription of a highly structured, 2-2.5-kb intron RNA with high processivity and fidelity. Although the latter properties are potentially useful for applications in cDNA synthesis and next-generation RNA sequencing (RNA-seq), group II intron RTs have been difficult to purify free of the intron RNA, and their utility as research tools has not been investigated systematically. Here, we developed general methods for the high-level expression and purification of group II intron-encoded RTs as fusion proteins with a rigidly linked, noncleavable solubility tag, and we applied them to group II intron RTs from bacterial thermophiles. We thus obtained thermostable group II intron RT fusion proteins that have higher processivity, fidelity, and thermostability than retroviral RTs, synthesize cDNAs at temperatures up to 81°C, and have significant advantages for qRT-PCR, capillary electrophoresis for RNA-structure mapping, and next-generation RNA sequencing. Further, we find that group II intron RTs differ from the retroviral enzymes in template switching with minimal base-pairing to the 3' ends of new RNA templates, making it possible to efficiently and seamlessly link adaptors containing PCR-primer binding sites to cDNA ends without an RNA ligase step. This novel template-switching activity enables facile and less biased cloning of nonpolyadenylated RNAs, such as miRNAs or protein-bound RNA fragments. Our findings demonstrate novel biochemical activities and inherent advantages of group II intron RTs for research, biotechnological, and diagnostic methods, with potentially wide applications.

Inactivation of human norovirus and Tulane virus in simple media and fresh whole strawberries by ionizing radiation.

PubMed

DiCaprio, Erin; Phantkankum, Nuttapong; Culbertson, Doug; Ma, Yuanmei; Hughes, John H; Kingsley, David; Uribe, Roberto M; Li, Jianrong

2016-09-02

Human norovirus (NoV) is a major cause of fresh produce-associated outbreaks and human NoV in irrigation water can potentially lead to viral internalization in fresh produce. Therefore, there is a need to develop novel intervention strategies to target internalized viral pathogens while maintaining fresh produce quality. In this study electron beam (E-beam) and gamma radiation were evaluated for efficacy against a human NoV GII.4 strain and Tulane virus (TV). Virus survival following ionizing radiation treatments was determined using direct quantitative reverse transcriptase PCR (RT-qPCR), the porcine gastric mucin magnetic bead (PGM-MB) binding assay followed by RT-qPCR, and plaque assay. In simple media, a high dose of E-beam treatment was required to completely abolish the receptor binding ability of human NoV (35.3kGy) and TV (19.5-24.1kGy), as assessed using the PGM-MB binding assay. Both human NoV and TV were more susceptible to gamma irradiation than E-beam, requiring 22.4kGy to achieve complete inactivation. In whole strawberries, no human NoV or TV RNA was detected following 28.7kGy of E-beam treatment using the PGM-MB binding assay. Overall, human NoV and TV are highly resistant to ionizing radiation and therefore the technology may not be suitable to eliminate viruses in fresh produce at the currently approved levels. In addition, the PGM-MB binding assay is an improved method to detect viral infectivity compared to direct RT-qPCR. Copyright © 2016. Published by Elsevier B.V.

Antigenicity of mesenchymal stem cells in an inflamed joint environment.

PubMed

Hill, Jacqueline A; Cassano, Jennifer M; Goodale, Margaret B; Fortier, Lisa A

2017-07-01

OBJECTIVE To determine whether major histocompatability complex (MHC) class II expression in equine mesenchymal stem cells (MSCs) changes with exposure to a proinflammatory environment reflective of an inflamed joint. SAMPLE Cryopreserved bone marrow-derived MSCs from 12 horses and cartilage and synovium samples from 1 horse euthanized for reasons other than lameness. PROCEDURES In part 1 of a 3-part study, the suitability of a quantitative reverse transcriptase PCR (qRT-PCR) assay for measurement of MHC class II expression in MSCs following stimulation with interferon (IFN)-γ was assessed. In part 2, synoviocyte-cartilage cocultures were or were not stimulated with interleukin (IL)-1β (10 ng/mL) to generate conditioned media that did and did not (control) mimic an inflamed joint environment. In part 3, a qRT-PCR assay was used to measure MSC MHC class II expression after 96 hours of incubation with 1 of 6 treatments (control-conditioned medium, IL-1β-conditioned medium, and MSC medium alone [untreated control] or with IL-1β [10 ng/mL], tumor necrosis factor-α [10 ng/mL], or IFN-γ [100 ng/mL]). RESULTS The qRT-PCR assay accurately measured MHC class II expression. Compared with MHC class II expression for MSCs exposed to the untreated control medium, that for MSCs exposed to IL-1β was decreased, whereas that for MSCs exposed to IFN-γ was increased. Neither the control-conditioned nor tumor necrosis factor-α medium altered MHC class II expression. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that MSC exposure to proinflammatory cytokine IL-1β decreased MHC class II expression and antigenicity. Treatment of inflamed joints with allogeneic MSCs might not be contraindicated, but further investigation is warranted.

Comprehensive analysis of the MYB-NFIB gene fusion in salivary adenoid cystic carcinoma: Incidence, variability, and clinicopathologic significance.

PubMed

Mitani, Yoshitsugu; Li, Jie; Rao, Pulivarthi H; Zhao, Yi-Jue; Bell, Diana; Lippman, Scott M; Weber, Randal S; Caulin, Carlos; El-Naggar, Adel K

2010-10-01

The objectives of this study were to determine the incidence of the MYB-NFIB fusion in salivary adenoid cystic carcinoma (ACC), to establish the clinicopathologic significance of the fusion, and to analyze the expression of MYB in ACCs in the context of the MYB-NFIB fusion. We did an extensive analysis involving 123 cancers of the salivary gland, including primary and metastatic ACCs, and non-ACC salivary carcinomas. MYB-NFIB fusions were identified by reverse transcriptase-PCR (RT-PCR) and sequencing of the RT-PCR products, and confirmed by fluorescence in situ hybridization. MYB RNA expression was determined by quantitative RT-PCR and protein expression was analyzed by immunohistochemistry. The MYB-NFIB fusion was detected in 28% primary and 35% metastatic ACCs, but not in any of the non-ACC salivary carcinomas analyzed. Different exons in both the MYB and NFIB genes were involved in the fusions, resulting in expression of multiple chimeric variants. Notably, MYB was overexpressed in the vast majority of the ACCs, although MYB expression was significantly higher in tumors carrying the MYB-NFIB fusion. The presence of the MYB-NFIB fusion was significantly associated (P = 0.03) with patients older than 50 years of age. No correlation with other clinicopathologic markers, factors, and survival was found. We conclude that the MYB-NFIB fusion characterizes a subset of ACCs and contributes to MYB overexpression. Additional mechanisms may be involved in MYB overexpression in ACCs lacking the MYB-NFIB fusion. These findings suggest that MYB may be a specific novel target for tumor intervention in patients with ACC. ©2010 AACR.

RT-PCR standardization and bone mineralization after low-level laser therapy on adult osteoblast cells

NASA Astrophysics Data System (ADS)

do Bomfim, Fernando R. C.; Sella, Valéria R. G.; Zanaga, Jéssica Q.; Pereira, Nayara S.; Nouailhetas, Viviane L. A.; Plapler, Hélio

2014-03-01

Purpose: Osteoblasts are capable to produce different compounds directly connected to bone mineralization process. This study aims to standardize the reverse transcriptase polymerase chain reaction (RT-PCR) for adult osteoblasts to observe the effect of low level laser therapy on bone mineralization. Methods: Five-millimeter long fragments obtained from the mead femoral region of male Wistar rats were assigned into group A (n=10, laser) and group B (n=10, no laser), submitted to mechanic and enzymatic digestion. After 7 days, cultures of group A were irradiated daily on a single spot with a GaInAs laser, λ=808nm, 200mW/cm2, 2J/cm2, bean diameter of 0,02mm, 5 seconds for 6 days. Group B was manipulated but received no laser irradiation. After 13 days the cells were trypsinized for 15 minute and stabilized with RNA later® for RNA extraction with Trizol®. cDNA synthesis used 10μg of RNA and M-MLV® enzyme. PCR was accomplished using the β-actin gene as a control. Another aliquot was fixed for Hematoxylin-Eosin and Von Kossa staining to visualize bone mineralization areas. Results: Under UV light we observed clearly the amplification of β-actin gene around 400bp. HE and Von Kossa staining showed osteoblast clusters, a higher number of bone cells and well defined mineralization areas in group A. Conclusion: The cell culture, RNA extraction and RT-PCR method for adult osteoblasts was effective, allowing to use these methods for bone mineralization studies. Laser improved bone mineralization and further studies are needed involving osteogenesis, calcium release mechanisms and calcium related channels.

RT-PCR amplification of RNA extracted from formalin-fixed, paraffin-embedded oral cancer sections: analysis of p53 pathway.

PubMed

Tachibana, Masatsugu; Shinagawa, Yasuhiro; Kawamata, Hitoshi; Omotehara, Fumie; Horiuchi, Hideki; Ohkura, Yasuo; Kubota, Keiichi; Imai, Yutaka; Fujibayashi, Takashi; Fujimori, Takahiro

2003-01-01

We present a new approach towards the detection of the mRNAs in formalin-fixed, paraffin-embedded samples using a reverse transcriptase (RT)-polymerase chain reaction (PCR). The total RNAs were extracted from 10-micron-thick sections and were reverse-transcribed, then the RT-products were subjected to PCR amplification of GAPDH mRNA for screening the mRNA degradation. Next, nested PCR was performed for examining the expression of p53-related genes, p21WAF1, MDM2, p33ING1 and p14ARF. GAPDH mRNA expression was detectable in 12 out of 21 oral squamous cell carcinoma (SCC) samples. p21WAF1 mRNA expression was detectable in 5 out of 12 SCC samples, MDM2 mRNA expression was detectable in 5 our of 12 SCC samples and p33ING1 mRNA expression was detectable in 6 out of 12 SCC samples. However, the expression of p14ARF mRNA was not detectable in any of the samples. Seven out of 12 oral SCC samples showed abnormal nuclear accumulation of p53 protein by immunohistochemical staining, whereas 5 out of 12 oral SCCs showed negative staining for p53 protein. Of of p33ING1 mRNA. One of these was a verrucous carcinoma in which the p53 gene products might be inactivated by the oncoprotein E6 of human papilloma virus. Thus, the p53 tumor suppressor pathway was disrupted in most oral SCCs at the cellular levels, due to either an abnormality in p53 itself or loss of expression of p53 regulatory factors. This method would assist in making diagnosis, determining therapeutic strategy and predicting the prognosis of various cancers including oral SCCs.

Use of FTA sampling cards for molecular detection of avian influenza virus in wild birds.

PubMed

Keeler, Shamus P; Ferro, Pamela J; Brown, Justin D; Fang, Xingwang; El-Attrache, John; Poulson, Rebecca; Jackwood, Mark W; Stallknecht, David E

2012-03-01

Current avian influenza (AI) virus surveillance programs involving wild birds rely on sample collection methods that require refrigeration or low temperature freezing to maintain sample integrity for virus isolation and/or reverse-transcriptase (RT) PCR. Maintaining the cold chain is critical for the success of these diagnostic assays but is not always possible under field conditions. The aim of this study was to test the utility of Finders Technology Associates (FTA) cards for reliable detection of AI virus from cloacal and oropharyngeal swabs of wild birds. The minimum detectable titer was determined, and the effect of room temperature storage was evaluated experimentally using multiple egg-propagated stock viruses (n = 6). Using real time RT-PCR, we compared results from paired cloacal swab and samples collected on FTA cards from both experimentally infected mallards (Anasplatyrhynchos) and hunter-harvested waterfowl sampled along the Texas Gulf Coast. Based on the laboratory trials, the average minimal detectable viral titer was determined to be 1 x 10(4.7) median embryo infectious dose (EID50)/ml (range: 1 x 10(4.3) to 1 x 10(5.4) EID50/ml), and viral RNA was consistently detectable on the FTA cards for a minimum of 20 days and up to 30 days for most subtypes at room temperature (23 C) storage. Real-time RT-PCR of samples collected using the FTA cards showed fair to good agreement in live birds when compared with both real-time RT-PCR and virus isolation of swabs. AI virus detection rates in samples from several wild bird species were higher when samples were collected using the FTA cards compared with cloacal swabs. These results suggest that FTA cards can be used as an alternative sample collection method when traditional surveillance methods are not possible, especially in avian populations that have historically received limited testing or situations in which field conditions limit the ability to properly store or ship swab samples.

Molecular pathological analysis for determining the possible mechanism of piperonyl butoxide-induced hepatocarcinogenesis in mice.

PubMed

Muguruma, Masako; Nishimura, Jihei; Jin, Meilan; Kashida, Yoko; Moto, Mitsuyoshi; Takahashi, Miwa; Yokouchi, Yusuke; Mitsumori, Kunitoshi

2006-12-07

Piperonyl butoxide (PBO), alpha-[2-(2-butoxyethoxy)ethoxy]-4,5-methylene-dioxy-2-propyltoluene, is widely used as a synergist for pyrethrins. In order to clarify the possible mechanism of non-genotoxic hepatocarcinogenesis induced by PBO, molecular pathological analyses consisting of low-density microarray analysis and real-time reverse transcriptase (RT)-PCR were performed in male ICR mice fed a basal powdered diet containing 6000 or 0 ppm PBO for 1, 4, or 8 weeks. The animals were sacrificed at weeks 1, 4, and 8, and the livers were histopathologically examined and analyzed for gene expression using the microarray at weeks 1 and 4 followed by real-time RT-PCR at each time point. Reactive oxygen species (ROS) products were also measured using liver microsomes. At each time point, the hepatocytes of PBO-treated mice showed centrilobular hypertrophy and increased lipofuscin deposition in Schmorl staining. The ROS products were significantly increased in the liver microsomes of PBO-treated mice. In the microarray analysis, the expression of oxidative and metabolic stress-related genes--cytochrome P450 (Cyp) 1A1, Cyp2A5 (week 1 only), Cyp2B9, Cyp2B10, and NADPH-cytochrome P450 oxidoreductase (Por) was over-expressed in mice given PBO at weeks 1 and 4. Fluctuations of these genes were confirmed by real-time RT-PCR in PBO-treated mice at each time point. In additional real-time RT-PCR, the expression of Cyclin D1 gene, key regulator of cell-cycle progression, and Xrcc5 gene, DNA damage repair-related gene, was significantly increased at each time point and at week 8, respectively. These results suggest the possibility that PBO has the potential to generate ROS via the metabolic pathway and to induce oxidative stress, including oxidative DNA damage, resulting in the induction of hepatocellular tumors in mice.

Active surveillance for congenital rubella syndrome in Yangon, Myanmar.

PubMed Central

Thant, Kyaw-Zin; Oo, Win-Mar; Myint, Thein-Thein; Shwe, Than-Nu; Han, Aye-Maung; Aye, Khin-Mar; Aye, Kay-Thi; Moe, Kyaw; Thein, Soe; Robertson, Susan E.

2006-01-01

OBJECTIVE: Rubella vaccine is not included in the immunization schedule in Myanmar. Although surveillance for outbreaks of measles and rubella is conducted nationwide, there is no routine surveillance for congenital rubella syndrome (CRS). Therefore, we organized a study to assess the burden of CRS. METHODS: From 1 December 2000 to 31 December 2002 active surveillance for CRS was conducted among children aged 0-17 months at 13 hospitals and 2 private clinics in Yangon, the capital city. Children with suspected CRS had a standard examination and a blood sample was obtained. All serum samples were tested for rubella-specific IgM; selected samples were tested for rubella-specific IgG and for rubella RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). FINDINGS: A total of 81 children aged 0-17 months were suspected of having CRS. Of these, 18 children had laboratory-confirmed CRS (7 were IgM positive; 7 were RT-PCR positive; and 10 were IgG positive at > 6 months of age). One additional child who tested positive by RT-PCR and whose mother had had rubella during pregnancy but who had a normal clinical examination was classified as having congenital rubella infection. During 2001-02 no rubella outbreaks were detected in Yangon Division. In the 31 urban townships of Yangon Division, the annual incidence was 0.1 laboratory-confirmed cases of CRS per 1000 live births. CONCLUSION: This is the first population-based study of CRS incidence from a developing country during a rubella-endemic period; the incidence of CRS is similar to endemic rates found in industrialized countries during the pre-vaccine era. Rubella-specific IgG tests proved practical for diagnosing CRS in children aged > 6 months. This is one of the first studies to report on the use of rubella-specific RT-PCR directly on serum samples; further studies are warranted to confirm the utility of this method as an additional means of diagnosing CRS. PMID:16501710

Novel, In-House, SYBR Green Based One-Step rRT-PCR: Rapid and Accurate Diagnosis of Crimean-Congo Hemorrhagic Fever Virus in Suspected Patients From Iran.

PubMed

Zahraei, Bentolhoda; Hashemzadeh, Mohammad Sadegh; Najarasl, Mohammad; Zahiriyeganeh, Samaneh; Tat, Mahdi; Metanat, Maliheh; Sepehri Rad, Nahid; Khansari-Nejad, Behzad; Zafari, Ehsan; Sharti, Mojtaba; Dorostkar, Ruhollah

2016-01-01

The Crimean-Congo hemorrhagic fever (CCHF) virus causes severe disease in humans, with a high mortality rate. Since, there is no approved vaccine or specific treatment for CCHF, an early and accurate diagnosis, as well as reliable surveillance, is essential for case management and patient improvement. For this research, our aim was to evaluate the application of a novel SYBR Green based one-step real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) assay for the in-house diagnosis of the CCHF virus. In this experimental study, the highly conserved S-region sequence of the CCHF viral genome was first adapted from GenBank, and the specific primers targeting this region were designed. Then, the viral RNA was extracted from 75 serum samples from different patients in eastern Iran. The sensitivity and specificity of the primers were also evaluated in positive serum samples previously confirmed to have the CCHF virus, by this one-step rRT-PCR assay, as well as a DNA sequencing analysis. From a total of 75 suspected serum samples, 42 were confirmed to be positive for CCHF virus, with no false-positives detected by the sequencing results. After 40 amplification cycles, the melting curve analysis revealed a mean melting temperature (Tm) of 86.5 ± 0.6°C (quite different from those of the primer-dimers), and the positive samples showed only a small variation in the parameters. In all of the positive samples, the predicted length of 420 bp was confirmed by electrophoresis. Moreover, the sensitivity test showed that this assay can detect less than 20 copies of viral RNA per reaction. This study showed that this novel one-step rRT-PCR assay is a rapid, reliable, repeatable, specific, sensitive, and simple tool for the detection of the CCHF virus.

Feasibility of cytological specimens for ALK fusion detection in patients with advanced NSCLC using the method of RT-PCR.

PubMed

Wang, Yan; Liu, Yu; Zhao, Chao; Li, Xuefei; Wu, Chunyan; Hou, Likun; Zhang, Shijia; Jiang, Tao; Chen, Xiaoxia; Su, Chunxia; Gao, Guanghui; Li, Wei; Wu, Fengying; Li, Aiwu; Ren, Shengxiang; Zhou, Caicun; Zhang, Jun

2016-04-01

Histological tissues are preferred for anaplastic lymphoma kinase (ALK) fusion detection in non-small cell lung cancer (NSCLC). The aim of this study was to evaluate the feasibility of cytological sample as an alternative specimen for ALK fusion testing in patients with advanced NSCLC. Advanced NSCLC patients with cytological specimens or tumor tissue who had their ALK fusion status detected by the method of reverse transcriptase polymerase chain reaction (RT-PCR) in Shanghai Pulmonary Hospital, Tongji University were included into this study. The efficacy was evaluated in those with ALK fusion positive and received the therapy of crizotinib. 1274 patients were included in this study. Among them, 108 patients were ALK RT-PCR positive and 69 of them received crizotinib treatment. Among 1002 patients with cytological specimens, the average concentration of RNA extracted from cytological specimens was 60.99 ng/μl (95% confidence interval [CI], 55.56-66.60) and the incidence rate of ALK fusion was 8.3% (83/1002), which were similar to 63.16 ng/μl (95% CI, 51.88-76.34) (p=0.727) and 9.2% (25/272, p=0.624) in 272 patients with tumor tissue. Also, there were no statistically significant differences regarding to the objective response rate (ORR) (62.0% vs. 42.1%, p=0.177) and the median progression free survival (mPFS) [8.6 months (95% CI 7.30-9.84) vs. 7.0 months (95% CI 4.54-9.47), p=0.736] in patients of cytological group and tissue group after the treatment of crizotinib. Cytological specimens showed a high feasibility to detect ALK fusion status, which could be regarded as alternative samples for ALK fusion detection by the method of RT-PCR in patients with advanced NSCLC. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Malaria prevalence defined by microscopy, antigen detection, DNA amplification and total nucleic acid amplification in a malaria-endemic region during the peak malaria transmission season.

PubMed

Waitumbi, John N; Gerlach, Jay; Afonina, Irina; Anyona, Samuel B; Koros, Joseph N; Siangla, Joram; Ankoudinova, Irina; Singhal, Mitra; Watts, Kate; Polhemus, Mark E; Vermeulen, Nicolaas M; Mahoney, Walt; Steele, Matt; Domingo, Gonzalo J

2011-07-01

To determine the malaria prevalence by microscopy, antigen detection and nucleic acid detection in a defined subpopulation in a Plasmodium falciparum-endemic region during the peak transmission season. Blood specimens were collected in a cross-sectional study involving children aged 5-10 years (n = 195) presenting with acute fever to two clinics in Western Kenya. All specimens underwent microscopy, HRP2 and aldolase antigen detection by enzyme immunoassay (EIA), parasite-specific DNA and total nucleic acid (RNA and DNA) by real-time PCR (qPCR) and reverse-transcriptase PCR (qRT-PCR). Microscopy detected 65/195 cases of malaria infection [95% confidence interval (CI) 52-78]. HRP2 and aldolase EIA had similar sensitivity levels detecting antigen in 65/195 (95% CI, 52-78) and 57/195 (95% CI, 45-70) cases. Discordants in antigen detection vs. microscopy occurred at <470 parasites/μl and <4900 parasites/μl for HRP2 and aldolase, respectively. Detection of total nucleic acid allowed a 3 log lower limit of detection than just DNA detection by real-time PCR in vitro. In clinical specimens, 114/195 (95% CI, 100-127) were qPCR positive (DNA), and 187/195 (95% CI, 179-191) were qRT-PCR positive (DNA plus RNA). The prevalence of submicroscopic malaria infection was significantly higher when detecting total nucleic acid than just DNA in this outpatient population during the high transmission season. Defining standards for submicroscopic infection will be important for control programmes, diagnostics development efforts and molecular epidemiology studies. © 2011 Blackwell Publishing Ltd.

Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis.

PubMed

Enyeart, Peter J; Mohr, Georg; Ellington, Andrew D; Lambowitz, Alan M

2014-01-13

Mobile group II introns are bacterial retrotransposons that combine the activities of an autocatalytic intron RNA (a ribozyme) and an intron-encoded reverse transcriptase to insert site-specifically into DNA. They recognize DNA target sites largely by base pairing of sequences within the intron RNA and achieve high DNA target specificity by using the ribozyme active site to couple correct base pairing to RNA-catalyzed intron integration. Algorithms have been developed to program the DNA target site specificity of several mobile group II introns, allowing them to be made into 'targetrons.' Targetrons function for gene targeting in a wide variety of bacteria and typically integrate at efficiencies high enough to be screened easily by colony PCR, without the need for selectable markers. Targetrons have found wide application in microbiological research, enabling gene targeting and genetic engineering of bacteria that had been intractable to other methods. Recently, a thermostable targetron has been developed for use in bacterial thermophiles, and new methods have been developed for using targetrons to position recombinase recognition sites, enabling large-scale genome-editing operations, such as deletions, inversions, insertions, and 'cut-and-pastes' (that is, translocation of large DNA segments), in a wide range of bacteria at high efficiency. Using targetrons in eukaryotes presents challenges due to the difficulties of nuclear localization and sub-optimal magnesium concentrations, although supplementation with magnesium can increase integration efficiency, and directed evolution is being employed to overcome these barriers. Finally, spurred by new methods for expressing group II intron reverse transcriptases that yield large amounts of highly active protein, thermostable group II intron reverse transcriptases from bacterial thermophiles are being used as research tools for a variety of applications, including qRT-PCR and next-generation RNA sequencing (RNA-seq). The high processivity and fidelity of group II intron reverse transcriptases along with their novel template-switching activity, which can directly link RNA-seq adaptor sequences to cDNAs during reverse transcription, open new approaches for RNA-seq and the identification and profiling of non-coding RNAs, with potentially wide applications in research and biotechnology.

Comprehensive phylogenetic analysis of bacterial reverse transcriptases.

PubMed

Toro, Nicolás; Nisa-Martínez, Rafael

2014-01-01

Much less is known about reverse transcriptases (RTs) in prokaryotes than in eukaryotes, with most prokaryotic enzymes still uncharacterized. Two surveys involving BLAST searches for RT genes in prokaryotic genomes revealed the presence of large numbers of diverse, uncharacterized RTs and RT-like sequences. Here, using consistent annotation across all sequenced bacterial species from GenBank and other sources via RAST, available from the PATRIC (Pathogenic Resource Integration Center) platform, we have compiled the data for currently annotated reverse transcriptases from completely sequenced bacterial genomes. RT sequences are broadly distributed across bacterial phyla, but green sulfur bacteria and cyanobacteria have the highest levels of RT sequence diversity (≤85% identity) per genome. By contrast, phylum Actinobacteria, for which a large number of genomes have been sequenced, was found to have a low RT sequence diversity. Phylogenetic analyses revealed that bacterial RTs could be classified into 17 main groups: group II introns, retrons/retron-like RTs, diversity-generating retroelements (DGRs), Abi-like RTs, CRISPR-Cas-associated RTs, group II-like RTs (G2L), and 11 other groups of RTs of unknown function. Proteobacteria had the highest potential functional diversity, as they possessed most of the RT groups. Group II introns and DGRs were the most widely distributed RTs in bacterial phyla. Our results provide insights into bacterial RT phylogeny and the basis for an update of annotation systems based on sequence/domain homology.

Comprehensive Phylogenetic Analysis of Bacterial Reverse Transcriptases

PubMed Central

Toro, Nicolás; Nisa-Martínez, Rafael

2014-01-01

Much less is known about reverse transcriptases (RTs) in prokaryotes than in eukaryotes, with most prokaryotic enzymes still uncharacterized. Two surveys involving BLAST searches for RT genes in prokaryotic genomes revealed the presence of large numbers of diverse, uncharacterized RTs and RT-like sequences. Here, using consistent annotation across all sequenced bacterial species from GenBank and other sources via RAST, available from the PATRIC (Pathogenic Resource Integration Center) platform, we have compiled the data for currently annotated reverse transcriptases from completely sequenced bacterial genomes. RT sequences are broadly distributed across bacterial phyla, but green sulfur bacteria and cyanobacteria have the highest levels of RT sequence diversity (≤85% identity) per genome. By contrast, phylum Actinobacteria, for which a large number of genomes have been sequenced, was found to have a low RT sequence diversity. Phylogenetic analyses revealed that bacterial RTs could be classified into 17 main groups: group II introns, retrons/retron-like RTs, diversity-generating retroelements (DGRs), Abi-like RTs, CRISPR-Cas-associated RTs, group II-like RTs (G2L), and 11 other groups of RTs of unknown function. Proteobacteria had the highest potential functional diversity, as they possessed most of the RT groups. Group II introns and DGRs were the most widely distributed RTs in bacterial phyla. Our results provide insights into bacterial RT phylogeny and the basis for an update of annotation systems based on sequence/domain homology. PMID:25423096

Structure of the HIV-1 reverse transcriptase Q151M mutant: insights into the inhibitor resistance of HIV-1 reverse transcriptase and the structure of the nucleotide-binding pocket of Hepatitis B virus polymerase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakamura, Akiyoshi; Tamura, Noriko; Yasutake, Yoshiaki, E-mail: y-yasutake@aist.go.jp

The structure of the HIV-1 reverse transcriptase Q151M mutant was determined at a resolution of 2.6 Å in space group P321. Hepatitis B virus polymerase (HBV Pol) is an important target for anti-HBV drug development; however, its low solubility and stability in vitro has hindered detailed structural studies. Certain nucleotide reverse transcriptase (RT) inhibitors (NRTIs) such as tenofovir and lamivudine can inhibit both HBV Pol and Human immunodeficiency virus 1 (HIV-1) RT, leading to speculation on structural and mechanistic analogies between the deoxynucleotide triphosphate (dNTP)-binding sites of these enzymes. The Q151M mutation in HIV-1 RT, located at the dNTP-binding site,more » confers resistance to various NRTIs, while maintaining sensitivity to tenofovir and lamivudine. The residue corresponding to Gln151 is strictly conserved as a methionine in HBV Pol. Therefore, the structure of the dNTP-binding pocket of the HIV-1 RT Q151M mutant may reflect that of HBV Pol. Here, the crystal structure of HIV-1 RT Q151M, determined at 2.6 Å resolution, in a new crystal form with space group P321 is presented. Although the structure of HIV-1 RT Q151M superimposes well onto that of HIV-1 RT in a closed conformation, a slight movement of the β-strands (β2–β3) that partially create the dNTP-binding pocket was observed. This movement might be caused by the introduction of the bulky thioether group of Met151. The structure also highlighted the possibility that the hydrogen-bonding network among amino acids and NRTIs is rearranged by the Q151M mutation, leading to a difference in the affinity of NRTIs for HIV-1 RT and HBV Pol.« less

Evaluation of reference genes for quantitative real-time RT-PCR analysis of gene expression in Nile tilapia (Oreochromis niloticus).

PubMed

Yang, Chang Geng; Wang, Xian Li; Tian, Juan; Liu, Wei; Wu, Fan; Jiang, Ming; Wen, Hua

2013-09-15

Quantitative real-time reverse-transcriptase polymerase chain reaction (RT-qPCR) has been used frequently to study gene expression related to fish immunology. In such studies, a stable reference gene should be selected to correct the expression of the target gene. In this study, seven candidate reference genes (glyceraldehyde-3-phosphate dehydrogenase (GADPH), ubiquitin-conjugating enzyme (UBCE), 18S ribosomal RNA (18S rRNA), beta-2-microglobulin (B2M), elongation factor 1 alpha (EF1A), tubulin alpha chain-like (TUBA) and beta actin (ACTB)), were selected to analyze their stability and normalization in seven tissues (liver, spleen, kidney, brain, heart, muscle and intestine) of Nile tilapia (Oreochromis niloticus) challenged with Streptococcus agalactiae or Streptococcus iniae, respectively. The results showed that all the candidate reference genes exhibited tissue-dependent transcriptional variations. With PBS injection as a control, UBCE was the most stable and suitable single reference gene in the intestine, liver, brain, kidney, and spleen after S. iniae infection, and in the liver, kidney, and spleen after S. agalactiae infection. EF1A was the most suitable in heart and muscle after S. iniae or S. agalactiae infection. GADPH was the most suitable gene in intestine and brain after S. agalactiae infection. In normal conditions, UBCE and 18S rRNA were the most stably expressed genes across the various tissues. These results showed that for RT-qPCR analysis of tilapia, selecting two or more reference genes may be more suitable for cross-tissue analysis of gene expression. Copyright © 2013 Elsevier B.V. All rights reserved.

Microbial water quality before and after the repair of a failing onsite wastewater treatment system adjacent to coastal waters

USGS Publications Warehouse

Conn, K.E.; Habteselassie, M.Y.; Denene, Blackwood A.; Noble, R.T.

2012-01-01

Aims: The objective was to assess the impacts of repairing a failing onsite wastewater treatment system (OWTS, i.e., septic system) as related to coastal microbial water quality. Methods and Results: Wastewater, groundwater and surface water were monitored for environmental parameters, faecal indicator bacteria (total coliforms, Escherichia coli, enterococci) and the viral tracer MS2 before and after repairing a failing OWTS. MS2 results using plaque enumeration and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) often agreed, but inhibition limited the qRT-PCR assay sensitivity. Prerepair, MS2 persisted in groundwater and was detected in the nearby creek; postrepair, it was not detected. In groundwater, total coliform concentrations were lower and E.??coli was not detected, while enterococci concentrations were similar to prerepair levels. E.??coli and enterococci surface water concentrations were elevated both before and after the repair. Conclusions: Repairing the failing OWTS improved groundwater microbial water quality, although persistence of bacteria in surface water suggests that the OWTS was not the singular faecal contributor to adjacent coastal waters. A suite of tracers is needed to fully assess OWTS performance in treating microbial contaminants and related impacts on receiving waters. Molecular methods like qRT-PCR have potential but require optimization. Significance and Impact of Study: This is the first before and after study of a failing OWTS and provides guidance on selection of microbial tracers and methods. ?? 2011 The Authors. Journal of Applied Microbiology ?? 2011 The Society for Applied Microbiology.

Quantitative estimation of Nipah virus replication kinetics in vitro

PubMed Central

Chang, Li-Yen; Ali, AR Mohd; Hassan, Sharifah Syed; AbuBakar, Sazaly

2006-01-01

Background Nipah virus is a zoonotic virus isolated from an outbreak in Malaysia in 1998. The virus causes infections in humans, pigs, and several other domestic animals. It has also been isolated from fruit bats. The pathogenesis of Nipah virus infection is still not well described. In the present study, Nipah virus replication kinetics were estimated from infection of African green monkey kidney cells (Vero) using the one-step SYBR® Green I-based quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) assay. Results The qRT-PCR had a dynamic range of at least seven orders of magnitude and can detect Nipah virus from as low as one PFU/μL. Following initiation of infection, it was estimated that Nipah virus RNA doubles at every ~40 minutes and attained peak intracellular virus RNA level of ~8.4 log PFU/μL at about 32 hours post-infection (PI). Significant extracellular Nipah virus RNA release occurred only after 8 hours PI and the level peaked at ~7.9 log PFU/μL at 64 hours PI. The estimated rate of Nipah virus RNA released into the cell culture medium was ~0.07 log PFU/μL per hour and less than 10% of the released Nipah virus RNA was infectious. Conclusion The SYBR® Green I-based qRT-PCR assay enabled quantitative assessment of Nipah virus RNA synthesis in Vero cells. A low rate of Nipah virus extracellular RNA release and low infectious virus yield together with extensive syncytial formation during the infection support a cell-to-cell spread mechanism for Nipah virus infection. PMID:16784519

Prevalence of influenza virus among the paediatric population in Mumbai during 2007-2009.

PubMed

Roy, S; Patil, D; Dahake, R; Mukherjee, S; Athlekar, S V; Deshmukh, R A; Chowdhary, A

2012-01-01

Influenza has a major impact on public heath, annually affecting 15-20% of the global population. Information on the activity of influenza virus in Mumbai is limited. The present study was carried out to determine the prevalence of influenza viruses causing acute respiratory infections in children by molecular methods. To study the prevalence of influenza viruses among the paediatric population in Mumbai by real-time reverse-transcriptase polymerase chain reaction (rRT-PCR). From July 2007 to July 2009, 100 respiratory samples (nasal and throat swabs) were collected from paediatric patients with acute respiratory symptoms. attending out patients department, and admitted to the paediatric wards of B. J. Wadia Hospital for Children, Mumbai. The samples were collected and processed as per World Health Organization (WHO) guidelines. Viral RNA was extracted and one-step rRT-PCR was performed to detect influenza type A (H1 and H3) and influenza type B virus. Out of 100 samples processed by rRT-PCR, a total of 11 samples (11%) were positive for influenza virus. The typing for influenza A subtypes showed 1% (1) positivity for H1 and 5% (5) positivity for H3 subtypes and 5% (5) samples tested positive for influenza type B virus. It was observed that both influenza type A and B viruses were prevalent in Mumbai during the study period. Such surveillance data are important in the early detection of any antigenic variants that may be helpful in global influenza vaccine preparation and for any pandemic preparedness activity.

TH and DCX mRNAs in peripheral blood and bone marrow predict outcome in metastatic neuroblastoma patients.

PubMed

Yáñez, Yania; Hervás, David; Grau, Elena; Oltra, Silvestre; Pérez, Gema; Palanca, Sarai; Bermúdez, Mar; Márquez, Catalina; Cañete, Adela; Castel, Victoria

2016-03-01

In metastatic neuroblastoma (NB) patients, accurate risk stratification and disease monitoring would reduce relapse probabilities. This study aims to evaluate the independent prognostic significance of detecting tyrosine hydroxylase (TH) and doublecortin (DCX) mRNAs by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) in peripheral blood (PB) and bone marrow (BM) samples from metastatic NB patients. RT-qPCR was performed on PB and BM samples from metastatic NB patients at diagnosis, post-induction therapy and at the end of treatment for TH and DCX mRNAs detection. High levels of TH and DCX mRNAs when detected in PB and BM at diagnosis independently predicted worse outcome in a cohort of 162 metastatic NB. In the subgroup of high-risk metastatic NB, TH mRNA detected in PB remained as independent predictor of EFS and OS at diagnosis. After the induction therapy, high levels of TH mRNA in PB and DCX mRNA in BM independently predicted poor EFS and OS. Furthermore TH mRNA when detected in BM predicted worse EFS. TH mRNA in PB samples at the end of treatment is an independent predictor of worse outcome. TH and DCX mRNAs levels in PB and BM assessed by RT-qPCR should be considered in new pre-treatment risk stratification strategies to reliable estimate outcome differences in metastatic NB patients. In those high-risk metastatic NB, TH and DCX mRNA quantification could be used for the assessment of response to treatment and for early detection of progressive disease or relapses.

Cloning and Expression Analysis of a PISTILLATA Homologous Gene from Pineapple (Ananas comosus L. Merr)

PubMed Central

Lv, Ling-Ling; Duan, Jun; Xie, Jiang-Hui; Liu, Yu-Ge; Wei, Chang-Bin; Liu, Sheng-Hui; Zhang, Jian-Xia; Sun, Guang-Ming

2012-01-01

PISTILLATA (PI)-like genes are crucial regulators of flowering in angiosperms. A homologue of PI, designated as AcPI (Genbank accession number HQ717796), was isolated from pineapple cultivar Comte de Paris by reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The cDNA sequence of AcPI is 907 bp in length and contains an open reading frame of 594 bp, which encodes a protein of 197 amino acids. The molecular weight was 2.29 kDa and the isoelectric point was 9.28. The alignment showed that AcPI had a high identity with CsPIC2 (78.6%), AoPI (77.4%), OrcPI (75.7%) and HPI2 (72.4%). Quantitative real-time polymerase chain reaction (qRT-PCR) analyses in different tissues showed that the expression pattern of AcPI was different from the B-class genes in eudicots. AcPI was expressed in all the tissues investigated. The expression level was very low in fruit stems, bracts, leaves and sepals, high in petals and carpels, and moderate in apical meristems, flesh and stamens. The qRT-PCR analyses in different stages indicated that the expression of AcPI reached the highest level at 40 days after flower inducement, when the multiple fruit and floral organs were forming. It proved the important role of AcPI in floral organs and fruit development. The 35S::AcPI transgenic Arabidopsis plants flowered earlier and had more inflorescences or branches than wild type plants. PMID:22312303

Cloning and expression analysis of a PISTILLATA homologous gene from pineapple (Ananas comosus L. Merr).

PubMed

Lv, Ling-Ling; Duan, Jun; Xie, Jiang-Hui; Liu, Yu-Ge; Wei, Chang-Bin; Liu, Sheng-Hui; Zhang, Jian-Xia; Sun, Guang-Ming

2012-01-01

PISTILLATA (PI)-like genes are crucial regulators of flowering in angiosperms. A homologue of PI, designated as AcPI (Genbank accession number HQ717796), was isolated from pineapple cultivar Comte de Paris by reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The cDNA sequence of AcPI is 907 bp in length and contains an open reading frame of 594 bp, which encodes a protein of 197 amino acids. The molecular weight was 2.29 kDa and the isoelectric point was 9.28. The alignment showed that AcPI had a high identity with CsPIC2 (78.6%), AoPI (77.4%), OrcPI (75.7%) and HPI2 (72.4%). Quantitative real-time polymerase chain reaction (qRT-PCR) analyses in different tissues showed that the expression pattern of AcPI was different from the B-class genes in eudicots. AcPI was expressed in all the tissues investigated. The expression level was very low in fruit stems, bracts, leaves and sepals, high in petals and carpels, and moderate in apical meristems, flesh and stamens. The qRT-PCR analyses in different stages indicated that the expression of AcPI reached the highest level at 40 days after flower inducement, when the multiple fruit and floral organs were forming. It proved the important role of AcPI in floral organs and fruit development. The 35S::AcPI transgenic Arabidopsis plants flowered earlier and had more inflorescences or branches than wild type plants.

Powassan Virus and Other Arthropod-Borne Viruses in Wildlife and Ticks in Ontario, Canada.

PubMed

Smith, Kathryn; Oesterle, Paul T; Jardine, Claire M; Dibernardo, Antonia; Huynh, Chris; Lindsay, Robbin; Pearl, David L; Bosco-Lauth, Angela M; Nemeth, Nicole M

2018-06-04

Powassan virus (POWV) is a tick-borne zoonosis maintained in natural enzootic cycles between ixodid ticks and wild mammals. Reported human cases have increased in recent years; these infections can be fatal or lead to long-term neurologic sequelae. However, both the geographic distribution and the role of common, potential mammalian hosts in POWV transmission are poorly understood, creating challenges to public health surveillance. We looked for evidence of POWV infection among candidate wildlife host species and ticks collected from mammals and birds in southern Ontario. Tissues (including blood) and ticks from trapped wild mammals were collected in the summers of 2015 and 2016. Ticks removed from dogs in 2015-2016 and wildlife diagnostic cases from 2011 to 2013 were also included. Tissue and tick ( Ixodes spp.) homogenates were tested for POWV by reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, sera from wild mammals were tested for antibodies to POWV, West Nile virus (WNV), and heartland virus (HRTV) by plaque reduction neutralization test. All 724 tissue samples were negative for POWV by RT-PCR. One of 53 pools of Ixodes cookei (among 98 total tick pools) was RT-PCR positive for deer tick virus (POWV) lineage. Antibodies to POWV and WNV were detected in 0.4% of 265 and 6.1% of 264 samples, respectively, and all of 219 serum samples tested negative for anti-HRTV antibodies. These results reveal low POWV detection rates in southern Ontario, while highlighting the challenges and need for continued efforts into understanding POWV epidemiology and targeted surveillance strategies.

Development of a multiplex probe combination-based one-step real-time reverse transcription-PCR for NA subtype typing of avian influenza virus.

PubMed

Sun, Zhihao; Qin, Tao; Meng, Feifei; Chen, Sujuan; Peng, Daxin; Liu, Xiufan

2017-10-18

Nine influenza virus neuraminidase (NA) subtypes have been identified in poultry and wild birds. Few methods are available for rapid and simple NA subtyping. Here we developed a multiplex probe combination-based one-step real-time reverse transcriptase PCR (rRT-PCR) to detect nine avian influenza virus NA subtypes. Nine primer-probe pairs were assigned to three groups based on the different fluorescent dyes of the probes (FAM, HEX, or Texas Red). Each probe detected only one NA subtype, without cross reactivity. The detection limit was less than 100 EID 50 or 100 copies of cDNA per reaction. Data obtained using this method with allantoic fluid samples isolated from live bird markets and H9N2-infected chickens correlated well with data obtained using virus isolation and sequencing, but was more sensitive. This new method provides a specific and sensitive alternative to conventional NA-subtyping methods.

Prevalence and clinicopathological characteristics of ALK fusion subtypes in lung adenocarcinomas from Chinese populations.

PubMed

Zheng, Difan; Wang, Rui; Zhang, Yang; Pan, Yunjian; Cheng, Xinghua; Cheng, Chao; Zheng, Shanbo; Li, Hang; Gong, Ranxia; Li, Yuan; Shen, Xuxia; Sun, Yihua; Chen, Haiquan

2016-04-01

We performed this retrospective study to have a comprehensive investigation of the clinicopathological characteristics of ALK fusion-positive lung adenocarcinoma in Chinese populations. We screened 1407 patients with primary lung adenocarcinoma from October 2007 to May 2013. Quantitative real-time PCR (qRT-PCR), reverse transcriptase PCR (RT-PCR), and fluorescence in situ hybridization were performed to detect ALK fusion genes, with validation of positive results using immunohistochemistry. Clinicopathological characteristics were collected to assess prognosis in ALK fusion-positive patients. Of 1407 patients with lung adenocarcinoma, there were 74 (5.3 %) ALK fusion-positive patients. Patients harboring ALK fusion were significantly younger (56.0 years vs. 59.8 years p = 0.002) and were more likely to have advanced stages (stage III or stage IV) (OR 1.761; 95 % CI 1.10-2.82, p = 0.017). Lepidic predominant adenocarcinoma was rarely found in ALK fusion patients (2.7 vs. 13.5 % p = 0.025), while IMA (invasive mucinous adenocarcinoma) predominant adenocarcinoma was more frequently found (21.6 vs. 5.0 % p < 0.001). ALK fusion was neither a risk factor nor protective factor in relapse-free survival and overall survival. Male, current smoker, and EML4-ALK variant 3 indicated poor prognosis among ALK fusion-positive lung adenocarcinomas. ALK fusion was detected in 5.3 % (74/1407) of the Chinese patients with lung adenocarcinoma. ALK fusion defines a molecular subset of lung adenocarcinoma with unique clinicopathological characteristics. Different ALK fusion variants determine distinct prognoses.

Detection of betanodaviruses in apparently healthy aquarium fishes and invertebrates.

PubMed

Gomez, Dennis Kaw; Lim, Dong Joo; Baeck, Gun Wook; Youn, Hee Jeong; Shin, Nam Shik; Youn, Hwa Young; Hwang, Cheol Yong; Park, Jun Hong; Park, Se Chang

2006-12-01

Betanodaviruses are the causative agents of viral nervous necrosis (VNN) in cultured marine fish. A total of 237 apparently healthy aquarium fish, marine (65 species) and freshwater (12 species) fishes and marine invertebrates (4 species), which were stocked in a commercial aquarium in Seoul, South Korea, were collected from November 2005 to February 2006. The brains of the fish and other tissues of the invertebrates were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR to detect betanodavirus. Positive nested PCR results were obtained from the brains of 8 marine fish species (shrimp fish Aeoliscus strigatus, milkfish Chanos chanos, three spot damsel Dascyllus trimaculatus, Japanese anchovy Engraulis japonicus, pinecone fish Monocentris japonica, blue ribbon eel Rhinomuraena quaesita, look down fish Selene vomer, yellow tang Zebrasoma flavesenes), 1 marine invertebrate species (spiny lobster Pamulirus versicolor), and 2 freshwater fish species (South American leaf fish Monocirrhus polyacanthus and red piranha Pygocentrus nattereri). The detection rate in nested PCR was 11/237 (4.64%). These subclinically infected aquarium fish and invertebrates may constitute an inoculum source of betanodaviruses for cultured fishes in the Korean Peninsula.

Detection of betanodaviruses in apparently healthy aquarium fishes and invertebrates

PubMed Central

Gomez, Dennis Kaw; Lim, Dong Joo; Baeck, Gun Wook; Youn, Hee Jeong; Shin, Nam Shik; Youn, Hwa Young; Hwang, Cheol Yong; Park, Jun Hong

2006-01-01

Betanodaviruses are the causative agents of viral nervous necrosis (VNN) in cultured marine fish. A total of 237 apparently healthy aquarium fish, marine (65 species) and freshwater (12 species) fishes and marine invertebrates (4 species), which were stocked in a commercial aquarium in Seoul, South Korea, were collected from November 2005 to February 2006. The brains of the fish and other tissues of the invertebrates were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR to detect betanodavirus. Positive nested PCR results were obtained from the brains of 8 marine fish species (shrimp fish Aeoliscus strigatus, milkfish Chanos chanos, three spot damsel Dascyllus trimaculatus, Japanese anchovy Engraulis japonicus, pinecone fish Monocentris japonica, blue ribbon eel Rhinomuraena quaesita, look down fish Selene vomer, yellow tang Zebrasoma flavesenes), 1 marine invertebrate species (spiny lobster Pamulirus versicolor), and 2 freshwater fish species (South American leaf fish Monocirrhus polyacanthus and red piranha Pygocentrus nattereri). The detection rate in nested PCR was 11/237 (4.64%). These subclinically infected aquarium fish and invertebrates may constitute an inoculum source of betanodaviruses for cultured fishes in the Korean Peninsula. PMID:17106229

SYBR green-based real-time reverse transcription-PCR for typing and subtyping of all hemagglutinin and neuraminidase genes of avian influenza viruses and comparison to standard serological subtyping tests

USGS Publications Warehouse

Tsukamoto, K.; Javier, P.C.; Shishido, M.; Noguchi, D.; Pearce, J.; Kang, H.-M.; Jeong, O.M.; Lee, Y.-J.; Nakanishi, K.; Ashizawa, T.

2012-01-01

Continuing outbreaks of H5N1 highly pathogenic (HP) avian influenza virus (AIV) infections of wild birds and poultry worldwide emphasize the need for global surveillance of wild birds. To support the future surveillance activities, we developed a SYBR green-based, real-time reverse transcriptase PCR (rRT-PCR) for detecting nucleoprotein (NP) genes and subtyping 16 hemagglutinin (HA) and 9 neuraminidase (NA) genes simultaneously. Primers were improved by focusing on Eurasian or North American lineage genes; the number of mixed-base positions per primer was set to five or fewer, and the concentration of each primer set was optimized empirically. Also, 30 cycles of amplification of 1:10 dilutions of cDNAs from cultured viruses effectively reduced minor cross- or nonspecific reactions. Under these conditions, 346 HA and 345 NA genes of 349 AIVs were detected, with average sensitivities of NP, HA, and NA genes of 10 1.5, 10 2.3, and 10 3.1 50% egg infective doses, respectively. Utility of rRT-PCR for subtyping AIVs was compared with that of current standard serological tests by using 104 recent migratory duck virus isolates. As a result, all HA genes and 99% of the NA genes were genetically subtyped, while only 45% of HA genes and 74% of NA genes were serologically subtyped. Additionally, direct subtyping of AIVs in fecal samples was possible by 40 cycles of amplification: approximately 70% of HA and NA genes of NP gene-positive samples were successfully subtyped. This validation study indicates that rRT-PCR with optimized primers and reaction conditions is a powerful tool for subtyping varied AIVs in clinical and cultured samples. Copyright ?? 2012, American Society for Microbiology. All Rights Reserved.

Comparison of RNA extraction kits for the purification and detection of an enteric virus surrogate on green onions via RT-PCR.

PubMed

Xu, Ruoyang; Shieh, Y Carol; Stewart, Diana S

2017-01-01

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) offers a rapid and sensitive molecular method for detection of enteric viruses. Unfortunately, these assays are often hampered by the low virus titre found in foods and PCR inhibition due to matrix carryover during RNA extraction. Four commercial RNA extraction kits (Qiagen's QIAamp Viral RNA Mini and UltraSens Virus kits, MoBio UltraClean Tissue & Cells RNA Isolation kit, and Ambion MagMAX Viral RNA Isolation kit) were evaluated for their ability to extract and purify MS2 bacteriophage RNA, an enteric virus surrogate, from inoculated green onions, a food which has been associated with viral gastroenteritis outbreaks. Inoculated green onion wash concentrates and green onion pieces with and without Qiagen QIAshredder homogenization were assayed in the kit comparison. MS2 detection and PCR inhibition were evaluated using a duplex real-time RT-PCR for MS2 and an exogenous internal amplification control (IAC) assay. Without homogenization, MS2 inoculated at 40pfu/g was detected in at least 4 lots of green onion wash concentrates using the silica-membrane spin-column kits. Inhibition was a factor for the magnetic silica-based MagMAX kit, which resulted in detection of MS2 in 1 of 5. Addition of QIAshredder homogenization prior to extraction did not adversely affect the silica-membrane kit results but improved the MS2 detection by MagMAX to 5 of 5 lots. Use of a 1:10 dilution of primary RNA extracts also improved detection. The QIAamp Viral RNA Mini and MagMAX kits were further compared for detection of MS2 from green onion pieces inoculated at 20 and 5pfu/g. Using homogenization, the MagMAX kit detected 20pfu/g in only 1 of 2 green onion lots, whereas the QIAamp Viral RNA kit detected 2 of 2 lots at 5 pfu/g without homogenization. Published by Elsevier B.V.

Identification and Characterization of a Porcine Torovirus

PubMed Central

Kroneman, A.; Cornelissen, L. A. H. M.; Horzinek, M. C.; de Groot, R. J.; Egberink, H. F.

1998-01-01

A porcine torovirus (PoTV) was identified and characterized; it is a novel member of the genus Torovirus (family Coronaviridae, order Nidovirales), closely related to but clearly distinct from the already recognized equine torovirus (ETV) and bovine torovirus (BoTV) representatives. Immunoelectron microscopy of feces from piglets revealed elongated, 120- by 55-nm particles which were recognized by a torovirus-specific antiserum. Amplification by reverse transcriptase (RT) PCR with primers designed to detect conserved regions (on the basis of the genomes of BoTV strain Breda and ETV strain Berne) resulted in the identification of the 489-bp nucleocapsid gene, encoding a 18.7-kDa protein. The sequence identity in this region between PoTV and both ETV and BoTV was only about 68%, whereas the latter two show 81% identity. Neutralizing antibodies directed against ETV were found in sera of adult and young pigs. In all 10 herds sampled, seropositive animals were present, and 81% of randomly selected adult sows possessed antibodies. A longitudinal study with RT PCR showed that piglets shed virus in the feces for 1 or more days, starting 4 to 14 days after weaning. PMID:9557628

Detection of Puumala virus in the tissue of infected naturally rodent hosts in the area of central Dinarides.

PubMed

Dervović, Edina; Hukić, Mirsada

2016-04-01

Hantaviruses are the causative agents of haemorrhagic fever with renal syndrome (HFRS) in Euroasia and of hantavirus cardiopulmonary syndrome (HCPS) in the North, Central and South America. HFRS is endemic in the Balkan Peninsula, where sporadic cases or outbreaks have been reported. Puumala virus (PUUV) is the causative agent of nephropathia epidemica (NE), a mild form of HFRS. PUUV is carried by the bank voles (Myodes glareolus). In this study, we investigated viral RNA from 76 tissues samples (lung n=30, heart n=6, liver n=18 and kidney n=22) of infected naturally rodent hosts in the area of Central Dinarides caught in live traps. Puumala virus was extracted from 34,7% (16/46) rodents by nested reverse transcriptase polymerase chain reaction (RT-PCR) and real-time RT-PCR. Overall, 18 (21,4%) specimens of internal organs (kidney n=8, liver n=6, heart n=2 and lung n=2) were positive for PUUV. It was shown a high rodent infestation rate in a relatively low number of rodent and their organs, although mice were not caught during the time of high density population of host rodents. Copyright © 2016 Elsevier B.V. All rights reserved.

Coronavirus infection in intensively managed cattle with respiratory disease.

PubMed

Hick, P M; Read, A J; Lugton, I; Busfield, F; Dawood, K E; Gabor, L; Hornitzky, M; Kirkland, P D

2012-10-01

A detailed laboratory investigation identified bovine coronavirus (BCoV) as the aetiological agent in an outbreak of respiratory disease at a semi-intensive beef cattle feedlot in south-east Australia. The outbreak caused 30% morbidity in the resident population and also affected two cohorts of cattle that were newly introduced to the property. At slaughter, pulmonary consolidation and inflammatory lesions in the trachea were identified in 15 of 49 animals. Pasteurella multocida or Histophilus somni was cultured from 3 of 7 animals with lesions. Histopathological examination revealed multifocal non-suppurative bronchointerstitial pneumonia with formation of epithelial syncytial cells, sometimes associated with suppurative bronchopneumonia. BCoV was detected in nasal swabs and pulmonary lesions using real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) assay and virus isolation. There was serological evidence of previous exposure to bovine viral diarrhoea virus, bovine respiratory syncytial virus and bovine parainfluenza virus type 3, but not to bovine herpesvirus type 1. None of these viral pathogens or Mycoplasma bovis was identified by qRT-PCR. This is believed to be the first report of BCoV in association with bovine respiratory disease complex in Australia. © 2012 The Authors. Australian Veterinary Journal © 2012 Australian Veterinary Association.

La Crosse Encephalitis Virus Infection in Field-Collected Aedes albopictus, Aedes japonicus, and Aedes triseriatus in Tennessee.

PubMed

Westby, Katie M; Fritzen, Charissa; Paulsen, Dave; Poindexter, Stephanie; Moncayo, Abelardo C

2015-09-01

La Crosse virus (LACV) is a mosquito-borne virus and a major cause of pediatric encephalitis in the USA. La Crosse virus emerged in Tennessee and other states in the Appalachian region in 1997. We investigated LACV infection rates and seasonal abundances of the native mosquito vector, Aedes triseriatus, and 2 recently introduced mosquito species, Ae. albopictus and Ae. japonicus, in an emerging disease focus in Tennessee. Mosquitoes were collected using multiple trapping methods specific for Aedes mosquitoes at recent human case sites. Mosquito pools were tested via reverse transcriptase-polymerase chain reaction (RT-PCR) of the S segment to detect multiple Bunyamwera and California serogroup viruses, including LACV, as well as real-time RT-PCR of the M segment. A total of 54 mosquito pools were positive, including wild-caught adult females and laboratory-reared adults, demonstrating transovarial transmission in all 3 species. Maximum likelihood estimates (per 1,000 mosquitoes) were 2.72 for Ae. triseriatus, 3.01 for Ae. albopictus, and 0.63 for Ae. japonicus. We conclude that Ae. triseriatus and Ae. albopictus are important LACV vectors and that Ae. japonicus also may be involved in virus maintenance and transmission.

Stable Reference Gene Selection for RT-qPCR Analysis in Nonviruliferous and Viruliferous Frankliniella occidentalis.

PubMed

Yang, Chunxiao; Li, Hui; Pan, Huipeng; Ma, Yabin; Zhang, Deyong; Liu, Yong; Zhang, Zhanhong; Zheng, Changying; Chu, Dong

2015-01-01

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for measuring and evaluating gene expression during variable biological processes. To facilitate gene expression studies, normalization of genes of interest relative to stable reference genes is crucial. The western flower thrips Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), the main vector of tomato spotted wilt virus (TSWV), is a destructive invasive species. In this study, the expression profiles of 11 candidate reference genes from nonviruliferous and viruliferous F. occidentalis were investigated. Five distinct algorithms, geNorm, NormFinder, BestKeeper, the ΔCt method, and RefFinder, were used to determine the performance of these genes. geNorm, NormFinder, BestKeeper, and RefFinder identified heat shock protein 70 (HSP70), heat shock protein 60 (HSP60), elongation factor 1 α, and ribosomal protein l32 (RPL32) as the most stable reference genes, and the ΔCt method identified HSP60, HSP70, RPL32, and heat shock protein 90 as the most stable reference genes. Additionally, two reference genes were sufficient for reliable normalization in nonviruliferous and viruliferous F. occidentalis. This work provides a foundation for investigating the molecular mechanisms of TSWV and F. occidentalis interactions.

Nonnucleoside reverse transcriptase inhibitor phenotypic hypersusceptibility can be demonstrated in different assays.

PubMed

Shulman, Nancy S; Delgado, Jamael; Bosch, Ronald J; Winters, Mark A; Johnston, Elizabeth; Shafer, Robert W; Katzenstein, David A; Merigan, Thomas C

2005-05-01

HIV-1 isolates harboring multiple nucleoside reverse transcriptase inhibitor (NRTI) resistance mutations are more susceptible ("hypersusceptible") to the nonnucleoside reverse transcriptase inhibitors (NNRTIs) than isolates lacking NRTI resistance mutations, but this has only been reported with a single-cycle replication phenotypic assay. In fact, there was a report that a commercial multicycle assay did not readily detect hypersusceptibility. To see whether NNRTI hypersusceptibility can be demonstrated in other types of phenotypic assays, including multicycle assays and enzyme inhibition assays. The susceptibility of HIV-1 clones derived from different patients in multicycle assays was tested in peripheral blood mononuclear cells (PBMCs) and in an established cell line. In addition, the reverse transcriptase (RT) of many of these clones was expressed and their susceptibility tested in an RT inhibition assay. Nevirapine and efavirenz susceptibilities were tested and compared with a control wild-type virus or RT. Hypersusceptibility to nevirapine and efavirenz was detected using each of the methods described above. R values correlating the other methods with single-cycle assay values were between 0.66 and 0.96. In addition to the high correlations, the different methods gave similar numeric results. NNRTI hypersusceptibility is readily seen in multicycle susceptibility assays and in enzyme inhibition assays.

Induction of anti-viral genes during acute infection with Viral hemorrhagic septicemia virus (VHSV) genogroup IVa in Pacific herring (Clupea pallasii)

USGS Publications Warehouse

Hansen, John D.; Woodson, James C.; Hershberger, Paul K.; Grady, Courtney; Gregg, Jacob L.; Purcell, Maureen K.

2012-01-01

Infection with the aquatic rhabdovirus Viral hemorrhagic septicemia virus (VHSV) genogroup IVa results in high mortality in Pacific herring (Clupea pallasii) and is hypothesized to be a potential limiting factor for herring recovery. To investigate anti-viral immunity in the Pacific herring, four immune response genes were identified: the myxovirus resistance (Clpa-Mx), a major histocompatibility complex IB (named Clpa-UAA.001), the inducible immunoproteosome subunit 9 (Clpa-PSMB9) and the neutrophil chemotactic factor (Clpa-LECT2). Reverse transcriptase quantitative PCR (RT-qPCR) assays were developed based on these gene sequences to investigate the host immune response to acute VHSV infection following both injection and immersion challenge. Virus levels were measured by both plaque assay and RT-qPCR and peaked at day 6 during the 10-day exposure period for both groups of fish. The interferon stimulated genes (Clpa-Mx, −UAA.001, and −PSMB9) were significantly up-regulated in response to VHSV infection at both 6 and 10 days post-infection in both spleen and fin. Results from this study indicate that Pacific herring mount a robust, early antiviral response in both fin and spleen tissues. The immunological tools developed in this study will be useful for future studies to investigate antiviral immunity in Pacific herring.

Plastic Polymers for Efficient DNA Microarray Hybridization: Application to Microbiological Diagnostics▿

PubMed Central

Zhao, Zhengshan; Peytavi, Régis; Diaz-Quijada, Gerardo A.; Picard, Francois J.; Huletsky, Ann; Leblanc, Éric; Frenette, Johanne; Boivin, Guy; Veres, Teodor; Dumoulin, Michel M.; Bergeron, Michel G.

2008-01-01

Fabrication of microarray devices using traditional glass slides is not easily adaptable to integration into microfluidic systems. There is thus a need for the development of polymeric materials showing a high hybridization signal-to-background ratio, enabling sensitive detection of microbial pathogens. We have developed such plastic supports suitable for highly sensitive DNA microarray hybridizations. The proof of concept of this microarray technology was done through the detection of four human respiratory viruses that were amplified and labeled with a fluorescent dye via a sensitive reverse transcriptase PCR (RT-PCR) assay. The performance of the microarray hybridization with plastic supports made of PMMA [poly(methylmethacrylate)]-VSUVT or Zeonor 1060R was compared to that with high-quality glass slide microarrays by using both passive and microfluidic hybridization systems. Specific hybridization signal-to-background ratios comparable to that obtained with high-quality commercial glass slides were achieved with both polymeric substrates. Microarray hybridizations demonstrated an analytical sensitivity equivalent to approximately 100 viral genome copies per RT-PCR, which is at least 100-fold higher than the sensitivities of previously reported DNA hybridizations on plastic supports. Testing of these plastic polymers using a microfluidic microarray hybridization platform also showed results that were comparable to those with glass supports. In conclusion, PMMA-VSUVT and Zeonor 1060R are both suitable for highly sensitive microarray hybridizations. PMID:18784318

Membrane expression of MRP-1, but not MRP-1 splicing or Pgp expression, predicts survival in patients with ESFT.

PubMed

Roundhill, E; Burchill, S

2013-07-09

Primary Ewing's sarcoma family of tumours (ESFTs) may respond to chemotherapy, although many patients experience subsequent disease recurrence and relapse. The survival of ESFT cells following chemotherapy has been attributed to the development of resistant disease, possibly through the expression of ABC transporter proteins. MRP-1 and Pgp mRNA and protein expression in primary ESFTs was determined by quantitative reverse-transcriptase PCR (RT-qPCR) and immunohistochemistry, respectively, and alternative splicing of MRP-1 by RT-PCR. We observed MRP-1 protein expression in 92% (43 out of 47) of primary ESFTs, and cell membrane MRP-1 was highly predictive of both overall survival (P<0.0001) and event-free survival (P<0.0001). Alternative splicing of MRP-1 was detected in primary ESFTs, although the pattern of splicing variants was not predictive of patient outcome, with the exception of loss of exon 9 in six patients, which predicted relapse (P=0.041). Pgp protein was detected in 6% (38 out of 44) of primary ESFTs and was not associated with patient survival. For the first time we have established that cell membrane expression of MRP-1 or loss of exon 9 is predictive of outcome but not the number of splicing events or expression of Pgp, and both may be valuable factors for the stratification of patients for more intensive therapy.

Contamination of infectious RD-114 virus in vaccines produced using non-feline cell lines.

PubMed

Yoshikawa, Rokusuke; Sato, Eiji; Miyazawa, Takayuki

2011-01-01

All domestic cats have a replication-competent endogenous retrovirus, termed RD-114 virus, in their genome and several feline cell lines produce RD-114 viruses. Recently, we found that a portion of live attenuated feline and canine vaccines produced using feline cell lines was contaminated with infectious RD-114 viruses. In this study, we expanded our survey and examined canine vaccines produced using 'non-feline' cell lines. Consequently, we found two vaccines containing RD-114 viral RNA by reverse transcriptase (RT)-polymerase chain reaction (PCR) and real-time RT-PCR. We also confirmed the presence of infectious RD-114 virus in the vaccines by the LacZ marker rescue assay and PCR to detect proviral DNA in TE671 cells (human rhabdomyosarcoma cells) inoculated with the vaccines. It is impossible to investigate the definitive cause of contamination with RD-114 virus; however, we suspect that a seed canine parvovirus type 2 was contaminated with RD-114 virus, because many canine parvoviruses have been isolated and attenuated using feline cell lines. To exclude RD-114 virus from live attenuated vaccines, we must pay attention to the contamination of seed viruses with RD-114 virus in addition to avoiding feline cell lines producing RD-114 virus when manufacturing vaccines. Copyright © 2010 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.

3'-terminal sequence of a small round structured virus (SRSV) in Japan.

PubMed

Utagawa, E T; Takeda, N; Inouye, S; Kasuga, K; Yamazaki, S

1994-01-01

We determined the nucleotide sequence of about 1,000 bases from the 3'-terminus of a small round structured virus (SRSV), which caused a gastroenteritis outbreak in Chiba Prefecture, Japan, in 1987. The sequence was compared with the corresponding sequence region of Norwalk virus; it consisted of a part of the open reading frame 2 (ORF2), whole ORF3, and 3'-noncoding region (NCR). The 624-base-long ORF3 had sequence homology of 68% with the corresponding region of Norwalk virus. (The amino acid sequence homology was 74%.) The 94-base-long NCR had 65% homology with Norwalk virus. We then selected two consensus-sequence portions in the above sequence between Chiba and Norwalk viruses for primers in the reverse transcriptase-polymerase chain reaction (RT-PCR). Using this primer set, we detected 669-bp bands in agarose gel electrophoresis of RT-PCR products from feces containing Chiba or Norwalk viruses. Furthermore, in Southern hybridization with Chiba probes which were labeled with digoxigenin-dUTP in PCR, the bands of the two viruses were clearly stained under a low stringency condition. Since both Chiba and Norwalk viruses were detected by the above primer set although they are geographically and chronologically different viruses, our primer-pair may be useful for detection of a broad range of SRSVs which cause gastroenteritis in different areas.

Novel Codon Insert in HIV Type 1 Clade B Reverse Transcriptase Associated with Low-Level Viremia During Antiretroviral Therapy

PubMed Central

Gianella, Sara; Vazquez, Homero; Ignacio, Caroline; Zweig, Adam C.; Richman, Douglas D.; Smith, Davey M.

2014-01-01

Abstract We investigated the pol genotype in two phylogenetically and epidemiologically linked partners, who were both experiencing persistent low-level viremia during antiretroviral therapy. In one partner we identified a new residue insertion between codon 248 and 249 of the HIV-1 RNA reverse transcriptase (RT) coding region (HXB2 numbering). We then investigated the potential impact of identified mutations in RT and antiretroviral binding affinity using a novel computational approach. PMID:24020934

Evidence for airborne transmission of Norwalk-like virus (NLV) in a hotel restaurant.

PubMed

Marks, P J; Vipond, I B; Carlisle, D; Deakin, D; Fey, R E; Caul, E O

2000-06-01

An outbreak of gastroenteritis followed a meal in a large hotel during which one of the diners vomited. The clinical features of the illness suggested Norwalk-like virus (NLV, small round structured virus) infection, and this was confirmed by electron microscopy and reverse transcriptase polymerase chain reaction (RT-PCR) of stool samples. Further characterization of the virus by nucleotide sequence analysis of the PCR amplicons revealed identical strains in all the affected individuals. The foods served at the meal could not be demonstrated to be the cause of the outbreak. Analysis of attack rates by dining table showed an inverse relationship with the distance from the person who vomited. No one eating in a separate restaurant reported illness. Transmission from person-to-person or direct contamination of food seems unlikely in this outbreak. However, the findings are consistent with airborne spread of NLV with infection by inhalation with subsequent ingestion of virus particles.

Borna Disease Virus Antibody and RNA from Peripheral Blood Mononuclear Cells of Race Horses and Jockeys in Korea

PubMed Central

Song, Jin-Won; Na, Kyoung-Sae; Tae, Seong-Ho

2011-01-01

Objective During the last two decades, Borna disease virus (BDV) has received much attention as a possible zoonotic agent, particularly as a cause of psychiatric disease. Although several studies have shown that BDV is present in Asia, BDV has not been detected in Korea. This study was designed to further investigate the presence of BDV infection in Korea. Methods Blood samples were taken from 39 race horses and 48 jockeys. Antibody to BDV was detected by indirect immunofluorescence antibody test and RNA of BDV by real time reverse transcriptase PCR (rRT-PCR). Results No evidence of BDV was detected in either the horses or the jockeys group. Conclusion Our results suggest that BDV infection may not be endemic in Korea. Further studies with novel diagnostic tools are required to clarify the prevalence of BDV infection in Korea. PMID:21519538

[Expression of gamma interferon during HPV and Chlamydia trachomatis infection in cervical samples].

PubMed

Colín-Ferreyra, María Del Carmen; Mendieta-Zerón, Hugo; Romero-Figueroa, María Del Socorro; Martínez-Madrigal, Migdania; Martínez-Pérez, Sergio; Domínguez-García, María Victoria

2015-02-01

The aim of this study was to mesure the expression of gamma interferon in HPV and Chlamydia trachomatis infection in squamous intraepithelial lesions. Samples from 100 patients diagnosed by colposcopy with or without squamous intraepithelial lesions were used in the present study. Each patient was found to be infected by HPV and C.trachomatis. Relative gamma interferon mRNA expression was assessed using a real-time reverse transcriptase PCR assay (RT-PCR). The relative units of expression of gamma interferon mRNA were 13, 1.8 and 0.3, for HPV and C.trachomatis co-infection, or HPV or C.trachomatis infection, respectively. HPV and C.trachomatis could overstimulate the expression of gamma interferon. Copyright © 2014 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication.

PubMed

Herzig, Eytan; Voronin, Nickolay; Kucherenko, Nataly; Hizi, Amnon

2015-08-01

The process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting the in vitro data. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis. Reverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting the in vitro data. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Simultaneous Detection of Rift Valley Fever, Bluetongue, Rinderpest, and Peste des Petits Ruminants Viruses by a Single-Tube Multiplex Reverse Transcriptase-PCR Assay Using a Dual-Priming Oligonucleotide System▿

PubMed Central

Yeh, Jung-Yong; Lee, Ji-Hye; Seo, Hyun-Ji; Park, Jee-Yong; Moon, Jin-San; Cho, In-Soo; Choi, In-Soo; Park, Seung-Yong; Song, Chang-Seon; Lee, Joong-Bok

2011-01-01

The aim of this study was to develop a highly sensitive and specific one-step multiplex reverse transcriptase PCR assay for the simultaneous and differential detection of Rift Valley Fever virus (RVFV), bluetongue virus (BTV), rinderpest virus (RPV), and Peste des petits ruminants virus (PPRV). These viruses cause mucosal lesions in cattle, sheep, and goats, and they are difficult to differentiate from one another based solely on their clinical presentation in suspected disease cases. In this study, we developed a multiplex reverse transcriptase PCR to detect these viruses using a novel dual-priming oligonucleotide (DPO). The DPO contains two separate priming regions joined by a polydeoxyinosine linker, which blocks extension of nonspecifically primed templates and consistently allows high PCR specificity even under less-than-optimal PCR conditions. A total of 19 DPO primers were designed to detect and discriminate between RVFV, BTV, RPV, and PPRV by the generation of 205-, 440-, 115-, and 243-bp cDNA products, respectively. The multiplex reverse transcriptase PCR described here enables the early diagnosis of these four viruses and may also be useful as part of a testing regime for cattle, sheep, or goats exhibiting similar clinical signs, including mucosal lesions. PMID:21307219

Three cases of imported dengue virus infection from Madeira to Belgium, 2012.

PubMed

Cnops, Lieselotte; Franco, Leticia; Van Meensel, Britt; Van den Ende, Jef; Paz Sanchez-Seco, Maria; Van Esbroeck, Marjan

2014-01-01

We report three laboratory-confirmed dengue virus (DENV) infections imported to Belgium by travelers returning from Madeira (Portugal). Despite the use of a mosquito-repellent spray as reported by two patients, the infection could not be prevented. Diagnosis was made by antigen detection and real-time reverse transcriptase polymerase chain reaction (RT-PCR) in two cases and by serology 1 month after onset of symptoms in a third one. The responsible virus was identified as DENV serotype 1, American/African genotype (genotype V). The close relationship to isolates from Colombia supports the previous findings that a South American strain originated the outbreak in Madeira in 2012. © 2014 International Society of Travel Medicine.

Differentiation of isolated human umbilical cord mesenchymal stem cells into neural stem cells

PubMed Central

Chen, Song; Zhang, Wei; Wang, Ji-Ming; Duan, Hong-Tao; Kong, Jia-Hui; Wang, Yue-Xin; Dong, Meng; Bi, Xue; Song, Jian

2016-01-01

AIM To investigate whether umbilical cord human mesenchymal stem cell (UC-MSC) was able to differentiate into neural stem cell and neuron in vitro. METHODS The umbilical cords were obtained from pregnant women with their written consent and the approval of the Clinic Ethnics Committee. UC-MSC were isolated by adherent culture in the medium contains 20% fetal bovine serum (FBS), then they were maintained in the medium contain 10% FBS and induced to neural cells in neural differentiation medium. We investigated whether UC-MSC was able to differentiate into neural stem cell and neuron in vitro by using flow cytometry, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence (IF) analyzes. RESULTS A substantial number of UC-MSC was harvested using the tissue explants adherent method at about 2wk. Flow cytometric study revealed that these cells expressed common markers of MSCs, such as CD105 (SH2), CD73 (SH3) and CD90. After induction of differentiation of neural stem cells, the cells began to form clusters; RT-PCR and IF showed that the neuron specific enolase (NSE) and neurogenic differentiation 1-positive cells reached 87.3%±14.7% and 72.6%±11.8%, respectively. Cells showed neuronal cell differentiation after induced, including neuron-like protrusions, plump cell body, obviously and stronger refraction. RT-PCR and IF analysis showed that microtubule-associated protein 2 (MAP2) and nuclear factor-M-positive cells reached 43.1%±10.3% and 69.4%±19.5%, respectively. CONCLUSION Human umbilical cord derived MSCs can be cultured and proliferated in vitro and differentiate into neural stem cells, which may be a valuable source for cell therapy of neurodegenerative eye diseases. PMID:26949608

Serological Response in RT-PCR Confirmed H1N1-2009 Influenza A by Hemagglutination Inhibition and Virus Neutralization Assays: An Observational Study

PubMed Central

Chen, Mark I.; Barr, Ian G.; Koh, Gerald C. H.; Lee, Vernon J.; Lee, Caroline P. S.; Shaw, Robert; Lin, Cui; Yap, Jonathan; Cook, Alex R.; Tan, Boon Huan; Loh, Jin Phang; Barkham, Timothy; Chow, Vincent T. K.; Lin, Raymond T. P.; Leo, Yee-Sin

2010-01-01

Background We describe the serological response following H1N1-2009 influenza A infections confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). Methodology and Principal Findings The study included patients admitted to hospital, subjects of a seroepidemiologic cohort study, and participants identified from outbreak studies in Singapore. Baseline (first available blood sample) and follow-up blood samples were analyzed for antibody titers to H1N1-2009 and recently circulating seasonal influenza A virus strains by hemagglutination inhibition (HI) and virus micro-neutralization (VM) assays. 267 samples from 118 cases of H1N1-2009 were analyzed. Geometric mean titers by HI peaked at 123 (95% confidence interval, CI 43-356) between days 30 to 39. The chance of observing seroconversion (four-fold or greater increase of antibodies) was maximized when restricting analysis to 45 participants with baseline sera collected within 5 days of onset and follow-up sera collected 15 or more days after onset; for these participants, 82% and 89% seroconverted to A/California/7/2009 H1N1 by HI and VM respectively. A four-fold or greater increase in cross-reactive antibody titers to seasonal A/Brisbane/59/2007 H1N1, A/Brisbane/10/2007 H3N2 and A/Wisconsin/15/2009 H3N2 occurred in 20%, 18% and 16% of participants respectively. Conclusions and Significance Appropriately timed paired serology detects 80–90% RT-PCR confirmed H1N1-2009; Antibodies from infection with H1N1-2009 cross-reacted with seasonal influenza viruses. PMID:20814575

Detection of ALK rearrangements in malignant pleural effusion cell blocks from patients with advanced non-small cell lung cancer: a comparison of Ventana immunohistochemistry and fluorescence in situ hybridization.

PubMed

Wang, Weiya; Tang, Yuan; Li, Jinnan; Jiang, Lili; Jiang, Yong; Su, Xueying

2015-02-01

Surgical resections or tumor biopsies are often not available for patients with late-stage non-small cell lung cancer (NSCLC). Cytological specimens, such as malignant pleural effusion (MPE) cell blocks, are critical for molecular testing. Currently, diagnostic methods to identify anaplastic lymphoma kinase (ALK) rearrangements include fluorescence in situ hybridization (FISH), real-time reverse transcriptase-polymerase chain reaction (RT-PCR), and immunohistochemistry (IHC). In the current study, the authors compared Ventana ALK IHC assays and ALK FISH to detect ALK rearrangements in MPE cell blocks from patients with advanced NSCLC. The ALK IHC assay and ALK FISH were performed on 63 MPE cell blocks. RT-PCR analysis was performed as additional validation in cases in which a discrepancy was observed between the IHC assay and FISH results. The Ventana ALK IHC assay was found to be informative for all 63 samples, and 8 cases were positive. Fifty-eight cases were interpretable for FISH detection, and 6 were positive. The concordance between IHC and FISH was 100% among the 58 cases. Of the 5 uninterpretable ALK FISH cases, 2 cases and 3 cases, respectively, were ALK IHC positive and negative. One of the 2 ALK IHC-positive cases also demonstrated a positive result in the RT-PCR assay and the patient benefited from crizotinib treatment. MPE cell blocks can be used successfully for the detection of ALK rearrangement when tumor tissue is not available. The Ventana ALK IHC assay is an effective screening method for ALK rearrangement in MPE cell blocks from patients with advanced NSCLC, demonstrating high agreement with FISH results. © 2014 American Cancer Society.

Molecular analysis of alternative transcripts of equine AXL receptor tyrosine kinase gene.

PubMed

Park, Jeong-Woong; Song, Ki-Duk; Kim, Nam Young; Choi, Jae-Young; Hong, Seul A; Oh, Jin Hyeog; Kim, Si Won; Lee, Jeong Hyo; Park, Tae Sub; Kim, Jin-Kyoo; Kim, Jong Geun; Cho, Byung-Wook

2017-10-01

Since athletic performance is a most importance trait in horses, most research focused on physiological and physical studies of horse athletic abilities. In contrast, the molecular analysis as well as the regulatory pathway studies remain insufficient for evaluation and prediction of horse athletic abilities. In our previous study, we identified AXL receptor tyrosine kinase ( AXL ) gene which was expressed as alternative spliced isoforms in skeletal muscle during exercise. In the present study, we validated two AXL alternative splicing transcripts (named as AXLa for long form and AXLb for short form) in equine skeletal muscle to gain insight(s) into the role of each alternative transcript during exercise. We validated two isoforms of AXL transcripts in horse tissues by reverse transcriptase polymerase chain reaction (RT-PCR), and then cloned the transcripts to confirm the alternative locus and its sequences. Additionally, we examined the expression patterns of AXLa and AXLb transcripts in horse tissues by quantitative RT-PCR (qRT-PCR). Both of AXLa and AXLb transcripts were expressed in horse skeletal muscle and the expression levels were significantly increased after exercise. The sequencing analysis showed that there was an alternative splicing event at exon 11 between AXLa and AXLb transcripts. 3-dimentional (3D) prediction of the alternative protein structures revealed that the structural distance of the connective region between fibronectin type 3 (FN3) and immunoglobin (Ig) domain was different between two alternative isoforms. It is assumed that the expression patterns of AXLa and AXLb transcripts would be involved in regulation of exercise-induced stress in horse muscle possibly through an NF-κB signaling pathway. Further study is necessary to uncover biological function(s) and significance of the alternative splicing isoforms in race horse skeletal muscle.

Prevalence and molecular characterization of avian infectious bronchitis virus in poultry flocks in Morocco from 2010 to 2014 and first detection of Italy 02 in Africa.

PubMed

Fellahi, Siham; Ducatez, Mariette; El Harrak, Mehdi; Guérin, Jean-Luc; Touil, Nadia; Sebbar, Ghizlane; Bouaiti, El Arbi; Khataby, Khadija; Ennaji, My Mustapha; El-Houadfi, Mohammed

2015-01-01

The aim of this study was to investigate the prevalence and diversity of infectious bronchitis virus (IBV) genotypes in poultry flocks in 16 areas of Morocco between 2010 and 2014. A total of 360 chicken flocks suspected of being infected by IBV were screened for the IBV N gene using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Flocks were classified into four groups according to their IBV vaccination programme. Group 1 contained unvaccinated birds. Group 2 received a single application of live H120 vaccine. Groups 3 and 4 birds received one or two booster vaccination(s), respectively, mostly using the H120 vaccine. The real-time RT-PCR results showed that 51.7% of the flocks were positive for the IBV genome with geographical disparities. Molecular characterization of IBV was performed on 50 RT-PCR positive samples by partially sequencing the S1 gene, including the hypervariable regions (nucleotides 705-1097). Two predominant genotypes were detected, with the Massachusetts type dominating (66%), among which 25% of the samples were identical to the H120 vaccine. The second most common genotype (present in 32% of the flocks) was surprisingly Italy 02, revealing the first detection of this genotype in Morocco and also in Africa. 793B, the predominant genotype in the late 1990s in Morocco, was only detected on one occasion and was identical to the 4/91 vaccine strain. This study highlights the high prevalence of IBV in poultry farms in Morocco and confirms its continuous dynamic changes and evolution.

Epidemic outbreaks, diagnostics, and control measures of the H5N1 highly pathogenic avian influenza in the Kingdom of Saudi Arabia, 2007-08.

PubMed

Lu, Huaguang; Ismail, Mahmoud Moussa; Khan, Owais Ahmed; Al Hammad, Yousef; Abdel Rhman, Salah Shaban; Al-Blowi, Mohamed Hamad

2010-03-01

The first outbreak of H5N1 highly pathogenic avian influenza (HPAI) in the Kingdom of Saudi Arabia (KSA) occurred in two "backyard" flocks of Houbara bustards and falcons in February 2007. Subsequent outbreaks were seen through the end of 2007 in "backyard" birds including native chickens, ostriches, turkeys, ducks, and peacocks. From November 2007 through January 2008, H5N1 HPAI outbreaks occurred in 19 commercial poultry premises, including two broiler breeder farms, one layer breeder farm, one ostrich farm, and 15 commercial layer farms, with approximately 4.75 million birds affected. Laboratory diagnosis of all H5N1-positive cases was conducted at the Central Veterinary Diagnostic Laboratory (CVDL) in Riyadh, Saudi Arabia. A combination of diagnostic tests was used to confirm the laboratory diagnosis. A rapid antigen-capture test and real-time reverse transcriptase-PCR (rtRT-PCR) assay on clinical and field specimens were conducted initially. Meanwhile, virus isolation in specific-pathogen-free embryonating chicken eggs was performed and was followed by hemagglutinin (HA) and hemagglutination inhibition tests, then rapid antigen-capture and rtRT-PCR tests on HA-positive allantoic fluid samples. In most HPAI cases, a complete laboratory diagnosis was made within 24-48 hr at the CVDL. Saudi Arabian government officials made immediate decisions to depopulate all H5N1-affected and nonaffected flocks within a 5-km radius area and applied quarantine zones to prevent the virus from spreading to other areas. Other control measures, such as closure of live bird markets and intensive surveillance tests on all poultry species within quarantine zones, were in place during the outbreaks. As a result, the HPAI outbreaks were quickly controlled, and no positive cases were detected after January 29, 2008. The KSA was declared free of HPAI on April 30, 2008, by the World Animal Health Organization.

Characterization of potential antiviral resistance mutations in hepatitis B virus reverse transcriptase sequences in treatment-naïve Chinese patients.

PubMed

Liu, Bao-Ming; Li, Tong; Xu, Jie; Li, Xiao-Guang; Dong, Jian-Ping; Yan, Ping; Yang, Jing-Xian; Yan, Ling; Gao, Zhi-Yong; Li, Wen-Peng; Sun, Xie-Wen; Wang, Yu-Hua; Jiao, Xiu-Juan; Hou, Chun-Sheng; Zhuang, Hui

2010-03-01

Full-length hepatitis B virus (HBV) reverse transcriptase (RT) sequences were amplified and sequenced among 192 nucleos(t)ide analogue (NA)-naïve Chinese patients with chronic hepatitis B. Deduced amino acids (AAs) at 42 previously reported potential NA resistance (NAr) mutation positions in RT region were analyzed. Patients were found with either B-genotype (28.65%) or C-genotype (71.35%) infections. Rt53, rt91, rt124, rt134, rt221, rt224, rt238 and rt256 were identified as B- and C-genotype-dependent polymorphic AA positions. AA substitutions at 11 classical NAr mutation positions, i.e. rt80, rt169, rt173, rt180, rt181, rt184, rt194, rt202, rt204, rt236 and rt250, were not detected. However, potential NAr mutations were found in 30.73% (59/192) isolates, which involved 18 positions including rt53, rt207, rt229, rt238 and rt256, etc. The concomitant AA changes of HBsAg occurred in 16.67% (32/192) isolates including sG145R mutation. One-third of mutation positions were located in functional RT domains (e.g. rt207 and rt233), A-B interdomains (overlapping HBsAg 'a' determinant and showing most concomitant immune-associated mutations) and non-A-B interdomains (e.g. rt191 and rt213), respectively. Genotypes B and C each showed several preferred positions to mutate. These results might provide insights into understanding the evolution and selection basis of NAr HBV strains under antiviral therapy.

Swine Influenza Virus and Association with the Porcine Respiratory Disease Complex in Pig Farms in Southern Brazil.

PubMed

Schmidt, C; Cibulski, S P; Andrade, C P; Teixeira, T F; Varela, A P M; Scheffer, C M; Franco, A C; de Almeida, L L; Roehe, P M

2016-05-01

Despite the putative endemic status of swine influenza A virus (swIAV) infections, data on the occurrence of swine influenza outbreaks are scarce in Brazil. The aim of this study was to detect and subtype swIAVs from six outbreaks of porcine respiratory disease complex (PRDC) in southern Brazil. Nasal swabs were collected from 66 piglets with signs of respiratory disease in six herds. Lung tissue samples were collected from six necropsied animals. Virus detection was performed by PCR screening and confirmed by virus isolation and hemagglutination (HA). Influenza A subtyping was performed by a real-time reverse transcriptase PCR (rRT-PCR) to detect the A(H1N1)pdm09; other swIAV subtypes were determined by multiplex RT-PCR. In lung tissues, the major bacterial and viral pathogens associated with PRDC (Pasteurella multocida, Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Haemophilus parasuis and PCV2) were investigated. In some affected pigs, clinico-pathological evaluations were conducted. Influenza A was detected by screening PCR in 46 of 66 swab samples and from five of six lungs. Virus was recovered from pigs of all six herds. Subtype A(H1N1)pdm09 was detected in four of six herds and H1N2 in the other two herds. In lung tissues, further agents involved in PRDC were detected in all cases; Pasteurella multocida was identified in five of six samples and Mycoplasma hyopneumoniae in three of six. Actinobacillus pleuropneumoniae (1/6), Haemophilus parasuis (1/6) and PCV2 (1/6) were also detected. These findings indicate that subtypes A(H1N1)pdm09 and H1N2 were present in pigs in southern Brazil and were associated with PRDC outbreaks. © 2015 Blackwell Verlag GmbH.

Molecular docking of (5E)-3-(2-aminoethyl)-5-(2- thienylmethylene)-1, 3-thiazolidine-2, 4-dione on HIV-1 reverse transcriptase: novel drug acting on enzyme.

PubMed

Seniya, Chandrabhan; Yadav, Ajay; Uchadia, Kuldeep; Kumar, Sanjay; Sagar, Nitin; Shrivastava, Priyanka; Shrivastava, Shilpi; Wadhwa, Gulshan

2012-01-01

The study of Human immunodeficiency virus (HIV) in humans and animal models in last 31 years suggested that it is a causative agent of AIDS. This causes serious pandemic public health concern globally. It was reported that the HIV-1 reverse transcriptase (RT) played a critical role in the life cycle of HIV. Therefore, inhibition of HIV-1RT enzyme is one of the major and potential targets in the treatment of AIDS. The enzyme (HIV-1RT) was successfully targeted by non nucleotide reverse transcriptase inhibitors (NNRTIs). But frequent application of NNRTIs led drug resistance mutation on HIV infections. Therefore, there is a need to search new NNRTIs with appropriate pharmacophores. For the purpose, a virtually screened 3D model of unliganded HIV-1RT (1DLO) was explored. The unliganded HIV-1RT (1DLO) was docked with 4-thiazolidinone and its derivatives (ChemBank Database) by using AutoDock4. The best seven docking solutions complex were selected and analyzed by Ligplot. The analysis showed that derivative (5E)-3-(2- aminoethyl)-5-(2- thienylmethylene)-1, 3-thiazolidine-2, 4-dione (CID 3087795) has maximum potential against unliganded HIV-1RT (1DLO). The analysis was done on the basis of scoring and binding ability. The derivative (5E)-3-(2- aminoethyl)-5-(2- thienylmethylene)-1, 3-thiazolidine-2, 4-dione (CID 3087795) indicated minimum energy score and highest number of interactions with active site residue and could be a promising inhibitor for HIV-1 RT as Drug target.

Structure-based methods to predict mutational resistance to diarylpyrimidine non-nucleoside reverse transcriptase inhibitors.

PubMed

Azeem, Syeda Maryam; Muwonge, Alecia N; Thakkar, Nehaben; Lam, Kristina W; Frey, Kathleen M

2018-01-01

Resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) is a leading cause of HIV treatment failure. Often included in antiviral therapy, NNRTIs are chemically diverse compounds that bind an allosteric pocket of enzyme target reverse transcriptase (RT). Several new NNRTIs incorporate flexibility in order to compensate for lost interactions with amino acid conferring mutations in RT. Unfortunately, even successful inhibitors such as diarylpyrimidine (DAPY) inhibitor rilpivirine are affected by mutations in RT that confer resistance. In order to aid drug design efforts, it would be efficient and cost effective to pre-evaluate NNRTI compounds in development using a structure-based computational approach. As proof of concept, we applied a residue scan and molecular dynamics strategy using RT crystal structures to predict mutations that confer resistance to DAPYs rilpivirine, etravirine, and investigational microbicide dapivirine. Our predictive values, changes in affinity and stability, are correlative with fold-resistance data for several RT mutants. Consistent with previous studies, mutation K101P is predicted to confer high-level resistance to DAPYs. These findings were further validated using structural analysis, molecular dynamics, and an enzymatic reverse transcription assay. Our results confirm that changes in affinity and stability for mutant complexes are predictive parameters of resistance as validated by experimental and clinical data. In future work, we believe that this computational approach may be useful to predict resistance mutations for inhibitors in development. Published by Elsevier Inc.

A Pragmatic Approach to HIV-1 Drug Resistance Determination in Resource-Limited Settings by Use of a Novel Genotyping Assay Targeting the Reverse Transcriptase-Encoding Region Only

PubMed Central

Bronze, Michelle; Wallis, Carole L.; Stuyver, Lieven; Steegen, Kim; Balinda, Sheila; Kityo, Cissy; Stevens, Wendy; Rinke de Wit, Tobias F.; Schuurman, Rob

2013-01-01

In resource-limited settings (RLS), reverse transcriptase (RT) inhibitors form the backbone of first-line treatment regimens. We have developed a simplified HIV-1 drug resistance genotyping assay targeting the region of RT harboring all major RT inhibitor resistance mutation positions, thus providing all relevant susceptibility data for first-line failures, coupled with minimal cost and labor. The assay comprises a one-step RT-PCR amplification reaction, followed by sequencing using one forward and one reverse primer, generating double-stranded coverage of RT amino acids (aa) 41 to 238. The assay was optimized for all major HIV-1 group M subtypes in plasma and dried blood spot (DBS) samples using a panel of reference viruses for HIV-1 subtypes A to D, F to H, and circulating recombinant form 01_AE (CRF01_AE) and applied to 212 clinical plasma samples and 25 DBS samples from HIV-1-infected individuals from Africa and Europe. The assay was subsequently transferred to Uganda and applied locally on clinical plasma samples. All major HIV-1 subtypes could be detected with an analytical sensitivity of 5.00E+3 RNA copies/ml for plasma and DBS. Application of the assay on 212 clinical samples from African subjects comprising subtypes A to D, F to H (rare), CRF01_AE, and CRF02_AG at a viral load (VL) range of 6.71E+2 to 1.00E+7 (median, 1.48E+5) RNA copies/ml was 94.8% (n = 201) successful. Application on clinical samples in Uganda demonstrated a comparable success rate. Genotyping of clinical DBS samples, all subtype C with a VL range of 1.02E+3 to 4.49E+5 (median, 1.42E+4) RNA copies/ml, was 84.0% successful. The described assay greatly reduces hands-on time and the costs required for genotyping and is ideal for use in RLS, as demonstrated in a reference laboratory in Uganda and its successful application on DBS samples. PMID:23536405

Assessment Effects of Resveratrol on Human Telomerase Reverse Transcriptase Messenger Ribonucleic Acid Transcript in Human Glioblastoma

PubMed Central

Mirzazadeh, Azin; Kheirollahi, Majid; Farashahi, Ehsan; Sadeghian-Nodoushan, Fatemeh; Sheikhha, Mohammad Hasan; Aflatoonian, Behrouz

2017-01-01

Background: Glioblastoma (GBM) is the most common and aggressive brain tumor, which has a poor prognosis despite the advent of different therapeutic strategies. There are numerous molecular biomarkers to contribute diagnosis, prognosis, and prediction of response to the current therapy in GBM. One of the most important markers that are potentially valuable is immortalization-specific or immortalization-associated marker named “hTERT messenger ribonucleic acid (mRNA)” the key subunit of telomerase enzyme, which is expressed in more than 85% of cancer cells, in spite of the majority of normal somatic cells. In this study, we investigated the effects of resveratrol (RSV) on this mRNA marker level, leading to cancer progression. Materials and Methods: U-87MG cell line was obtained from Pasteur Institute of Iran and treated with various concentrations of 0–160 μg/mL of RSV and at different time points (24, 48, and 72 h). To evaluate viability of U-87MG cells, standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed. Real-time polymerase chain reaction (RT-PCR) was used for comparative and quantitative assessment of human telomerase reverse transcriptase (hTERT) mRNA copy number versus control–untreated group. Results: The results of our investigation suggested that RSV effectively inhibited cell growth and caused cell death in dose-dependent (P < 0.05) and not in time-dependent manner (P > 0.05), in vitro. Interestingly, quantitative RT-PCR analysis demonstrated that at half inhibition concentration, RSV dramatically decreased mRNA expression of hTERT, the catalytic subunit of telomerase enzyme, which leads to prevention of cell division and tumor progression. Conclusion: With regard to downregulation of this immortalization-associated marker, RSV may potentially be used as a therapeutic agent against GBM. PMID:28706881

Advances in Developing HIV-1 Viral Load Assays for Resource-Limited Settings

PubMed Central

Wang, ShuQi; Xu, Feng; Demirci, Utkan

2010-01-01

Commercial HIV-1 RNA viral load assays have been routinely used in developed countries to monitor antiretroviral treatment (ART). However, these assays require expensive equipment and reagents, well-trained operators, and established laboratory infrastructure. These requirements restrict their use in resource-limited settings where people are most afflicted with the HIV-1 epidemic. Inexpensive alternatives such as the Ultrasensitive p24 assay, the Reverse Transcriptase (RT) assay and in-house reverse transcription quantitative polymerase chain reaction (RT-qPCR) have been developed. However, they are still time-consuming, technologically complex and inappropriate for decentralized laboratories as point-of-care (POC) tests. Recent advances in microfluidics and nanotechnology offer new strategies to develop low-cost, rapid, robust and simple HIV-1 viral load monitoring systems. We review state-of-the-art technologies used for HIV-1 viral load monitoring in both developed and developing settings. Emerging approaches based on microfluidics and nanotechnology, which have potential to be integrated into POC HIV-1 viral load assays, are also discussed. PMID:20600784

Unique clinical and imaging findings in a first ever documented PCR positive rabies survival patient: A case report.

PubMed

M, Netravathi; V, Udani; Rs, Mani; V, Gadad; M A, Ashwini; M, Bhat; S, Mehta; A, Chowdhary; P K, Pal; S N, Madhusudana; P, Satishchandra

2015-09-01

Rabies is fatal encephalitis which is a major public health problem in Asian and African countries. Till date, only 12 cases have been reported who have survived after rabies. In this communication we report a patient who is unique as the first documented long term rabies survivor with PCR positivity even after 4½ years of illness. Child sustained dog bite following which he received adequate prophylaxis. Within two weeks, child developed encephalopathy requiring evaluation. Child continues to have persistent myoclonic jerks, seizures, is dependent on all activities with severe neurological deficits. Nested reverse transcriptase PCR (RT-PCR) specific for rabies nucleoprotein gene in CSF and nuchal skin biopsy were positive for rabies viral RNA. The nuchal skin biopsy was also positive for rabies nucleoprotein antigen by fluorescent antibody test (FAT). We describe the clinical evolution and sequential MRI brain changes in this child. Despite the uniformly dismal prognosis of human rabies, these unusual reports of survival of rabies patients may provide an impetus to explore newer therapeutic strategies for this otherwise fatal disease. Copyright © 2015 Elsevier B.V. All rights reserved.

Generation of non-genomic oligonucleotide tag sequences for RNA template-specific PCR

PubMed Central

Pinto, Fernando Lopes; Svensson, Håkan; Lindblad, Peter

2006-01-01

Background In order to overcome genomic DNA contamination in transcriptional studies, reverse template-specific polymerase chain reaction, a modification of reverse transcriptase polymerase chain reaction, is used. The possibility of using tags whose sequences are not found in the genome further improves reverse specific polymerase chain reaction experiments. Given the absence of software available to produce genome suitable tags, a simple tool to fulfill such need was developed. Results The program was developed in Perl, with separate use of the basic local alignment search tool, making the tool platform independent (known to run on Windows XP and Linux). In order to test the performance of the generated tags, several molecular experiments were performed. The results show that Tagenerator is capable of generating tags with good priming properties, which will deliberately not result in PCR amplification of genomic DNA. Conclusion The program Tagenerator is capable of generating tag sequences that combine genome absence with good priming properties for RT-PCR based experiments, circumventing the effects of genomic DNA contamination in an RNA sample. PMID:16820068

Detection of Viral Hemorrhagic Septicemia virus (VHSV) from the leech Myzobdella lugubris Leidy, 1851

PubMed Central

Faisal, Mohamed; Schulz, Carolyn A

2009-01-01

The leech Myzobdella lugubris is widespread in the Lake Erie Watershed, especially Lake St. Clair. However, its role in pathogen transmission is not fully understood. In this same watershed, several widespread fish mortalities associated with the Viral Hemorrhagic Septicemia virus (VHSV) were recorded. Viral Hemorrhagic Septicemia is an emerging disease in the Great Lakes Basin that is deadly to the fish population, yet little is known about its mode of transmission. To assess the potential role of M. lugubris in VHSV transmission, leeches were collected from Lake St. Clair and Lake Erie and pooled into samples of five. Cell culture and reverse transcriptase polymerase chain reaction (RT-PCR) were used to determine the presence of the virus and its identity. Results showed that 57 of the 91 pooled leech samples were positive by cell culture for VHSV and 66 of the 91 pooled leech samples were positive by RT-PCR for the VHSV. Two representative virus isolates were sequenced for further genetic confirmation and genotype classification. VHSV detected within M. lugubris was homologous to the Great Lakes strain of VHSV genotype IVb. This is the first record of the VHSV being detected from within a leech, specifically M. lugubris, and suggests the potential of M. lugubris being involved in VHSV transmission. PMID:19785752

An epidemiological study of enteric viruses in sewage with molecular characterization by RT-PCR and sequence analysis.

PubMed

Arraj, A; Bohatier, J; Aumeran, C; Bailly, J L; Laveran, H; Traoré, O

2008-09-01

The aim of this study was to assess the presence and seasonal frequency of various enteric viruses in wastewater treatment. The detection of astrovirus, norovirus, enterovirus, hepatitis A virus (HAV) and rotavirus was carried out by molecular analyses in concentrated water samples collected over 18 months at the entrance and exit of an activated sludge sewage treatment plant. The reverse transcriptase-polymerase chain reaction (RT-PCR) results were confirmed by sequencing, and comparative phylogenetic analysis was performed on the isolated strains. Genomes of human astrovirus and human rotavirus were identified in 26/29 and 11/29 samples of raw sewage, respectively, and in 12/29 and 13/29 treated effluent samples, respectively. Some rotavirus sequences detected in environmental samples were very close to those of clinical strains. Noroviruses, enteroviruses and HAV were not detected during the study period. This could be related to the small sample volume, to the sensitivity of the detection methods or to local epidemiological situations. Frequent detection of viral RNA, whether infectious or not, in the exit effluent of sewage treatment indicates wide dispersion of enteric viruses in the environment. Consequently, viral contamination resulting from the use of these treated waters is a risk that needs to be addressed.

Evaluation of Energy Balance on Human Telomerase Reverse Transcriptase (hTERT) Alternative Splicing by Semi-quantitative RT-PCR in Human Umbilical Vein Endothelial Cells.

PubMed

Behjati, Mohaddeseh; Hashemi, Mohammad; Kazemi, Mohammad; Salehi, Mansoor; Javanmard, Shaghayegh Haghjooy

2017-01-01

Decreased high-energy phosphate level is involved in endothelial cell injury and dysfunction. Reduced telomerase activity in endothelial cells in parallel with reduced energy levels might be due to altered direction of alternative splicing machine as a complication of depleted energy during the process of atherosclerosis. Isolated human umbilical vein endothelial cells (HUVECs) were treated for 24 hours by oligomycine (OM) and 2-deoxy glucose (2-DG). After 24 hours, the effect of energy depletion on telomerase splicing pattern was evaluated using RT-PCR. Indeed, in both treated and untargeted cells, nitric oxide (NO) and von Willebrand factor (vWF) were measured. ATP was depleted in treated cells by 43.9% compared with control group. We observed a slight decrease in NO levels ( P = 0.09) and vWF ( P = 0.395) in the setting of 49.36% ATP depletion. In both groups, no telomerase gene expression was seen. Telomerase and housekeeping gene expression were found in positive control group (colon cancer tissue) and sample tissue. The absence of telomerase gene expression in HUVECs might be due to the mortality of these cells or the low level of telomerase gene expression in these cells under normal circumstances.

Analysis of Acquisition and Titer of Maize Mosaic Rhabdovirus in Its Vector, Peregrinus maidis (Hemiptera: Delphacidae)

PubMed Central

Barandoc-Alviar, Karen; Ramirez, Girly M.; Rotenberg, Dorith; Whitfield, Anna E.

2016-01-01

The corn planthopper, Peregrinus maidis (Ashmead) (Hemiptera: Delphacidae), transmits Maize mosaic rhabdovirus (MMV), an important pathogen of maize and sorghum, in a persistent propagative manner. To better understand the vectorial capacity of P. maidis, we determined the efficiency of MMV acquisition by nymphal and adult stages, and characterized MMV titer through development. Acquisition efficiency, i.e., proportion of insects that acquired the virus, was determined by reverse transcriptase polymerase chain reaction (RT-PCR) and virus titer of individual insects was estimated by quantitative RT-PCR. Acquisition efficiency of MMV differed significantly between nymphs and adults. MMV titer increased significantly over time and throughout insect development from nymphal to adult stage, indication of virus replication in the vector during development. There was a positive association between the vector developmental stage and virus titer. Also, the average titer in male insects was threefold higher than female titers, and this difference persisted up to 30 d post adult eclosion. Overall, our findings indicate that nymphs are more efficient than adults at acquiring MMV and virus accumulated in the vector over the course of nymphal development. Furthermore, sustained infection over the lifespan of P. maidis indicates a potentially high capacity of this vector to transmit MMV. PMID:28076276

Syncytial Hepatitis of Tilapia ( Oreochromis niloticus L.) is Associated With Orthomyxovirus-Like Virions in Hepatocytes.

PubMed

Del-Pozo, J; Mishra, N; Kabuusu, R; Cheetham, S; Eldar, A; Bacharach, E; Lipkin, W I; Ferguson, H W

2017-01-01

Using transmission electron microscopy (TEM), the presented work expands on the ultrastructural findings of an earlier report on "syncytial hepatitis," a novel disease of tilapia (SHT). Briefly, TEM confirmed the presence of an orthomyxovirus-like virus within the diseased hepatocytes but not within the endothelium. This was supported by observing extracellular and intracellular (mostly intraendosomal), 60-100 nm round virions with a trilaminar capsid containing up to 7 electron-dense aggregates. Other patterns noted included enveloped or filamentous virions and virion-containing cytoplasmic membrane folds, suggestive of endocytosis. Patterns atypical for orthymyxovirus included the formation of syncytia and the presence of virions within the perinuclear cisternae (suspected to be the Golgi apparatus). The ultrastructural morphology of SHT-associated virions is similar to that previously reported for tilapia lake virus (TiLV). A genetic homology was investigated using the available reverse transcriptase polymerase chain reaction (RT-PCR) probes for TiLV and comparing clinically sick with clinically normal fish and negative controls. By RT-PCR analysis, viral nucleic acid was detected only in diseased fish. Taken together, these findings strongly suggest that a virus is causally associated with SHT, that this virus shares ultrastructural features with orthomyxoviruses, and it presents with partial genetic homology with TiLV (190 nucleotides).

Submicroscopic malaria infections in pregnant women from six departments in Haiti.

PubMed

Elbadry, Maha A; Tagliamonte, Massimiliano S; Raccurt, Christian P; Lemoine, Jean F; Existe, Alexandre; Boncy, Jacques; Weppelmann, Thomas A; Dame, John B; Okech, Bernard A

2017-08-01

To describe the epidemiology of malaria in pregnancy in Haiti. Cross-sectional study among pregnant women in six departments of Haiti. After obtaining informed consent, whole blood samples and demographic surveys were collected to investigate malaria prevalence, anaemia and socio-behavioural risk factors for infection, respectively. A total of 311 pregnant women were screened for Plasmodium falciparum infection using a rapid diagnostic test (RDT), microscopy and a novel, quantitative reverse transcriptase polymerase chain reaction method (qRT-PCR). Overall, 1.2% (4/311) of pregnant women were tested positive for malaria infection by both microscopy and RDT. However, using the qRT-PCR, 16.4% (51/311) of pregnant women were positive. The prevalence of malaria infection varied with geographical locations ranging between 0% and 46.4%. Additionally, 53% of pregnant women had some form of anaemia; however, no significant association was found between anaemia and submicroscopic malaria infection. The socio-behavioural risk factors identified to be protective of malaria infection were marital status (P < 0.05) and travel within one month prior to screening (P < 0.05). This study is the first to document the high prevalence of submicroscopic malaria infections among pregnant women in Haiti and identify social and behavioural risk factors for disease transmission. © 2017 John Wiley & Sons Ltd.

Leptin influences estrogen metabolism and accelerates prostate cell proliferation.

PubMed

Habib, Christine N; Al-Abd, Ahmed M; Tolba, Mai F; Khalifa, Amani E; Khedr, Alaa; Mosli, Hisham A; Abdel-Naim, Ashraf B

2015-01-15

The present study was designed to investigate the effect of leptin on estrogen metabolism in prostatic cells. Malignant (PC-3) and benign (BPH-1) human prostate cells were treated with 17-β-hydroxyestradiol (1 μM) alone or in combination with leptin (0.4, 4, 40 ng/ml) for 72 h. Cell proliferation assay, immunocytochemical staining of estrogen receptor (ER), liquid chromatography-tandem mass spectrometry method (LC-MS) and semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) were used. Cell proliferation assay demonstrated that leptin caused significant growth potentiation in both cells. Immunocytochemical staining showed that leptin significantly increased the expression of ER-α and decreased that of ER-β in PC-3 cells. LC-MS method revealed that leptin increased the concentration 4-hydroxyestrone and/or decreased that of 2-methoxyestradiol, 4-methoxyestradiol and 2-methoxyestrone. Interestingly, RT-PCR showed that leptin significantly up-regulated the expression of aromatase and cytochrome P450 1B1 (CYP1B1) enzymes; however down-regulated the expression of catechol-o-methyltransferase (COMT) enzyme. These data indicate that leptin-induced proliferative effect in prostate cells might be partly attributed to estrogen metabolism. Thus, leptin might be a novel target for therapeutic intervention in prostatic disorders. Copyright © 2014 Elsevier Inc. All rights reserved.

Stable Reference Gene Selection for RT-qPCR Analysis in Nonviruliferous and Viruliferous Frankliniella occidentalis

PubMed Central

Pan, Huipeng; Ma, Yabin; Zhang, Deyong; Liu, Yong; Zhang, Zhanhong; Zheng, Changying; Chu, Dong

2015-01-01

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for measuring and evaluating gene expression during variable biological processes. To facilitate gene expression studies, normalization of genes of interest relative to stable reference genes is crucial. The western flower thrips Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), the main vector of tomato spotted wilt virus (TSWV), is a destructive invasive species. In this study, the expression profiles of 11 candidate reference genes from nonviruliferous and viruliferous F. occidentalis were investigated. Five distinct algorithms, geNorm, NormFinder, BestKeeper, the ΔC t method, and RefFinder, were used to determine the performance of these genes. geNorm, NormFinder, BestKeeper, and RefFinder identified heat shock protein 70 (HSP70), heat shock protein 60 (HSP60), elongation factor 1 α, and ribosomal protein l32 (RPL32) as the most stable reference genes, and the ΔC t method identified HSP60, HSP70, RPL32, and heat shock protein 90 as the most stable reference genes. Additionally, two reference genes were sufficient for reliable normalization in nonviruliferous and viruliferous F. occidentalis. This work provides a foundation for investigating the molecular mechanisms of TSWV and F. occidentalis interactions. PMID:26244556

Up-regulation of proproliferative genes and the ligand/receptor pair placental growth factor and vascular endothelial growth factor receptor 1 in hepatitis C cirrhosis.

PubMed

Huang, Xiao X; McCaughan, Geoffrey W; Shackel, Nicholas A; Gorrell, Mark D

2007-09-01

Cirrhosis can lead to hepatocellular carcinoma (HCC). Non-diseased liver and hepatitis C virus (HCV)-associated cirrhosis with or without HCC were compared. Proliferation pathway genes, immune response genes and oncogenes were analysed by a quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and immunostaining. Real-time RT-PCR showed up-regulation of genes in HCV cirrhosis including the proliferation-associated genes bone morphogenetic protein 3 (BMP3), placental growth factor 3 (PGF3), vascular endothelial growth factor receptor 1 (VEGFR1) and soluble VEGFR1, the oncogene FYN, and the immune response-associated genes toll-like receptor 9 (TLR9) and natural killer cell transcript 4 (NK4). Expressions of TLR2 and the oncogenes B-cell CLL/lymphoma 9 (BCL9) and PIM2 were decreased in HCV cirrhosis. In addition, PIM2 and TLR2 were increased in HCV cirrhosis with HCC compared with HCV cirrhosis. The ligand/receptor pair PGF and VEGFR1 was intensely expressed by the portal tract vascular endothelium. VEGFR1 was expressed in reactive biliary epithelial structures in fibrotic septum and in some stellate cells and macrophages. PGF and VEGFR1 may have an important role in the pathogenesis of the neovascular response in cirrhosis.

Development and evaluation of a culture-independent method for source determination of fecal wastes in surface and storm waters using reverse transcriptase-PCR detection of FRNA coliphage genogroup gene sequences.

EPA Science Inventory

A complete method, incorporating recently improved reverse transcriptase-PCR primer/probe assays and including controls for determining interferences to phage recoveries from water sample concentrates and for detecting interferences to their analysis, was developed for the direct...

Development and evaluation of a culture-independent method for source determination of fecal wastes in surface and storm waters using reverse transcriptase-PCR detection of FRNA coliphage genogroup gene sequences

EPA Science Inventory

A complete method, incorporating recently improved reverse transcriptase-PCR primer/probe assays and including controls for determining interferences to phage recoveries from water sample concentrates and for detecting interferences to their analysis, was developed for the direct...

A Novel High-Throughput Method for Molecular Detection of Human Pathogenic Viruses Using a Nanofluidic Real-Time PCR System

PubMed Central

Coudray-Meunier, Coralie; Fraisse, Audrey; Martin-Latil, Sandra; Delannoy, Sabine; Fach, Patrick; Perelle, Sylvie

2016-01-01

Human enteric viruses are recognized as the main causes of food- and waterborne diseases worldwide. Sensitive and quantitative detection of human enteric viruses is typically achieved through quantitative RT-PCR (RT-qPCR). A nanofluidic real-time PCR system was used to develop novel high-throughput methods for qualitative molecular detection (RT-qPCR array) and quantification of human pathogenic viruses by digital RT-PCR (RT-dPCR). The performance of high-throughput PCR methods was investigated for detecting 19 human pathogenic viruses and two main process controls used in food virology. The conventional real-time PCR system was compared to the RT-dPCR and RT-qPCR array. Based on the number of genome copies calculated by spectrophotometry, sensitivity was found to be slightly better with RT-qPCR than with RT-dPCR for 14 viruses by a factor range of from 0.3 to 1.6 log10. Conversely, sensitivity was better with RT-dPCR than with RT-qPCR for seven viruses by a factor range of from 0.10 to 1.40 log10. Interestingly, the number of genome copies determined by RT-dPCR was always from 1 to 2 log10 lower than the expected copy number calculated by RT-qPCR standard curve. The sensitivity of the RT-qPCR and RT-qPCR array assays was found to be similar for two viruses, and better with RT-qPCR than with RT-qPCR array for eighteen viruses by a factor range of from 0.7 to 3.0 log10. Conversely, sensitivity was only 0.30 log10 better with the RT-qPCR array than with conventional RT-qPCR assays for norovirus GIV detection. Finally, the RT-qPCR array and RT-dPCR assays were successfully used together to screen clinical samples and quantify pathogenic viruses. Additionally, this method made it possible to identify co-infection in clinical samples. In conclusion, given the rapidity and potential for large numbers of viral targets, this nanofluidic RT-qPCR assay should have a major impact on human pathogenic virus surveillance and outbreak investigations and is likely to be of benefit to public health. PMID:26824897

Selection of reference genes for gene expression studies in heart failure for left and right ventricles.

PubMed

Li, Mengmeng; Rao, Man; Chen, Kai; Zhou, Jianye; Song, Jiangping

2017-07-15

Real-time quantitative reverse transcriptase-PCR (qRT-PCR) is a feasible tool for determining gene expression profiles, but the accuracy and reliability of the results depends on the stable expression of selected housekeeping genes in different samples. By far, researches on stable housekeeping genes in human heart failure samples are rare. Moreover the effect of heart failure on the expression of housekeeping genes in right and left ventricles is yet to be studied. Therefore we aim to provide stable housekeeping genes for both ventricles in heart failure and normal heart samples. In this study, we selected seven commonly used housekeeping genes as candidates. By using the qRT-PCR, the expression levels of ACTB, RAB7A, GAPDH, REEP5, RPL5, PSMB4 and VCP in eight heart failure and four normal heart samples were assessed. The stability of candidate housekeeping genes was evaluated by geNorm and Normfinder softwares. GAPDH showed the least variation in all heart samples. Results also indicated the difference of gene expression existed in heart failure left and right ventricles. GAPDH had the highest expression stability in both heart failure and normal heart samples. We also propose using different sets of housekeeping genes for left and right ventricles respectively. The combination of RPL5, GAPDH and PSMB4 is suitable for the right ventricle and the combination of GAPDH, REEP5 and RAB7A is suitable for the left ventricle. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression Patterns and Identified Protein-Protein Interactions Suggest That Cassava CBL-CIPK Signal Networks Function in Responses to Abiotic Stresses.

PubMed

Mo, Chunyan; Wan, Shumin; Xia, Youquan; Ren, Ning; Zhou, Yang; Jiang, Xingyu

2018-01-01

Cassava is an energy crop that is tolerant of multiple abiotic stresses. It has been reported that the interaction between Calcineurin B-like (CBL) protein and CBL-interacting protein kinase (CIPK) is implicated in plant development and responses to various stresses. However, little is known about their functions in cassava. Herein, 8 CBL ( MeCBL ) and 26 CIPK ( MeCIPK ) genes were isolated from cassava by genome searching and cloning of cDNA sequences of Arabidopsis CBL s and CIPK s. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that the expression levels of MeCBL and MeCIPK genes were different in different tissues throughout the life cycle. The expression patterns of 7 CBL and 26 CIPK genes in response to NaCl, PEG, heat and cold stresses were analyzed by quantitative real-time PCR (qRT-PCR), and it was found that the expression of each was induced by multiple stimuli. Furthermore, we found that many pairs of CBLs and CIPKs could interact with each other via investigating the interactions between 8 CBL and 25 CIPK proteins using a yeast two-hybrid system. Yeast cells co-transformed with cassava MeCIPK24, MeCBL10 , and Na + /H + antiporter MeSOS1 genes exhibited higher salt tolerance compared to those with one or two genes. These results suggest that the cassava CBL-CIPK signal network might play key roles in response to abiotic stresses.

Expression Patterns and Identified Protein-Protein Interactions Suggest That Cassava CBL-CIPK Signal Networks Function in Responses to Abiotic Stresses

PubMed Central

Mo, Chunyan; Wan, Shumin; Xia, Youquan; Ren, Ning; Zhou, Yang; Jiang, Xingyu

2018-01-01

Cassava is an energy crop that is tolerant of multiple abiotic stresses. It has been reported that the interaction between Calcineurin B-like (CBL) protein and CBL-interacting protein kinase (CIPK) is implicated in plant development and responses to various stresses. However, little is known about their functions in cassava. Herein, 8 CBL (MeCBL) and 26 CIPK (MeCIPK) genes were isolated from cassava by genome searching and cloning of cDNA sequences of Arabidopsis CBLs and CIPKs. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that the expression levels of MeCBL and MeCIPK genes were different in different tissues throughout the life cycle. The expression patterns of 7 CBL and 26 CIPK genes in response to NaCl, PEG, heat and cold stresses were analyzed by quantitative real-time PCR (qRT-PCR), and it was found that the expression of each was induced by multiple stimuli. Furthermore, we found that many pairs of CBLs and CIPKs could interact with each other via investigating the interactions between 8 CBL and 25 CIPK proteins using a yeast two-hybrid system. Yeast cells co-transformed with cassava MeCIPK24, MeCBL10, and Na+/H+ antiporter MeSOS1 genes exhibited higher salt tolerance compared to those with one or two genes. These results suggest that the cassava CBL-CIPK signal network might play key roles in response to abiotic stresses. PMID:29552024

Membrane expression of MRP-1, but not MRP-1 splicing or Pgp expression, predicts survival in patients with ESFT

PubMed Central

Roundhill, E; Burchill, S

2013-01-01

Background: Primary Ewing's sarcoma family of tumours (ESFTs) may respond to chemotherapy, although many patients experience subsequent disease recurrence and relapse. The survival of ESFT cells following chemotherapy has been attributed to the development of resistant disease, possibly through the expression of ABC transporter proteins. Methods: MRP-1 and Pgp mRNA and protein expression in primary ESFTs was determined by quantitative reverse-transcriptase PCR (RT-qPCR) and immunohistochemistry, respectively, and alternative splicing of MRP-1 by RT-PCR. Results: We observed MRP-1 protein expression in 92% (43 out of 47) of primary ESFTs, and cell membrane MRP-1 was highly predictive of both overall survival (P<0.0001) and event-free survival (P<0.0001). Alternative splicing of MRP-1 was detected in primary ESFTs, although the pattern of splicing variants was not predictive of patient outcome, with the exception of loss of exon 9 in six patients, which predicted relapse (P=0.041). Pgp protein was detected in 6% (38 out of 44) of primary ESFTs and was not associated with patient survival. Conclusion: For the first time we have established that cell membrane expression of MRP-1 or loss of exon 9 is predictive of outcome but not the number of splicing events or expression of Pgp, and both may be valuable factors for the stratification of patients for more intensive therapy. PMID:23799853

Glycosylation-related genes in NS0 cells are insensitive to moderately elevated ammonium concentrations

PubMed Central

Brodsky, Arthur Nathan; Caldwell, Mary; Bae, Sooneon; Harcum, Sarah W.

2014-01-01

NS0 and Chinese hamster ovary (CHO) cell lines are used to produce recombinant proteins for human therapeutics; however, ammonium accumulation can negatively impact cell growth, recombinant protein production, and protein glycosylation. To improve product quality and decrease costs, the relationship between ammonium and protein glycosylation needs to be elucidated. While ammonium has been shown to adversely affect glycosylation-related gene expression in CHO cells, NS0 studies have not been performed. Therefore, this study sought to determine if glycosylation in NS0 cells were ammonium-sensitive at the gene expression level. Using a DNA microarray that contained mouse glycosylation-related and housekeeping genes, the of these genes was analysed in response to various culture conditions – elevated ammonium, elevated salt, and elevated ammonium with proline. Surprisingly, no significant differences in gene expression levels were observed between the control and these conditions. Further, the elevated ammonium cultures were analysed using real-time quantitative reverse transcriptase PCR (qRT-PCR) for key glycosylation genes, and the qRT-PCR results corroborated the DNA microarray results, demonstrating that NS0 cells are ammonium-insensitive at the gene expression level. Since NS0 are known to have elevated nucleotide sugar pools under ammonium stress, and none of the genes directly responsible for these metabolic pools were changed, consequently cellular control at the translational or substrate-level must be responsible for the universally observed decreased glycosylation quality under elevated ammonium. PMID:25062658

Structure-based virtual screening efforts against HIV-1 reverse transcriptase to introduce the new potent non-nucleoside reverse transcriptase inhibitor

NASA Astrophysics Data System (ADS)

Hosseini, Yaser; Mollica, Adriano; Mirzaie, Sako

2016-12-01

The human immunodeficiency virus (HIV) which is strictly related to the development of AIDS, is treated by a cocktail of drugs, but due its high propensity gain drug resistance, the rational development of new medicine is highly desired. Among the different mechanism of action we selected the reverse transcriptase (RT) inhibition, for our studies. With the aim to identify new chemical entities to be used for further rational drug design, a set of 3000 molecules from the Zinc Database have been screened by docking experiments using AutoDock Vina software. The best ranked compounds with respect of the crystallographic inhibitor MK-4965 resulted to be five compounds, and the best among them was further tested by molecular dynamics (MD) simulation. Our results indicate that comp1 has a stronger interaction with the subsite p66 of RT than MK-4965 and that both are able to stabilize specific conformational changes of the RT 3D structure, which may explain their activity as inhibitors. Therefore comp1 could be a good candidate for biological tests and further development.

Direct CRISPR spacer acquisition from RNA by a natural reverse-transcriptase-Cas1 fusion protein

PubMed Central

Sidote, David J.; Markham, Laura M.; Sanchez-Amat, Antonio; Bhaya, Devaki; Lambowitz, Alan M.; Fire, Andrew Z.

2016-01-01

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) systems mediate adaptive immunity in diverse prokaryotes. CRISPR-associated Cas1 and Cas2 proteins have been shown to enable adaptation to new threats in Type I and II CRISPR systems by the acquisition of short segments of DNA (“spacers”) from invasive elements. In several Type III CRISPR systems, Cas1 is naturally fused to a reverse transcriptase (RT). In the marine bacterium Marinomonas mediterranea (MMB-1), we show that an RT-Cas1 fusion enables the acquisition of RNA spacers in vivo in an RT-dependent manner. In vitro, the MMB-1 RT-Cas1 and Cas2 proteins catalyze ligation of RNA segments into the CRISPR array, followed by reverse transcription. These observations outline a host-mediated mechanism for reverse information flow from RNA to DNA. PMID:26917774

Application of Coamplification at Lower Denaturation Temperature-PCR Sequencing for Early Detection of Antiviral Drug Resistance Mutations of Hepatitis B Virus

PubMed Central

Wong, Danny Ka-Ho; Tsoi, Ottilia; Huang, Fung-Yu; Seto, Wai-Kay; Fung, James; Lai, Ching-Lung

2014-01-01

Nucleoside/nucleotide analogue for the treatment of chronic hepatitis B virus (HBV) infection is hampered by the emergence of drug resistance mutations. Conventional PCR sequencing cannot detect minor variants of <20%. We developed a modified co-amplification at lower denaturation temperature-PCR (COLD-PCR) method for the detection of HBV minority drug resistance mutations. The critical denaturation temperature for COLD-PCR was determined to be 78°C. Sensitivity of COLD-PCR sequencing was determined using serially diluted plasmids containing mixed proportions of HBV reverse transcriptase (rt) wild-type and mutant sequences. Conventional PCR sequencing detected mutations only if they existed in ≥25%, whereas COLD-PCR sequencing detected mutations when they existed in 5 to 10% of the viral population. The performance of COLD-PCR was compared to conventional PCR sequencing and a line probe assay (LiPA) using 215 samples obtained from 136 lamivudine- or telbivudine-treated patients with virological breakthrough. Among these 215 samples, drug resistance mutations were detected in 155 (72%), 148 (69%), and 113 samples (53%) by LiPA, COLD-PCR, and conventional PCR sequencing, respectively. Nineteen (9%) samples had mutations detectable by COLD-PCR but not LiPA, while 26 (12%) samples had mutations detectable by LiPA but not COLD-PCR, indicating both methods were comparable (P = 0.371). COLD-PCR was more sensitive than conventional PCR sequencing. Thirty-five (16%) samples had mutations detectable by COLD-PCR but not conventional PCR sequencing, while none had mutations detected by conventional PCR sequencing but not COLD-PCR (P < 0.0001). COLD-PCR sequencing is a simple method which is comparable to LiPA and superior to conventional PCR sequencing in detecting minor lamivudine/telbivudine resistance mutations. PMID:24951803

Virtual screening studies on HIV-1 reverse transcriptase inhibitors to design potent leads.

PubMed

Vadivelan, S; Deeksha, T N; Arun, S; Machiraju, Pavan Kumar; Gundla, Rambabu; Sinha, Barij Nayan; Jagarlapudi, Sarma A R P

2011-03-01

The purpose of this study is to identify novel and potent inhibitors against HIV-1 reverse transcriptase (RT). The crystal structure of the most active ligand was converted into a feature-shaped query. This query was used to align molecules to generate statistically valid 3D-QSAR (r(2) = 0.873) and Pharmacophore models (HypoGen). The best HypoGen model consists of three Pharmacophore features (one hydrogen bond acceptor, one hydrophobic aliphatic and one ring aromatic) and further validated using known RT inhibitors. The designed novel inhibitors are further subjected to docking studies to reduce the number of false positives. We have identified and proposed some novel and potential lead molecules as reverse transcriptase inhibitors using analog and structure based studies. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Yellow Fever Vaccination of a Primary Vaccinee During Adalimumab Therapy.

PubMed

Nash, Esther R; Brand, Myron; Chalkias, Spyridon

2015-01-01

In this case report, we describe a 63-year-old female with Crohn's disease since age 16 years, and on adalimumab therapy, who inadvertently received a yellow fever vaccine (YFV) 4 days before her next dose of adalimumab. She had never received YFV. Her next dose of tumor necrosis factor (TNF) antagonist was held. She did not report any adverse effects referable to the vaccine. Reverse transcriptase-polymerase chain reaction (RT-PCR) for yellow fever (YF) viral RNA on days 12 and 18 postvaccination was negative. Neutralizing antibody to YF virus vaccine was immunoprotective on day 18 following vaccination, which further increased by day 26. A neutralizing antibody obtained 2 years following vaccination also remained immunoprotective. © 2015 International Society of Travel Medicine.

Comparison of electron microscopy, ELISA, real time RT-PCR and insulated isothermal RT-PCR for the detection of Rotavirus group A (RVA) in feces of different animal species.

PubMed

Soltan, Mohamed A; Tsai, Yun-Long; Lee, Pei-Yu A; Tsai, Chuan-Fu; Chang, Hsiao-Fen G; Wang, Hwa-Tang T; Wilkes, Rebecca P

2016-09-01

There is no gold standard for detection of Rotavirus Group A (RVA), one of the main causes of diarrhea in neonatal animals. Sensitive and specific real-time RT-PCR (rtRT-PCR) assays are available for RVA but require submission of the clinical samples to diagnostic laboratories. Patient-side immunoassays for RVA protein detection have shown variable results, particularly with samples from unintended species. A sensitive and specific test for detection of RVA on the farm would facilitate rapid management decisions. The insulated isothermal RT-PCR (RT-iiPCR) assay works in a portable machine to allow sensitive and specific on-site testing. The aim of this investigation was to evaluate a commercially available RT-iiPCR assay for RVA detection in feces from different animal species. This assay was compared to an in-house rtRT-PCR assay and a commercially available rtRT-PCR kit, as well as an ELISA and EM for RVA detection. All three PCR assays targeted the well-conserved NSP5 gene. Clinical fecal samples from 108 diarrheic animals (mainly cattle and horses) were tested. The percentage of positive samples by ELISA, EM, in-house rtRT-PCR, commercial rtRT-PCR, and RT-iiPCR was 29.4%, 31%, 36.7%, 51.4%, 56.9%, respectively. The agreement between different assays was high (81.3-100%) in samples containing high viral loads. The sensitivity of the RT-iiPCR assay appeared to be higher than the commercially available rtRT-PCR assay, with a limit of detection (95% confidence index) of 3-4 copies of in vitro transcribed dsRNA. In conclusion, the user-friendly, field-deployable RT-iiPCR system holds substantial promise for on-site detection of RVA. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

A novel and highly sensitive real-time nested RT-PCR assay in a single closed tube for detection of enterovirus.

PubMed

Shen, Xin-Xin; Qiu, Fang-Zhou; Zhao, Huai-Long; Yang, Meng-Jie; Hong, Liu; Xu, Song-Tao; Zhou, Shuai-Feng; Li, Gui-Xia; Feng, Zhi-Shan; Ma, Xue-Jun

2018-03-01

The sensitivity of qRT-PCR assay is not adequate for the detection of the samples with lower viral load, particularly in the cerebrospinal fluid (CSF) of patients. Here, we present the development of a highly sensitive real-time nested RT-PCR (RTN RT-PCR) assay in a single closed tube for detection of human enterovirus (HEV). The clinical performance of both RTN RT-PCR and qRT-PCR was also tested and compared using 140 CSF and fecal specimens. The sensitivities of RTN RT-PCR assay for EV71, Coxsackievirus A (CVA)16, CVA6 and CVA10 achieved 10 -8 dilution with a corresponding Ct value of 38.20, 36.45, 36.75, and 36.45, respectively, which is equal to traditional two-step nested RT-PCR assay and approximately 2-10-fold lower than that of qRT-PCR assay. The specificity of RTN RT-PCR assay was extensively analyzed insilico and subsequently verified using the reference isolates and clinical samples. Sixteen qRT-PCR-negative samples were detected by RTN RT-PCR and a variety of enterovirus serotypes was identified by sequencing of inner PCR products. We conclude RTN RT-PCR is more sensitive than qRT-PCR for the detection of HEV in clinical samples. Copyright © 2017 Elsevier Inc. All rights reserved.

Functional analysis of a viroid RNA motif mediating cell-to-cell movement in Nicotiana benthamiana.

PubMed

Jiang, Dongmei; Wang, Meng; Li, Shifang

2017-01-01

Cell-to-cell trafficking through different cellular layers is a key process for various RNAs including those of plant viruses and viroids, but the regulatory mechanisms involved are still not fully elucidated and good model systems are important. Here, we analyse the function of a simple RNA motif (termed 'loop19') in potato spindle tuber viroid (PSTVd) which is required for trafficking in Nicotiana benthamiana leaves. Northern blotting, reverse transcriptase PCR (RT-PCR) and in situ hybridization analyses demonstrated that unlike wild-type PSTVd, which was present in the nuclei in all cell types, the trafficking-defective loop19 mutants were visible only in the nuclei of upper epidermal and palisade mesophyll cells, which shows that PSTVd loop19 plays a role in mediating RNA trafficking from palisade to spongy mesophyll cells in N.benthamiana leaves. Our findings and approaches have broad implications for studying the RNA motifs mediating trafficking of RNAs across specific cellular boundaries in other biological systems.

Evidence for airborne transmission of Norwalk-like virus (NLV) in a hotel restaurant.

PubMed Central

Marks, P. J.; Vipond, I. B.; Carlisle, D.; Deakin, D.; Fey, R. E.; Caul, E. O.

2000-01-01

An outbreak of gastroenteritis followed a meal in a large hotel during which one of the diners vomited. The clinical features of the illness suggested Norwalk-like virus (NLV, small round structured virus) infection, and this was confirmed by electron microscopy and reverse transcriptase polymerase chain reaction (RT-PCR) of stool samples. Further characterization of the virus by nucleotide sequence analysis of the PCR amplicons revealed identical strains in all the affected individuals. The foods served at the meal could not be demonstrated to be the cause of the outbreak. Analysis of attack rates by dining table showed an inverse relationship with the distance from the person who vomited. No one eating in a separate restaurant reported illness. Transmission from person-to-person or direct contamination of food seems unlikely in this outbreak. However, the findings are consistent with airborne spread of NLV with infection by inhalation with subsequent ingestion of virus particles. PMID:10982072

Canine distemper of vaccine origin in European mink, Mustela lutreola--a case report.

PubMed

Ek-Kommonen, C; Rudbäck, E; Anttila, M; Aho, M; Huovilainen, A

2003-04-02

Cases of canine distemper (CD) related to vaccination of exotic carnivores extend over three decades and have been described in at least nine different species. Our report describes a case of acute CD in a European mink, Mustela lutreola, vaccinated with live attenuated CD vaccine licensed for use in fur-farmed mink. The male mink died of an acute grey matter disease with an unusually long incubation period. A female vaccinated at the same time showed no obvious signs of illness. The diagnosis was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and by subsequent sequencing of the PCR products. The sequenced products of the virus isolated from the mink and of the vaccine batch showed 100% identity. This is the first report in which molecular methods were used to confirm that the disease was caused by the vaccine strain. Based on our findings, it is clearly evident that current CD vaccines cannot be safely used in exotic species.

Expression of very low density lipoprotein receptor mRNA in circulating human monocytes: its up-regulation by hypoxia.

PubMed

Nakazato, K; Ishibashi, T; Nagata, K; Seino, Y; Wada, Y; Sakamoto, T; Matsuoka, R; Teramoto, T; Sekimata, M; Homma, Y; Maruyama, Y

2001-04-01

Although very low density lipoprotein (VLDL) receptor expression by macrophages has been shown in the vascular wall, it is not clear whether or not circulating monocytes express the VLDL receptor. We investigated the expression of VLDL receptor mRNA in human peripheral blood monocytes and monocyte-derived macrophages by reverse transcriptase polymerase chain reaction (RT-PCR) and nucleotide sequencing after subcloning of PCR product. VLDL receptor mRNA was detected both in peripheral blood monocytes and monocyte-derived macrophages. Expression of VLDL receptor mRNA was upregulated by hypoxia in monocytes, whereas treatment with oxidized LDL, interleukin-1beta or monocyte chemoattractant protein-1 did not affect the levels of VLDL receptor mRNA in monocytes and macrophages. The present study shows a novel response of VLDL receptor mRNA to hypoxia, suggesting a role for VLDL receptor in the metabolism of lipoproteins in the vascular wall and the development of atherosclerosis.

The use of short and long PCR products for improved detection of prunus necrotic ringspot virus in woody plants.

PubMed

Rosner, A; Maslenin, L; Spiegel, S

1997-09-01

The reverse transcriptase-polymerase chain reaction (RT-PCR) was used for detection of prunus necrotic ringspot virus (PNRSV) in dormant peach and almond trees by the application of two different pairs of primers yielding a short and a long product, respectively. The relative amount of the short (200 base pair, bp) product was higher than the longer (785 bp) product. PNRSV was detected better in plant tissues with a low virus concentration (e.g. dormant trees) by amplification of the short PCR product, whereas the long product was product was produced at higher virus titers. Simultaneous amplification of both short and long products was demonstrated using a three-primer mixture in a single reaction tube. In this assay, amplification of either PCR product indicated the presence of PNRSV-specific sequences in the plant tissue examined, thus covering a wide range of virus concentrations in a single test. Dilution of the RNA extracted from infected plant material resulted in a steep decline in the amplification of both short and long PCR products. In contrast, serial dilutions of the intermediate cDNA template differentially affected the amplification patterns: the relative amount of the short product increased whereas that of the long product decreased. These results may explain the preferential amplification of the short PCR product observed in samples containing low virus concentrations.

Diversity, dynamics, and activity of bacterial communities during production of an artisanal Sicilian cheese as evaluated by 16S rRNA analysis.

PubMed

Randazzo, Cinzia L; Torriani, Sandra; Akkermans, Antoon D L; de Vos, Willem M; Vaughan, Elaine E

2002-04-01

The diversity and dynamics of the microbial communities during the manufacturing of Ragusano cheese, an artisanal cheese produced in Sicily (Italy), were investigated by a combination of classical and culture-independent approaches. The latter included PCR, reverse transcriptase-PCR (RT-PCR), and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes (rDNA). Bacterial and Lactobacillus group-specific primers were used to amplify the V6 to V8 and V1 to V3 regions of the 16S rRNA gene, respectively. DGGE profiles from samples taken during cheese production indicated dramatic shifts in the microbial community structure. Cloning and sequencing of rDNA amplicons revealed that mesophilic lactic acid bacteria (LAB), including species of Leuconostoc, Lactococcus lactis, and Macrococcus caseolyticus were dominant in the raw milk, while Streptococcus thermophilus prevailed during lactic fermentation. Other thermophilic LAB, especially Lactobacillus delbrueckii and Lactobacillus fermentum, also flourished during ripening. Comparison of the rRNA-derived patterns obtained by RT-PCR to the rDNA DGGE patterns indicated a substantially different degree of metabolic activity for the microbial groups detected. Identification of cultivated LAB isolates by phenotypic characterization and 16S rDNA analysis indicated a variety of species, reflecting to a large extent the results obtained from the 16S rDNA clone libraries, with the significant exception of the Lactobacillus delbrueckii species, which dominated in the ripening cheese but was not detected by cultivation. The present molecular approaches combined with culture can effectively describe the complex ecosystem of natural fermented dairy products, giving useful information for starter culture design and preservation of artisanal fermented food technology.

Presence and distribution of leptin and leptin receptor in the canine gallbladder.

PubMed

Lee, Sungin; Lee, Aeri; Kweon, Oh-Kyeong; Kim, Wan Hee

2016-09-01

The hormone leptin is produced by mature adipocytes and plays an important role in regulating food intake and energy metabolism through its interaction with the leptin receptor. In addition to roles in obesity and obesity-related diseases, leptin has been reported to affect the components and secretion of bile in leptin-deficient mice. Furthermore, gallbladder diseases such as cholelithiasis are known to be associated with serum leptin concentrations in humans. We hypothesized that the canine gallbladder is a source of leptin and that the leptin receptor may be localized in the gallbladder, where it plays a role in regulating the function of this organ. The aim of this study was to demonstrate the presence and expression patterns of leptin and its receptors in normal canine gallbladders using reverse transcriptase-PCR (RT-PCR) and immunohistochemistry. Clinically normal gallbladder tissue samples were obtained from four healthy beagle dogs with similar body condition scores. RT-PCR and sequencing of the amplified PCR products revealed the presence of leptin mRNA and its receptors in the gallbladder. Immunohistochemical investigations demonstrated the expression of leptin and its receptors in the luminal single columnar and tubuloalveolar glandular epithelial cells. In conclusion, the results of this study demonstrated the presence of leptin and its receptors in the gallbladders of dogs. Leptin and its receptor were both localized throughout the cytoplasm of luminal and glandular epithelial cells. These results suggested that the gallbladder is not only a source of leptin, but also a target of leptin though autocrine/paracrine mechanisms. The results of this study could increase the understanding of both the normal physiological functions of the gallbladder and the pathophysiological mechanisms of gallbladder diseases characterized by leptin system dysfunction. Copyright © 2016 The Authors. Published by Elsevier GmbH.. All rights reserved.

Nitrate reduction mechanisms and rates in an unconfined eogenetic karst aquifer in two sites with different redox potential

NASA Astrophysics Data System (ADS)

Henson, W. R.; Huang, L.; Graham, W. D.; Ogram, A.

2017-05-01

This study integrates push-pull tracer tests (PPTT) with microbial characterization of extracted water via quantitative polymerase chain reaction (qPCR) and reverse transcriptase qPCR (RT-qPCR) of selected functional N transformation genes to quantify nitrate reduction mechanisms and rates in sites with different redox potential in a karst aquifer. PPTT treatments with nitrate (AN) and nitrate-fumarate (ANC) were executed in two wells representing anoxic and oxic geochemical end-members. Oxic aquifer zero-order nitrate loss rates (mmol L-1 h-1) were similar for AN and ANC treatment, ranging from 0.03 ± 0.01 to 0.05 ± 0.01. Anoxic aquifer zero-order nitrate loss rates ranged from 0.03 ± 0.02 (AN) to 0.13 ± 0.02 (ANC). Microbial characterization indicates mechanisms influencing these rates were dissimilatory nitrate reduction to ammonium (DNRA) at the anoxic site with AN treatment, assimilatory reduction of nitrate to ammonium (ANRA) with ANC treatment in the water column at both sites, and additional documented nitrate reduction that occurred in unsampled biofilms. With carbon treatment, total numbers of microbes (16S rRNA genes) significantly increased (fourteenfold to thirtyfold), supporting stimulated growth with resulting ANRA. Decreased DNRA gene concentrations (nrfA DNA) and increased DNRA activity ratio (nrfA-cDNA/DNA) supported the assertion that DNRA occurred in the anoxic zone with AN and ANC treatment. Furthermore, decreased DNRA gene copy numbers at the anoxic site with ANC treatment suggests that DNRA microbes in the anoxic site are chemolithoautotrophic. Increased RT-qPCR denitrification gene expression (nirK and nirS) was not observed in water samples, supporting that any observed NO3-N loss due to denitrification may be occurring in unsampled microbial biofilms.

Measurement of indicator genes using global complementary DNA (cDNA) amplification, by polyadenylic acid reverse transcriptase polymerase chain reaction (poly A RT-PCR): A feasibility study using paired samples from tissue and ductal juice in patients undergoing pancreatoduodenectomy.

PubMed

Sanyal, Sudip; Siriwardena, Ajith K; Byers, Richard

2018-06-01

The aim of this study is to compare gene expression profiles in RNA isolated from pancreatic ductal juice with the RNA expression profiles of the same genes from matched intra-operative tissue samples from pancreatic tumours. Intra-operative sampling of pancreatic juice and collection of matched tissue samples was undertaken in patients undergoing pancreatoduodenectomy for clinically suspected pancreatic cancer and a precursor lesion, main-duct intraductal papillary mucinous neoplasm. RNA was isolated and Poly A PCR was used to globally amplify the RNA. Real-time polymerase chain reaction (RT-PCR) was used to measure expression levels of 17 genes selected from microarray studies. Spearman's rank correlation test was used to examine the relationship of gene expression between pancreatic juice and tissue. The study was approved by Regional Ethics Committee. Mesothelin (MSLN) showed significant correlation (p < 0.008) in expression levels between paired pancreatic juice and tissue samples in pancreas cancer. In intraductal papillary mucinous neoplasms (IPMN), Matrix Metalloproteinase 7 (MMP7), showed significant correlation (p < 0.01) in the expression levels between paired pancreatic juice and tissue samples. This study confirms that RNA analysis of paired pancreatic juice and tissue samples and establishment of cDNA using poly A PCR is technically feasible. Application of the technique to non-invasively obtained pancreatic juice during endoscopic assessment of tumours and the use of gene arrays of cancer indicator genes are the next steps in development of this technique. Copyright © 2018 IAP and EPC. Published by Elsevier B.V. All rights reserved.

Clinical characterization of acute and convalescent illness of confirmed chikungunya cases from Chiapas, S. Mexico: A cross sectional study

PubMed Central

Trujillo-Murillo, Karina del Carmen; Caballero-Sosa, Sandra; Sepúlveda-Delgado, Jesús; Malo-García, Iliana Rosalía; Canseco-Ávila, Luis Miguel; Salgado-Corsantes, Luis Manuel; Domínguez-Arrevillaga, Sergio; Torres-Zapata, Raúl; Gómez-Cruz, Omar; Fernández-Salas, Ildefonso

2017-01-01

Background The emerging chikungunya virus (CHIKV), is an arbovirus causing intense outbreaks in North America. The situation in Mexico is alarming, and CHIKV threatens to spread further throughout North America. Clinical and biological features of CHIKF outbreaks in Mexico have not been well described; thus, we conducted a cross sectional study of a CHIKV outbreak in Chiapas, Southern Mexico to further characterize these features. Methodology/Principal findings We collected blood samples from patients suspected of having chikungunya fever (CHIKF) who presented to Clinical Hospital ISSSTE Dr. Roberto Nettel in Tapachula, Chiapas, Mexico. In addition to the clinical examination, real-time polymerase chain reaction (PCR) standardized for the Asian Chikungunya lineage and/or enzyme-linked immunosorbent assay for immunoglobulin M (IgM) were used to confirm CHIKV diagnosis. Of a total of 850 patients who presented with probably CHIKV at Hospital “Dr. Roberto Nettel”, 112 probable CHIKF cases were enrolled in this study from November 2014- June 2015, of which 95 patients (84.8%) were CHIKV positive and 17 were negative (15.2%). Of these 95 CHIKV positive patients, 62 were positive by real-time reverse transcriptase PCR (+qRT-PCR); and 33 were seropositive to +IgM with a negative qRT-PCR. The most frequent symptoms reported were fever (100%), headache (82.3%), polyarthralgia (72.1%), and exanthem (82.3%). Biological abnormalities observed during CHIKV infection were lymphopenia (41.1%), leukopenia (51.6%), elevated transaminases (30.5%-46.3%) and high LDH (46.3%) and CRP (60.0%). Conclusion Clinical and biological data obtained from this study is providing more useful information for benchmarking purposes with outbreaks from different parts of the world and would be helpful for better patient care and treatment. PMID:29065182

Clinical characterization of acute and convalescent illness of confirmed chikungunya cases from Chiapas, S. Mexico: A cross sectional study.

PubMed

Danis-Lozano, Rogelio; Díaz-González, Esteban Eduardo; Trujillo-Murillo, Karina Del Carmen; Caballero-Sosa, Sandra; Sepúlveda-Delgado, Jesús; Malo-García, Iliana Rosalía; Canseco-Ávila, Luis Miguel; Salgado-Corsantes, Luis Manuel; Domínguez-Arrevillaga, Sergio; Torres-Zapata, Raúl; Gómez-Cruz, Omar; Fernández-Salas, Ildefonso

2017-01-01

The emerging chikungunya virus (CHIKV), is an arbovirus causing intense outbreaks in North America. The situation in Mexico is alarming, and CHIKV threatens to spread further throughout North America. Clinical and biological features of CHIKF outbreaks in Mexico have not been well described; thus, we conducted a cross sectional study of a CHIKV outbreak in Chiapas, Southern Mexico to further characterize these features. We collected blood samples from patients suspected of having chikungunya fever (CHIKF) who presented to Clinical Hospital ISSSTE Dr. Roberto Nettel in Tapachula, Chiapas, Mexico. In addition to the clinical examination, real-time polymerase chain reaction (PCR) standardized for the Asian Chikungunya lineage and/or enzyme-linked immunosorbent assay for immunoglobulin M (IgM) were used to confirm CHIKV diagnosis. Of a total of 850 patients who presented with probably CHIKV at Hospital "Dr. Roberto Nettel", 112 probable CHIKF cases were enrolled in this study from November 2014- June 2015, of which 95 patients (84.8%) were CHIKV positive and 17 were negative (15.2%). Of these 95 CHIKV positive patients, 62 were positive by real-time reverse transcriptase PCR (+qRT-PCR); and 33 were seropositive to +IgM with a negative qRT-PCR. The most frequent symptoms reported were fever (100%), headache (82.3%), polyarthralgia (72.1%), and exanthem (82.3%). Biological abnormalities observed during CHIKV infection were lymphopenia (41.1%), leukopenia (51.6%), elevated transaminases (30.5%-46.3%) and high LDH (46.3%) and CRP (60.0%). Clinical and biological data obtained from this study is providing more useful information for benchmarking purposes with outbreaks from different parts of the world and would be helpful for better patient care and treatment.

Crystal structures of HIV-1 nonnucleoside reverse transcriptase inhibitors: N-benzyl-4-methyl-benzimidazoles

NASA Astrophysics Data System (ADS)

Ziółkowska, Natasza E.; Michejda, Christopher J.; Bujacz, Grzegorz D.

2009-07-01

HIV-1 nonnucleoside reverse transcriptase inhibitors are potentially specific and effective drugs in AIDS therapy. The presence of two aromatic systems with an angled orientation in the molecule of the inhibitor is crucial for interactions with HIV-1 RT. The inhibitor drives like a wedge into the cluster of aromatic residues of RT HIV-1 and restrains the enzyme in a conformation that blocks the chemical step of nucleotide incorporation. Structural studies provide useful information for designing new, more active inhibitors. The crystal structures of four NNRTIs are presented here. The investigated compounds are derivatives of N-benzyl-4-methyl-benzimidazole with various aliphatic and aromatic substituents at carbon 2 positions and a 2,6-dihalogeno-substituted N-benzyl moiety. Structural data reported here show that the conformation of the investigated compounds is relatively rigid. Such feature is important for the nonnucleoside inhibitor binding to HIV-1 reverse transcriptase.

A Combination Microbicide Gel Protects Macaques Against Vaginal Simian Human Immunodeficiency Virus-Reverse Transcriptase Infection, But Only Partially Reduces Herpes Simplex Virus-2 Infection After a Single High-Dose Cochallenge

PubMed Central

Hsu, Mayla; Aravantinou, Meropi; Menon, Radhika; Seidor, Samantha; Goldman, Daniel; Kenney, Jessica; Derby, Nina; Gettie, Agegnehu; Blanchard, James; Piatak, Michael; Lifson, Jeffrey D.; Fernández-Romero, Jose A.; Zydowsky, Thomas M.

2014-01-01

Abstract Herpes simplex virus-2 (HSV-2) infection increases HIV susceptibility. We previously established a rhesus macaque model of vaginal HSV-2 preexposure followed by cochallenge with HSV-2 and simian/human immunodeficiency virus-reverse transcriptase (SHIV-RT). Using this model, we showed that a gel containing the nonnucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 in carrageenan (CG) reduced SHIV-RT infection. To evaluate the efficacy of new generation microbicides against both viruses, we first established dual infection after single vaginal cochallenge with SHIV-RT and HSV-2 in HSV-2-naive macaques. All animals (6/6) became HSV-2 infected, with 4/6 coinfected with SHIV-RT. In a control group cochallenged with SHIV-RT and UV-inactivated HSV-2, 2/4 became SHIV-RT infected, and none had detectable HSV-2. Low-level HSV-2-specific antibody and T cell responses were detected in some HSV-2-infected animals. To test a CG gel containing MIV-150 and zinc acetate (MZC), which provided naive animals full protection from SHIV-RT for at least 8 h, MZC (vs. CG) was applied daily for 14 days followed by cochallenge 8 h later. MZC prevented SHIV-RT infection (0/9 infected, p=0.04 vs. 3/6 in CG controls), but only reduced HSV-2 infection by 20% (6/9 infected vs. 5/6 in CG, p=0.6). In HSV-2-infected animals, none of the gel-treated animals seroconverted, and only the CG controls had measurable HSV-2-specific T cell responses. This study shows the promise of MZC to prevent immunodeficiency virus infection (even in the presence of HSV-2) and reduce HSV-2 infection after exposure to a high-dose inoculum. Additionally, it demonstrates the potential of a macaque coinfection model to evaluate broad-spectrum microbicides. PMID:24117013

A combination microbicide gel protects macaques against vaginal simian human immunodeficiency virus-reverse transcriptase infection, but only partially reduces herpes simplex virus-2 infection after a single high-dose cochallenge.

PubMed

Hsu, Mayla; Aravantinou, Meropi; Menon, Radhika; Seidor, Samantha; Goldman, Daniel; Kenney, Jessica; Derby, Nina; Gettie, Agegnehu; Blanchard, James; Piatak, Michael; Lifson, Jeffrey D; Fernández-Romero, Jose A; Zydowsky, Thomas M; Robbiani, Melissa

2014-02-01

Herpes simplex virus-2 (HSV-2) infection increases HIV susceptibility. We previously established a rhesus macaque model of vaginal HSV-2 preexposure followed by cochallenge with HSV-2 and simian/human immunodeficiency virus-reverse transcriptase (SHIV-RT). Using this model, we showed that a gel containing the nonnucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 in carrageenan (CG) reduced SHIV-RT infection. To evaluate the efficacy of new generation microbicides against both viruses, we first established dual infection after single vaginal cochallenge with SHIV-RT and HSV-2 in HSV-2-naive macaques. All animals (6/6) became HSV-2 infected, with 4/6 coinfected with SHIV-RT. In a control group cochallenged with SHIV-RT and UV-inactivated HSV-2, 2/4 became SHIV-RT infected, and none had detectable HSV-2. Low-level HSV-2-specific antibody and T cell responses were detected in some HSV-2-infected animals. To test a CG gel containing MIV-150 and zinc acetate (MZC), which provided naive animals full protection from SHIV-RT for at least 8 h, MZC (vs. CG) was applied daily for 14 days followed by cochallenge 8 h later. MZC prevented SHIV-RT infection (0/9 infected, p=0.04 vs. 3/6 in CG controls), but only reduced HSV-2 infection by 20% (6/9 infected vs. 5/6 in CG, p=0.6). In HSV-2-infected animals, none of the gel-treated animals seroconverted, and only the CG controls had measurable HSV-2-specific T cell responses. This study shows the promise of MZC to prevent immunodeficiency virus infection (even in the presence of HSV-2) and reduce HSV-2 infection after exposure to a high-dose inoculum. Additionally, it demonstrates the potential of a macaque coinfection model to evaluate broad-spectrum microbicides.

Soft shell clams Mya arenaria with disseminated neoplasia demonstrate reverse transcriptase activity

USGS Publications Warehouse

House, M.L.; Kim, C.H.; Reno, P.W.

1998-01-01

Disseminated neoplasia (DN), a proliferative cell disorder of the circulatory system of bivalves, was first reported in oysters in 1969. Since that time, the disease has been determined to be transmissible through water-borne exposure, but the etiological agent has not been unequivocally identified. In order to determine if a viral agent, possibly a retrovirus, could be the causative agent of DN, transmission experiments were performed, using both a cell-free filtrate and a sucrose gradient-purified preparation of a cell-free filtrate of DN positive materials. Additionally, a PCR-enhanced reverse transcriptase assay was used to determine if reverse transcriptase was present in tissues or hemolymph from DN positive soft shell clams Mya arenaria. DN was transmitted to healthy clams by injection with whole DN cells, but not with cell-free flitrates prepared from either tissues from DN positive clams, or DN cells. The cell-free preparations from DN-positive tissues and hemolymph having high levels of DN cells in circulation exhibited positive reactions in the PCR-enhanced reverse transcriptase assay. Cell-free preparations of hemolymph from clams having low levels of DN (<0.1% of cells abnormal), hemocytes from normal soft shell clams, and normal soft shell clam tissues did not produce a positive reaction in the PCR enhanced reverse transcriptase assay.

Evaluation of two singleplex reverse transcription-Insulated isothermal PCR tests and a duplex real-time RT-PCR test for the detection of porcine epidemic diarrhea virus and porcine deltacoronavirus.

PubMed

Zhang, Jianqiang; Tsai, Yun-Long; Lee, Pei-Yu Alison; Chen, Qi; Zhang, Yan; Chiang, Cheng-Jen; Shen, Yu-Han; Li, Fu-Chun; Chang, Hsiao-Fen Grace; Gauger, Phillip C; Harmon, Karen M; Wang, Hwa-Tang Thomas

2016-08-01

Recent outbreaks of porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) in multiple countries have caused significant economic losses and remain a serious challenge to the swine industry. Rapid diagnosis is critical for the implementation of efficient control strategies before and during PEDV and PDCoV outbreaks. Insulated isothermal PCR (iiPCR) on the portable POCKIT™ device is user friendly for on-site pathogen detection. In the present study, a singleplex PEDV RT-iiPCR, a singleplex PDCoV RT-iiPCR, and a duplex PEDV/PDCoV real-time RT-PCR (rRT-PCR) commercial reagents targeting the M gene were compared to an N gene-based PEDV rRT-PCR and an M gene-based PDCoV rRT-PCR that were previously published and used as reference PCRs. All PCR assays were highly specific and did not cross react with other porcine enteric pathogens. Analytical sensitivities of the PEDV RT-iiPCR, PDCoV RT-iiPCR and duplex PEDV/PDCoV rRT-PCR were determined using in vitro transcribed RNA as well as viral RNA extracted from ten-fold serial dilutions of PEDV and PDCoV cell culture isolates. Performance of each PCR assay was further evaluated using 170 clinical samples (86 fecal swabs, 24 feces, 19 intestines, and 41 oral fluids). Compared to the reference PEDV rRT-PCR, the sensitivity, specificity and accuracy of the PEDV RT-iiPCR were 97.73%, 98.78%, and 98.24%, respectively, and those of the duplex PEDV/PDCoV rRT-PCR were 98.86%, 96.34%, and 97.65%, respectively. Compared to the reference PDCoV rRT-PCR, the sensitivity, specificity and accuracy of the PDCoV RT-iiPCR were 100%, 100%, and 100%, respectively, and those of the PEDV/PDCoV duplex rRT-PCR were 96.34%, 100%, and 98.24%, respectively. Overall, all three new PCR assays were comparable to the reference rRT-PCRs for detection of PEDV and/or PDCoV. The PEDV and PDCoV RT-iiPCRs are potentially useful tools for on-site detection and the duplex PEDV/PDCoV rRT-PCR provides a convenient method to simultaneously detect the two viruses and differentiate PEDV from PDCoV. Copyright © 2016 Elsevier B.V. All rights reserved.

HuR interacts with human immunodeficiency virus type 1 reverse transcriptase, and modulates reverse transcription in infected cells

PubMed Central

Lemay, Julie; Maidou-Peindara, Priscilla; Bader, Thomas; Ennifar, Eric; Rain, Jean-Christophe; Benarous, Richard; Liu, Lang Xia

2008-01-01

Reverse transcription of the genetic material of human immunodeficiency virus type 1 (HIV-1) is a critical step in the replication cycle of this virus. This process, catalyzed by reverse transcriptase (RT), is well characterized at the biochemical level. However, in infected cells, reverse transcription occurs in a multiprotein complex – the reverse transcription complex (RTC) – consisting of viral genomic RNA associated with viral proteins (including RT) and, presumably, as yet uncharacterized cellular proteins. Very little is known about the cellular proteins interacting with the RTC, and with reverse transcriptase in particular. We report here that HIV-1 reverse transcription is affected by the levels of a nucleocytoplasmic shuttling protein – the RNA-binding protein HuR. A direct protein-protein interaction between RT and HuR was observed in a yeast two-hybrid screen and confirmed in vitro by homogenous time-resolved fluorescence (HTRF). We mapped the domain interacting with HuR to the RNAse H domain of RT, and the binding domain for RT to the C-terminus of HuR, partially overlapping the third RRM RNA-binding domain of HuR. HuR silencing with specific siRNAs greatly impaired early and late steps of reverse transcription, significantly inhibiting HIV-1 infection. Moreover, by mutagenesis and immunoprecipitation studies, we could not detect the binding of HuR to the viral RNA. These results suggest that HuR may be involved in and may modulate the reverse transcription reaction of HIV-1, by an as yet unknown mechanism involving a protein-protein interaction with HIV-1 RT. PMID:18544151

Novel Structure of Ty3 Reverse Transcriptase | Center for Cancer Research

Cancer.gov

Retrotransposons are mobile genetic elements that self amplify via a single-stranded RNA intermediate, which is converted to double-stranded DNA by an encoded reverse transcriptase (RT) with both DNA polymerase (pol) and ribonuclease H (RNase) activities. Categorized by whether they contain flanking long terminal repeat (LTR) sequences, retrotransposons play a critical role in

Evidence for retrovirus infections in green turtles Chelonia mydas from the Hawaiian islands

USGS Publications Warehouse

Casey, R.N.; Quackenbush, S.L.; Work, Thierry M.; Balazs, G.H.; Bowser, P.R.; Casey, J.W.

1997-01-01

Apparently normal Hawaiian green turtles Chelonia mydas and those displaying fibropapillomas were analyzed for infection by retroviruses. Strikingly, all samples were positive for polymerase enhanced reverse transcriptase (PERT) with levels high enough to quantitate by the conventional reverse transcriptase (RT) assay. However, samples of skin, even from asymptomatic turtles, were RT positive, although the levels of enzyme activity in healthy turtles hatched and raised in captivity were much lower than those observed in asymptomatic free-ranging turtles. Turtles with fibropapillomas displayed a broad range of reverse transcriptase activity. Skin and eye fibropapillomas and a heart tumor were further analyzed and shown to have reverse transcriptase activity that banded in a sucrose gradient at 1.17 g ml-1. The reverse transcriptase activity purified from the heart tumor displayed a temperature optimum of 37??C and showed a preference for Mn2+ over Mg2+. Sucrose gradient fractions of this sample displaying elevated reverse transcriptase activity contained primarily retrovitalsized particles with prominent envelope spikes, when negatively stained and examined by electron microscopy. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of gradient-purified virions revealed a conserved profile among 4 independent tumors and showed 7 prominent proteins having molecular weights of 116, 83, 51, 43, 40, 20 and 14 kDa. The data suggest that retroviral infections are widespread in Hawaiian green turtles and a comprehensive investigation is warranted to address the possibility that these agents cause green turtle fibropapillomatosis (GTFP).

Structure-Based Design of Novel Dihydroalkoxybenzyloxopyrimidine Derivatives as Potent Nonnucleoside Inhibitors of the Human Immunodeficiency Virus Reverse Transcriptase

PubMed Central

Sudbeck, Elise A.; Mao, Chen; Vig, Rakesh; Venkatachalam, T. K.; Tuel-Ahlgren, Lisa; Uckun, Fatih M.

1998-01-01

Two highly potent dihydroalkoxybenzyloxopyrimidine (DABO) derivatives targeting the nonnucleoside inhibitor (NNI) binding site of human immunodeficiency virus (HIV) reverse transcriptase (RT) have been designed based on the structure of the NNI binding pocket and tested for anti-HIV activity. Our lead DABO derivative, 5-isopropyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-one, elicited potent inhibitory activity against purified recombinant HIV RT and abrogated HIV replication in peripheral blood mononuclear cells at nanomolar concentrations (50% inhibitory concentration, <1 nM) but showed no detectable cytotoxicity at concentrations as high as 100 μM. PMID:9835518

Incorporation of deoxyribonucleotides and ribonucleotides by a dNTP-binding cleft mutated reverse transcriptase in hepatitis B virus core particles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Hee-Young; Kim, Hye-Young; Jung, Jaesung

2008-01-05

Our recent observation that hepatitis B virus (HBV) DNA polymerase (P) might initiate minus-strand DNA synthesis without primer [Kim et al., (2004) Virology 322, 22-30], raised a possibility that HBV P protein may have the potential to function as an RNA polymerase. Thus, we mutated Phe 436, a bulky amino acid with aromatic side chain, at the putative dNTP-binding cleft in reverse transcriptase (RT) domain of P protein to smaller amino acids (Gly or Val), and examined RNA polymerase activity. HBV core particles containing RT dNTP-binding cleft mutant P protein were able to incorporate {sup 32}P-ribonucleotides, but not HBV coremore » particles containing wild type (wt), priming-deficient mutant, or RT-deficient mutant P proteins. Since all the experiments were conducted with core particles isolated from transfected cells, our results indicate that the HBV RT mutant core particles containing RT dNTP-binding cleft mutant P protein could incorporate both deoxyribonucleotides and ribonucleotides in replicating systems.« less

HIV Resistance Prediction to Reverse Transcriptase Inhibitors: Focus on Open Data.

PubMed

Tarasova, Olga; Poroikov, Vladimir

2018-04-19

Research and development of new antiretroviral agents are in great demand due to issues with safety and efficacy of the antiretroviral drugs. HIV reverse transcriptase (RT) is an important target for HIV treatment. RT inhibitors targeting early stages of the virus-host interaction are of great interest for researchers. There are a lot of clinical and biochemical data on relationships between the occurring of the single point mutations and their combinations in the pol gene of HIV and resistance of the particular variants of HIV to nucleoside and non-nucleoside reverse transcriptase inhibitors. The experimental data stored in the databases of HIV sequences can be used for development of methods that are able to predict HIV resistance based on amino acid or nucleotide sequences. The data on HIV sequences resistance can be further used for (1) development of new antiretroviral agents with high potential for HIV inhibition and elimination and (2) optimization of antiretroviral therapy. In our communication, we focus on the data on the RT sequences and HIV resistance, which are available on the Internet. The experimental methods, which are applied to produce the data on HIV-1 resistance, the known data on their concordance, are also discussed.

Etravirine and Rilpivirine Drug Resistance Among HIV-1 Subtype C Infected Children Failing Non-Nucleoside Reverse Transcriptase Inhibitor-Based Regimens in South India.

PubMed

Saravanan, Shanmugam; Kausalya, Bagavathi; Gomathi, Selvamurthi; Sivamalar, Sathasivam; Pachamuthu, Balakrishnan; Selvamuthu, Poongulali; Pradeep, Amrose; Sunil, Solomon; Mothi, Sarvode N; Smith, Davey M; Kantor, Rami

2017-06-01

We have analyzed reverse transcriptase (RT) region of HIV-1 pol gene from 97 HIV-infected children who were identified as failing first-line therapy that included first-generation non-nucleoside RT inhibitors (Nevirapine and Efavirenz) for at least 6 months. We found that 54% and 65% of the children had genotypically predicted resistance to second-generation non-nucleoside RT inhibitors drugs Etravirine (ETR) and Rilpivirine, respectively. These cross-resistance mutations may compromise future NNRTI-based regimens, especially in resource-limited settings. To complement these investigations, we also analyzed the sequences in Stanford database, Monogram weighted score, and DUET weighted score algorithms for ETR susceptibility and found almost perfect agreement between the three algorithms in predicting ETR susceptibility from genotypic data.

Expression and characterization of a novel reverse transcriptase of the LTR retrotransposon Tf1.

PubMed

Kirshenboim, Noa; Hayouka, Zvi; Friedler, Assaf; Hizi, Amnon

2007-09-30

The LTR retrotransposon of Schizosacharomyces pombe, Tf1, has several distinctive properties that can be related to the unique properties of its reverse transcriptase (RT). Consequently, we expressed, purified and studied the recombinant Tf1 RT. This monomeric protein possesses all activities typical to RTs: DNA and RNA-dependent DNA polymerase as well as an inherent ribonuclease H. The DNA polymerase activity shows preference to Mn(+)(2) or Mg(+)(2), depending on the substrate used, whereas the ribonuclease H strongly prefers Mn(+)(2). The most outstanding feature of Tf1 RT is its capacity to add non-templated nucleotides to the 3'-ends of the nascent DNA. This is mainly apparent in the presence of Mn(+)(2), as is the noticeable low fidelity of DNA synthesis. In all, Tf1 RT has a marked infidelity in synthesizing DNA at template ends, a phenomenon that can explain, as discussed herein, some of the features of Tf1 replication in the host cells.

Discovery and partial characterization of a non-LTR retrotransposon that may be associated with abdominal segment deformity disease (ASDD) in the whiteleg shrimp Penaeus (Litopenaeus) vannamei.

PubMed

Sakaew, Waraporn; Pratoomthai, Benjamart; Pongtippatee, Pattira; Flegel, Timothy W; Withyachumnarnkul, Boonsirm

2013-09-30

Abdominal segment deformity disease (ASDD) of cultivated whiteleg shrimp Penaeus (Litopenaeus) vannamei causes economic loss of approximately 10% in affected specimens because of the unsightliness of distorted abdominal muscles. It is associated with the presence of viral-like particles seen by electron microscopy in the ventral nerve cords of affected shrimp. Thus, shotgun cloning was carried out to seek viral-like sequences in affected shrimp. A new retrovirus-like element of 5052 bp (named abdominal segment deformity element or ASDE) was compiled by shotgun cloning and 3' and 5' RACE using RNA and DNA extracted from ventral nerve cords of ASDD shrimp. ASDE contained 7 putative open reading frames (ORF). One ORF (called the PENS sub-domain), had a deduced amino acid (aa) sequence homologous to the GIY-YIG endonuclease domain of penelope-like retrotransposons while two others were homologous to the reverse transcriptase (RT) and RNaseH domains of the pol gene of non-long terminal repeat (non-LTR) retrotransposons (called the NLRS sub-domain). No single amplicon of 5 kb containing both these elements was obtained by PCR or RT-PCR from ASDD shrimp. Subsequent analysis indicated that PENS and NLRS were not contiguous and that NLRS was a host genetic element. In situ hybridization using a dioxygenin-labeled NLRS probe revealed that NLRS gave positive reactions in abdominal-ganglion neurons of ASDD shrimp but not normal shrimp. Preliminary analysis indicated that long-term use of female broodstock after eyestalk ablation in the hatchery increased the intensity of RT-PCR amplicons for NLRS and also the prevalence of ASDD in mysis 3 offspring of the broodstock. The deformities persist upon further cultivation until shrimp harvest but do not increase in prevalence and do not affect growth or survival. Our results suggested that NLRS is a shrimp genetic element associated with ASDD and that immediate preventative measures could include shorter-term use of broodstock after eyestalk ablation and/or discard of broodstock that give strong RT-PCR reactions for NLRS. In the longer term, it is recommended, if possible, that currently used, domesticated shrimp lines be selected for freedom from NLRS. The molecular tools developed in this work will facilitate the management and further study of ASDD.

Discovery and partial characterization of a non-LTR retrotransposon that may be associated with abdominal segment deformity disease (ASDD) in the whiteleg shrimp Penaeus (Litopenaeus) vannamei

PubMed Central

2013-01-01

Background Abdominal segment deformity disease (ASDD) of cultivated whiteleg shrimp Penaeus (Litopenaeus) vannamei causes economic loss of approximately 10% in affected specimens because of the unsightliness of distorted abdominal muscles. It is associated with the presence of viral-like particles seen by electron microscopy in the ventral nerve cords of affected shrimp. Thus, shotgun cloning was carried out to seek viral-like sequences in affected shrimp. Results A new retrovirus-like element of 5052 bp (named abdominal segment deformity element or ASDE) was compiled by shotgun cloning and 3′ and 5′ RACE using RNA and DNA extracted from ventral nerve cords of ASDD shrimp. ASDE contained 7 putative open reading frames (ORF). One ORF (called the PENS sub-domain), had a deduced amino acid (aa) sequence homologous to the GIY-YIG endonuclease domain of penelope-like retrotransposons while two others were homologous to the reverse transcriptase (RT) and RNaseH domains of the pol gene of non-long terminal repeat (non-LTR) retrotransposons (called the NLRS sub-domain). No single amplicon of 5 kb containing both these elements was obtained by PCR or RT-PCR from ASDD shrimp. Subsequent analysis indicated that PENS and NLRS were not contiguous and that NLRS was a host genetic element. In situ hybridization using a dioxygenin-labeled NLRS probe revealed that NLRS gave positive reactions in abdominal-ganglion neurons of ASDD shrimp but not normal shrimp. Preliminary analysis indicated that long-term use of female broodstock after eyestalk ablation in the hatchery increased the intensity of RT-PCR amplicons for NLRS and also the prevalence of ASDD in mysis 3 offspring of the broodstock. The deformities persist upon further cultivation until shrimp harvest but do not increase in prevalence and do not affect growth or survival. Conclusions Our results suggested that NLRS is a shrimp genetic element associated with ASDD and that immediate preventative measures could include shorter-term use of broodstock after eyestalk ablation and/or discard of broodstock that give strong RT-PCR reactions for NLRS. In the longer term, it is recommended, if possible, that currently used, domesticated shrimp lines be selected for freedom from NLRS. The molecular tools developed in this work will facilitate the management and further study of ASDD. PMID:24074037

Metagenomic analysis of the bioremediation of diesel-contaminated Canadian high arctic soils.

PubMed

Yergeau, Etienne; Sanschagrin, Sylvie; Beaumier, Danielle; Greer, Charles W

2012-01-01

As human activity in the Arctic increases, so does the risk of hydrocarbon pollution events. On site bioremediation of contaminated soil is the only feasible clean up solution in these remote areas, but degradation rates vary widely between bioremediation treatments. Most previous studies have focused on the feasibility of on site clean-up and very little attention has been given to the microbial and functional communities involved and their ecology. Here, we ask the question: which microorganisms and functional genes are abundant and active during hydrocarbon degradation at cold temperature? To answer this question, we sequenced the soil metagenome of an ongoing bioremediation project in Alert, Canada through a time course. We also used reverse-transcriptase real-time PCR (RT-qPCR) to quantify the expression of several hydrocarbon-degrading genes. Pseudomonas species appeared as the most abundant organisms in Alert soils right after contamination with diesel and excavation (t = 0) and one month after the start of the bioremediation treatment (t = 1m), when degradation rates were at their highest, but decreased after one year (t = 1y), when residual soil hydrocarbons were almost depleted. This trend was also reflected in hydrocarbon degrading genes, which were mainly affiliated with Gammaproteobacteria at t = 0 and t = 1m and with Alphaproteobacteria and Actinobacteria at t = 1y. RT-qPCR assays confirmed that Pseudomonas and Rhodococcus species actively expressed hydrocarbon degradation genes in Arctic biopile soils. Taken together, these results indicated that biopile treatment leads to major shifts in soil microbial communities, favoring aerobic bacteria that can degrade hydrocarbons.

Metagenomic Analysis of the Bioremediation of Diesel-Contaminated Canadian High Arctic Soils

PubMed Central

Yergeau, Etienne; Sanschagrin, Sylvie; Beaumier, Danielle; Greer, Charles W.

2012-01-01

As human activity in the Arctic increases, so does the risk of hydrocarbon pollution events. On site bioremediation of contaminated soil is the only feasible clean up solution in these remote areas, but degradation rates vary widely between bioremediation treatments. Most previous studies have focused on the feasibility of on site clean-up and very little attention has been given to the microbial and functional communities involved and their ecology. Here, we ask the question: which microorganisms and functional genes are abundant and active during hydrocarbon degradation at cold temperature? To answer this question, we sequenced the soil metagenome of an ongoing bioremediation project in Alert, Canada through a time course. We also used reverse-transcriptase real-time PCR (RT-qPCR) to quantify the expression of several hydrocarbon-degrading genes. Pseudomonas species appeared as the most abundant organisms in Alert soils right after contamination with diesel and excavation (t = 0) and one month after the start of the bioremediation treatment (t = 1m), when degradation rates were at their highest, but decreased after one year (t = 1y), when residual soil hydrocarbons were almost depleted. This trend was also reflected in hydrocarbon degrading genes, which were mainly affiliated with Gammaproteobacteria at t = 0 and t = 1m and with Alphaproteobacteria and Actinobacteria at t = 1y. RT-qPCR assays confirmed that Pseudomonas and Rhodococcus species actively expressed hydrocarbon degradation genes in Arctic biopile soils. Taken together, these results indicated that biopile treatment leads to major shifts in soil microbial communities, favoring aerobic bacteria that can degrade hydrocarbons. PMID:22253877

Investigation of Rotavirus with Various Methods in Children with Acute Gastroenteritis and Determination of Its Molecular Epidemiology in Kayseri Province, Turkey.

PubMed

Artiran, Sukran; Atalay, Altay; Gökahmetoglu, Selma; Ozturk, Mehmet Adnan; Balci, Nurgul; Cakir, Nuri; Kilic, Huseyin; Durmaz, Riza

2017-03-01

In this study, the fresh stool samples from 254 children under 5 years of age with acute gastroenteritis which were delivered between October 2012 and December 2013 were collected. In the stool samples, rotavirus antigens were investigated using two different immunochromatographic methods which are routinely used at different times, namely the RIDA ® QUICK Rotavirus/Adenovirus Combi Test (R-Biopharm AG, Germany) and the Genx ® Rotavirus Test (Diamed-Lab, Turkey), in addition to the Rotavirus Ag (Stool) ELISA (DRG, Germany) kit. The results were compared with reverse transcriptase PCR (RT-PCR). When the Genx ® Rotavirus Test and RIDA ® QUICK Rotavirus/Adenovirus Combi Test immunochromatographic methods were compared with RT-PCR, their sensitivity and specificity were found as 97.1%, 100%, and 80.4%, 72%, respectively. As to the Rotavirus Ag (Stool) ELISA method, on the other hand, its sensitivity was found to be 95.1% and its specificity was 86.5%. The most common genotype was G9P[8] (40%), which was followed by the G1P[8] (18.7%) and G3P[8] (9.6%) genotypes. Consequently, it was revealed that the sensitivity of ELISA and immunochromatographic methods, which provide results in a short time and are used in the investigation of rotavirus antigen, was high and their specificity was low; further studies to determine the distribution of G and P genotypes will contribute to establishing strategies for vaccine development for rotavirus in the world. © 2016 Wiley Periodicals, Inc.

Murine hepatocellular carcinoma derived stem cells reveal epithelial-to-mesenchymal plasticity.

PubMed

Jayachandran, Aparna; Shrestha, Ritu; Dhungel, Bijay; Huang, I-Tao; Vasconcelos, Marianna Yumi Kawashima; Morrison, Brian J; Ramlogan-Steel, Charmaine A; Steel, Jason C

2017-09-26

To establish a model to enrich and characterize stem-like cells from murine normal liver and hepatocellular carcinoma (HCC) cell lines and to further investigate stem-like cell association with epithelial-to-mesenchymal transition (EMT). In this study, we utilized a stem cell conditioned serum-free medium to enrich stem-like cells from mouse HCC and normal liver cell lines, Hepa 1-6 and AML12, respectively. We isolated the 3-dimensional spheres and assessed their stemness characteristics by evaluating the RNA levels of stemness genes and a cell surface stem cell marker by quantitative reverse transcriptase-PCR (qRT-PCR). Next, we examined the relationship between stem cells and EMT using qRT-PCR. Three-dimensional spheres were enriched by culturing murine HCC and normal hepatocyte cell lines in stem cell conditioned serum-free medium supplemented with epidermal growth factor, basic fibroblast growth factor and heparin sulfate. The 3-dimensional spheres had enhanced stemness markers such as Klf4 and Bmi1 and hepatic cancer stem cell (CSC) marker Cd44 compared to parental cells grown as adherent cultures. We report that epithelial markers E-cadherin and ZO-1 were downregulated, while mesenchymal markers Vimentin and Fibronectin were upregulated in 3-dimensional spheres. The 3-dimensional spheres also exhibited changes in expression of Snai , Zeb and Twist family of EMT transcription factors. Our novel method successfully enriched stem-like cells which possessed an EMT phenotype. The isolation and characterization of murine hepatic CSCs could establish a precise target for the development of more effective therapies for HCC.

Overexpression of TGF-β1 enhances chondrogenic differentiation and proliferation of human synovium-derived stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Yong Il; Ryu, Jae-Sung; Yeo, Jee Eun

2014-08-08

Highlights: • Continuous TGF-β1 overexpression in hSD-MSCs did not influence their phenotypes. • Retroviral-mediated transduction of TGFB1 in hSD-MSCs enhances cell proliferation. • TGF-β1 overexpression did not effect to adipo- or osteogenic potential of hSD-MSCs. • TGF-β1 overexpression in hSD-MSCs could stimulate and accelerate chondrogenesis. - Abstract: Transforming growth factor-beta (TGF-β) superfamily proteins play a critical role in proliferation, differentiation, and other functions of mesenchymal stem cells (MSCs). During chondrogenic differentiation of MSCs, TGF-β up-regulates chondrogenic gene expression by enhancing the expression of the transcription factor SRY (sex-determining region Y)-box9 (Sox9). In this study, we investigated the effect of continuousmore » TGF-β1 overexpression in human synovium-derived MSCs (hSD-MSCs) on immunophenotype, differentiation potential, and proliferation rate. hSD-MSCs were transduced with recombinant retroviruses (rRV) encoding TGF-β1. The results revealed that continuous overexpression of TGF-β1 did not affect their phenotype as evidenced by flow cytometry and reverse transcriptase PCR (RT-PCR). In addition, continuous TGF-β1 overexpression strongly enhanced cell proliferation of hSD-MSCs compared to the control groups. Also, induction of chondrogenesis was more effective in rRV-TGFB-transduced hSD-MSCs as shown by RT-PCR for chondrogenic markers, toluidine blue staining and glycosaminoglycan (GAG)/DNA ratio. Our data suggest that overexpression of TGF-β1 positively enhances the proliferation and chondrogenic potential of hSD-MSCs.« less

Inhibition of P-glycoprotein and glutathione S-transferase-pi mediated resistance by fluoxetine in MCF-7/ADM cells.

PubMed

Zhang, Ye; Zhou, Ting; Duan, Jingjing; Xiao, Zhijun; Li, Guihua; Xu, Feng

2013-10-01

Chemotherapy is important in the systematic treatment of breast cancer. While multidrug resistance (MDR) is the main obstacle in chemotherapy, a reversal reagent with high reversal effect but low toxicity is the hotspot issue at present to overcome MDR. Antidepressant fluoxetine (FLX) is a potential new highly effective chemosensitizer, however, the possible mechanism is unclear. In this study, the effect of FLX on multidrug resistance mediated by P-glycoprotein (P-gp) and glutathione S-transferase-pi (GST-π) were researched in resistant/sensitive breast cancer cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to determine the cells viability after being incubated with FLX/Adriamycin (ADM)/Paclitaxel (PTX) alone or FLX-ADM, FLX-PTX combination. Western blot was performed to assay the expression of P-gp and GST-π proteins. Reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR) were performed to assay the level of MDR1 mRNA. The results showed that pre-treatment with FLX enhance cytotoxicity significantly both on resistant and sensitive cells, downregulated the expression of P-gp and GST-π proteins in resistance cells, decreased the MDR1 mRNA by FLX-PTX combination only. No P-gp and GST-π were detected in sensitive cells. Our research thus indicated that FLX reverse the breast cancer cell's resistance and enhance the chemosensitivity by regulating P-gp and GST-π levels. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

High false-negative rate of anti-HCV among Egyptian patients on regular hemodialysis.

PubMed

El-Sherif, Assem; Elbahrawy, Ashraf; Aboelfotoh, Atef; Abdelkarim, Magdy; Saied Mohammad, Abdel-Gawad; Abdallah, Abdallah Mahmoud; Mostafa, Sadek; Elmestikawy, Amr; Elwassief, Ahmed; Salah, Mohamed; Abdelbaseer, Mohamed Ali; Abdelwahab, Kouka Saadeldin

2012-07-01

Routine serological testing for hepatitis C virus (HCV) infection among hemodialysis (HD) patients is currently recommended. A dilemma existed on the value of serology because some investigators reported a high rate of false-negative serologic testing. In this study, we aimed to detect the false-negative rate of anti-HCV among Egyptian HD patients. Seventy-eight HD patients, negative for anti-HCV, anti-HIV, and hepatitis B surface antigen, were tested for HCV RNA by reverse transcriptase polymerase chain reaction (RT-PCR). In the next step, the viral load was quantified by real-time PCR in RT-PCR-positive patients. Risk factors for HCV infection, as well as clinical and biochemical indicators of liver disease, were compared between false-negative and true-negative anti-HCV HD patients. The frequency of false-negative anti-HCV was 17.9%. Frequency of blood transfusion, duration of HD, dialysis at multiple centers, and diabetes mellitus were not identified as risk factors for HCV infection. The frequency of false-negative results had a linear relation to the prevalence of HCV infection in the HD units. Timely identification of HCV within dialysis units is needed in order to lower the risk of HCV spread within the HD units. The high false-negative rate of anti-HCV among HD patients in our study justifies testing of a large scale of patients for precious assessment of effectiveness of nucleic acid amplification technology testing in screening HD patient. © 2012 The Authors. Hemodialysis International © 2012 International Society for Hemodialysis.

Optimization of arsenic removal water treatment system through characterization of terminal electron accepting processes.

PubMed

Upadhyaya, Giridhar; Clancy, Tara M; Brown, Jess; Hayes, Kim F; Raskin, Lutgarde

2012-11-06

Terminal electron accepting process (TEAP) zones developed when a simulated groundwater containing dissolved oxygen (DO), nitrate, arsenate, and sulfate was treated in a fixed-bed bioreactor system consisting of two reactors (reactors A and B) in series. When the reactors were operated with an empty bed contact time (EBCT) of 20 min each, DO-, nitrate-, sulfate-, and arsenate-reducing TEAP zones were located within reactor A. As a consequence, sulfate reduction and subsequent arsenic removal through arsenic sulfide precipitation and/or arsenic adsorption on or coprecipitation with iron sulfides occurred in reactor A. This resulted in the removal of arsenic-laden solids during backwashing of reactor A. To minimize this by shifting the sulfate-reducing zone to reactor B, the EBCT of reactor A was sequentially lowered from 20 min to 15, 10, and 7 min. While 50 mg/L (0.81 mM) nitrate was completely removed at all EBCTs, more than 90% of 300 μg/L (4 μM) arsenic was removed with the total EBCT as low as 27 min. Sulfate- and arsenate-reducing bacteria were identified throughout the system through clone libraries and quantitative PCR targeting the 16S rRNA, dissimilatory (bi)sulfite reductase (dsrAB), and dissimilatory arsenate reductase (arrA) genes. Results of reverse transcriptase (RT) qPCR of partial dsrAB (i.e., dsrA) and arrA transcripts corresponded with system performance. The RT qPCR results indicated colocation of sulfate- and arsenate-reducing activities, in the presence of iron(II), suggesting their importance in arsenic removal.

Development of a one-step duplex RT-qPCR for the quantification of phocine distemper virus.

PubMed

Bogomolni, Andrea L; Frasca, Salvatore; Matassa, Keith A; Nielsen, Ole; Rogers, Kara; De Guise, Sylvain

2015-04-01

Worldwide, stranded marine mammals and the network personnel who respond to marine mammal mortality have provided much of the information regarding marine morbillivirus infections. An assay to determine the amount of virus present in tissue samples would be useful to assist in routine surveying of animal health and for monitoring large-scale die-off events. False negatives from poor-quality samples prevent determination of the true extent of infection, while only small amounts of tissue samples or archived RNA may be available at the time of collection for future retrospective analysis. We developed a one-step duplex real-time reverse transcriptase-quantitative-PCR assay (RT-qPCR) based on Taqman probe technology to quantify phocine distemper virus (PDV) isolated from an outbreak in harbor (Phoca vitulina concolor) and gray seals (Halichoerus grypus) along the northeast US coast in 2006. The glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) gene was selected to assess RNA quality. This duplex assay is specific for PDV and sensitive through a range of 10(0) to 10(9) copies ds-plasmid DNA. For the GAPDH target, the reaction in duplex amplified 10(0) to 10(9) copies of ds-plasmid DNA and was detectable in multiple seal species. This assay reduced the likelihood of false negative results due to degradation of tissues and well-to-well variability while providing sensitive and specific detection of PDV, which would be applicable in molecular epidemiologic studies and pathogen detection in field and laboratory investigations involving a variety of seal species.

Incidence of Enteric Viruses in Groundwater from Household Wells in Wisconsin

PubMed Central

Borchardt, Mark A.; Bertz, Phil D.; Spencer, Susan K.; Battigelli, David A.

2003-01-01

Recent studies on the contamination of groundwater with human enteric viruses have focused on public water systems, whereas little is known about the occurrence of viruses in private household wells. The objective of the present study was to estimate the incidence of viruses in Wisconsin household wells located near septage land application sites or in rural subdivisions served by septic systems. Fifty wells in seven hydrogeologic districts were sampled four times over a year, once each season. Reverse transcriptase PCR (RT-PCR), followed by Southern hybridization, was used to detect enteroviruses, rotavirus, hepatitis A virus (HAV), and Norwalk-like viruses (NLVs). In addition, cell culture was used to detect culturable enteroviruses. Companion water samples were collected for total coliforms, Escherichia coli, fecal enterococci, F-specific RNA coliphages, nitrate, and chloride analyses. Among the 50 wells, four (8%) were positive for viruses by RT-PCR. Three wells were positive for HAV, and the fourth well was positive for both rotavirus and NLV in one sample and an enterovirus in another sample. Contamination was transient, since none of the wells was virus positive for two sequential samples. Culturable enteroviruses were not detected in any of the wells. Water quality indicators were not statistically associated with virus occurrence, although some concordance was noted for chloride. The present study is the first in the United States to systematically monitor private household wells for virus contamination and, combined with data for public wells, provides further insight on the extent of groundwater contamination with human enteric viruses. PMID:12571044

Expression and distribution of the transient receptor potential cationic channel ankyrin 1 (TRPA1) in the human vagina.

PubMed

Ückert, S; Sonnenberg, J E; Albrecht, K; Kuczyk, M A; Hedlund, P

2015-01-01

The transient receptor potential cationic channel type A1 (TRPA1), belonging to a superfamily of cationic membrane channels, has been suggested to act as mechano- and pain sensor and, thus, to play a role in neurotransmission in the human body, including the urogenital tract. While the expression of TRPA1 has been investigated in a variety of tissues, up until today, no study has addressed the expression and distribution in the female genital tract. The present study aimed to investigate the expression and distribution of TRPA1 protein in human vaginal tissue. Reverse transcriptase PCR (RT-PCR) was applied in order to identify messenger ribonuleic acid specifically encoding for TRPA/A1. The distribution of TRPA1 in relation to the neuronal nitric oxide synthase (nNOS) and the signaling peptide calcitonin gene-related peptide (CGRP) was examined by means of immunohistochemical methods (double-antibody technique, laser fluorescence microscopy). RT-PCR analysis revealed the expression of mRNA encoding sequences specific for TRPA in the vaginal wall and epithelium. Immunostaining related to TRPA1 was observed in the basal epithelium and in slender varicose nerve fibers transversing the subepithelial and stromal space of the vaginal sections. In addition, these fibers presented immunoreactivity specific for nNOS or CGRP. The smooth musculature of the vaginal wall and small vessels interspersing the tissue did not present signals related to TRPA1. The findings indicate that TRPA1 might be involved in afferent neurotransmission in the vagina and work synergistically together with the nitric oxide/cyclic guanosine monophosphate pathway.

A comparative study of digital RT-PCR and RT-qPCR for quantification of Hepatitis A virus and Norovirus in lettuce and water samples.

PubMed

Coudray-Meunier, Coralie; Fraisse, Audrey; Martin-Latil, Sandra; Guillier, Laurent; Delannoy, Sabine; Fach, Patrick; Perelle, Sylvie

2015-05-18

Sensitive and quantitative detection of foodborne enteric viruses is classically achieved by quantitative RT-PCR (RT-qPCR). Recently, digital PCR (dPCR) was described as a novel approach to genome quantification without need for a standard curve. The performance of microfluidic digital RT-PCR (RT-dPCR) was compared to RT-qPCR for detecting the main viruses responsible for foodborne outbreaks (human Noroviruses (NoV) and Hepatitis A virus (HAV)) in spiked lettuce and bottled water. Two process controls (Mengovirus and Murine Norovirus) were used and external amplification controls (EAC) were added to examine inhibition of RT-qPCR and RT-dPCR. For detecting viral RNA and cDNA, the sensitivity of the RT-dPCR assays was either comparable to that of RT-qPCR (RNA of HAV, NoV GI, Mengovirus) or slightly (around 1 log10) decreased (NoV GII and MNV-1 RNA and of HAV, NoV GI, NoV GII cDNA). The number of genomic copies determined by dPCR was always from 0.4 to 1.7 log10 lower than the expected numbers of copies calculated by using the standard qPCR curve. Viral recoveries calculated by RT-dPCR were found to be significantly higher than by RT-qPCR for NoV GI, HAV and Mengovirus in water, and for NoV GII and HAV in lettuce samples. The RT-dPCR assay proved to be more tolerant to inhibitory substances present in lettuce samples. This absolute quantitation approach may be useful to standardize quantification of enteric viruses in bottled water and lettuce samples and may be extended to quantifying other human pathogens in food samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Regulation of expression and biochemical characterization of a beta-class carbonic anhydrase from the plant growth-promoting rhizobacterium, Azospirillum brasilense Sp7.

PubMed

Kaur, Simarjot; Mishra, Mukti Nath; Tripathi, Anil K

2009-10-01

Carbonic anhydrase (CA; [EC 4.2.1.1]) is a ubiquitous enzyme catalysing the reversible hydration of CO(2) to bicarbonate, a reaction that supports various biochemical and physiological functions. Genome analysis of Azospirillum brasilense, a nonphotosynthetic, nitrogen-fixing, rhizobacterium, revealed an ORF with homology to beta-class carbonic anhydrases (CAs). Biochemical characteristics of the beta-class CA of A. brasilense, analysed after cloning the gene (designated as bca), overexpressing in Escherichia coli and purifying the protein by affinity purification, revealed that the native recombinant enzyme is a homotetramer, inhibited by the known CA inhibitors. CA activity in A. brasilense cell extracts, reverse transcriptase (RT)-PCR and Western blot analyses showed that bca was constitutively expressed under aerobic conditions. Lower beta-galactosidase activity in A. brasilense cells harbouring bca promoter: lacZ fusion during the stationary phase or during growth on 3% CO(2) enriched air or at acidic pH indicated that the transcription of bca was downregulated by the stationary phase, elevated CO(2) levels and acidic pH conditions. These observations were also supported by RT-PCR analysis. Thus, beta-CA in A. brasilense seems to be required for scavenging CO(2) from the ambient air and the requirement of CO(2) hydration seems to be higher for the cultures growing exponentially at neutral to alkaline pH.

Characterization of tight junction proteins in cultured human urothelial cells.

PubMed

Rickard, Alice; Dorokhov, Nikolay; Ryerse, Jan; Klumpp, David J; McHowat, Jane

2008-01-01

Tight junctions (TJs) are essential for normal function of epithelia, restricting paracellular diffusion and contributing to the maintenance of cell surface polarity. Superficial cells of the urothelium develop TJs, the basis for the paracellular permeability barrier of the bladder against diffusion of urinary solutes. Focusing on the superficial cell layer of stratified cell cultures of an immortalized human ureteral cell line, TEU-2 cells, we have examined the presence of TJ and TJ-associated proteins. TEU-2 cells were treated with calcium chloride and fetal bovine serum culture conditions used to induce stratification that resembles the normal transitional epithelial phenotype. Cultures were examined for TJ and TJ-associated proteins by confocal immunofluorescence microscopy and evaluated for TJ mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR). TEU-2 cultures exhibited immunoreactivity at intercellular margins for claudins 1, 4, 5, 7, 14, and 16 whereas claudins 2, 8, and 12 were intracellular. RT-PCR corroborated the presence of these claudins at the mRNA level. The TJ-associated proteins occludin, JAM-1, and zonula occludens (ZO-1, ZO-2, and ZO-3) were localized at cell margins. We have found that numerous TJs and TJ-associated proteins are expressed in stratified TEU-2 cultures. Further, we propose TEU-2s provide a useful ureteral model for future studies on the involvement of TJs proteins in the normal and pathological physiology of the human urinary system.

Detection of bacteria in platelet concentrates prepared from spiked single donations using cultural and molecular genetic methods.

PubMed

Störmer, M; Cassens, U; Kleesiek, K; Dreier, J

2007-02-01

Bacteria show differences in their growth kinetics depending on the type of blood component. On to storage at 22 degrees C, platelet concentrates (PCs) seem to be more prone to bacterial multiplication than red cell concentrates. Knowledge of the potential for bacterial proliferation in blood components, which are stored at a range of temperatures, is essential before considering implementation of a detection strategy. The efficacy of bacterial detection was determined, using real-time reverse transcriptase-polymerase chain reaction (RT-PCR), following bacterial growth in blood components obtained from a deliberately contaminated whole-blood (WB) unit. Cultivation was used as the reference method. WB was spiked with 2 colony-forming units mL(-1)Staphylococcus epidermidis or Klebsiella pneumoniae, kept for 15 h at room temperature and component preparation was processed. Samples were drawn, at intervals throughout the whole separation process, from each blood component. Nucleic acids were extracted using an automated high-volume extraction method. The 15-h storage revealed an insignificant increase in bacterial titre. No bacterial growth was detected in red blood cell or plasma units. K. pneumoniae showed rapid growth in the pooled PC and could be detected immediately after preparation using RT-PCR. S. epidermidis grew slowly and was detected 24 h after separation. These experiments show that sampling is indicative at 24 h after preparation of PCs at the earliest to minimize the sampling error.

A Machine Learning Application Based in Random Forest for Integrating Mass Spectrometry-Based Metabolomic Data: A Simple Screening Method for Patients With Zika Virus

PubMed Central

Melo, Carlos Fernando Odir Rodrigues; Navarro, Luiz Claudio; de Oliveira, Diogo Noin; Guerreiro, Tatiane Melina; Lima, Estela de Oliveira; Delafiori, Jeany; Dabaja, Mohamed Ziad; Ribeiro, Marta da Silva; de Menezes, Maico; Rodrigues, Rafael Gustavo Martins; Morishita, Karen Noda; Esteves, Cibele Zanardi; de Amorim, Aline Lopes Lucas; Aoyagui, Caroline Tiemi; Parise, Pierina Lorencini; Milanez, Guilherme Paier; do Nascimento, Gabriela Mansano; Ribas Freitas, André Ricardo; Angerami, Rodrigo; Costa, Fábio Trindade Maranhão; Arns, Clarice Weis; Resende, Mariangela Ribeiro; Amaral, Eliana; Junior, Renato Passini; Ribeiro-do-Valle, Carolina C.; Milanez, Helaine; Moretti, Maria Luiza; Proenca-Modena, Jose Luiz; Avila, Sandra; Rocha, Anderson; Catharino, Rodrigo Ramos

2018-01-01

Recent Zika outbreaks in South America, accompanied by unexpectedly severe clinical complications have brought much interest in fast and reliable screening methods for ZIKV (Zika virus) identification. Reverse-transcriptase polymerase chain reaction (RT-PCR) is currently the method of choice to detect ZIKV in biological samples. This approach, nonetheless, demands a considerable amount of time and resources such as kits and reagents that, in endemic areas, may result in a substantial financial burden over affected individuals and health services veering away from RT-PCR analysis. This study presents a powerful combination of high-resolution mass spectrometry and a machine-learning prediction model for data analysis to assess the existence of ZIKV infection across a series of patients that bear similar symptomatic conditions, but not necessarily are infected with the disease. By using mass spectrometric data that are inputted with the developed decision-making algorithm, we were able to provide a set of features that work as a “fingerprint” for this specific pathophysiological condition, even after the acute phase of infection. Since both mass spectrometry and machine learning approaches are well-established and have largely utilized tools within their respective fields, this combination of methods emerges as a distinct alternative for clinical applications, providing a diagnostic screening—faster and more accurate—with improved cost-effectiveness when compared to existing technologies. PMID:29696139

Analysis of Acquisition and Titer of Maize Mosaic Rhabdovirus in Its Vector, Peregrinus maidis (Hemiptera: Delphacidae).

PubMed

Barandoc-Alviar, Karen; Ramirez, Girly M; Rotenberg, Dorith; Whitfield, Anna E

2016-01-01

The corn planthopper, Peregrinus maidis (Ashmead) (Hemiptera: Delphacidae), transmits Maize mosaic rhabdovirus (MMV), an important pathogen of maize and sorghum, in a persistent propagative manner. To better understand the vectorial capacity of P. maidis, we determined the efficiency of MMV acquisition by nymphal and adult stages, and characterized MMV titer through development. Acquisition efficiency, i.e., proportion of insects that acquired the virus, was determined by reverse transcriptase polymerase chain reaction (RT-PCR) and virus titer of individual insects was estimated by quantitative RT-PCR. Acquisition efficiency of MMV differed significantly between nymphs and adults. MMV titer increased significantly over time and throughout insect development from nymphal to adult stage, indication of virus replication in the vector during development. There was a positive association between the vector developmental stage and virus titer. Also, the average titer in male insects was threefold higher than female titers, and this difference persisted up to 30 d post adult eclosion. Overall, our findings indicate that nymphs are more efficient than adults at acquiring MMV and virus accumulated in the vector over the course of nymphal development. Furthermore, sustained infection over the lifespan of P. maidis indicates a potentially high capacity of this vector to transmit MMV. © The Author 2016. Published by Oxford University Press on behalf of the Entomological Society of America.

One-Step Reverse Transcription-Polymerase Chain Reaction for Ebola and Marburg Viruses.

PubMed

Park, Sun-Whan; Lee, Ye-Ji; Lee, Won-Ja; Jee, Youngmee; Choi, WooYoung

2016-06-01

Ebola and Marburg viruses (EBOVs and MARVs, respectively) are causative agents of severe hemorrhagic fever with high mortality rates in humans and nonhuman primates. In 2014, there was a major Ebola outbreak in various countries in West Africa, including Guinea, Liberia, Republic of Sierra Leone, and Nigeria. EBOV and MARV are clinically difficult to diagnose and distinguish from other African epidemic diseases. Therefore, in this study, we aimed to develop a method for rapid identification of the virus to prevent the spread of infection. We established a conventional one-step reverse transcription-polymerase chain reaction (RT-PCR) assay for these pathogens based on the Superscript Reverse Transcriptase-Platinum Taq polymerase enzyme mixture. All assays were thoroughly optimized using in vitro-transcribed RNA. We designed seven primer sets of nucleocapsid protein (NP) genes based on sequences from seven filoviruses, including five EBOVs and two MARVs. To evaluate the sensitivity of the RT-PCR assay for each filovirus, 10-fold serial dilutions of synthetic viral RNA transcripts of EBOV or MARV NP genes were used to assess detection limits of viral RNA copies. The potential for these primers to cross react with other filoviruses was also examined. The results showed that the primers were specific for individual genotype detection in the examined filoviruses. The assay established in this study may facilitate rapid, reliable laboratory diagnosis in suspected cases of Ebola and Marburg hemorrhagic fevers.

A Machine Learning Application Based in Random Forest for Integrating Mass Spectrometry-Based Metabolomic Data: A Simple Screening Method for Patients With Zika Virus.

PubMed

Melo, Carlos Fernando Odir Rodrigues; Navarro, Luiz Claudio; de Oliveira, Diogo Noin; Guerreiro, Tatiane Melina; Lima, Estela de Oliveira; Delafiori, Jeany; Dabaja, Mohamed Ziad; Ribeiro, Marta da Silva; de Menezes, Maico; Rodrigues, Rafael Gustavo Martins; Morishita, Karen Noda; Esteves, Cibele Zanardi; de Amorim, Aline Lopes Lucas; Aoyagui, Caroline Tiemi; Parise, Pierina Lorencini; Milanez, Guilherme Paier; do Nascimento, Gabriela Mansano; Ribas Freitas, André Ricardo; Angerami, Rodrigo; Costa, Fábio Trindade Maranhão; Arns, Clarice Weis; Resende, Mariangela Ribeiro; Amaral, Eliana; Junior, Renato Passini; Ribeiro-do-Valle, Carolina C; Milanez, Helaine; Moretti, Maria Luiza; Proenca-Modena, Jose Luiz; Avila, Sandra; Rocha, Anderson; Catharino, Rodrigo Ramos

2018-01-01

Recent Zika outbreaks in South America, accompanied by unexpectedly severe clinical complications have brought much interest in fast and reliable screening methods for ZIKV (Zika virus) identification. Reverse-transcriptase polymerase chain reaction (RT-PCR) is currently the method of choice to detect ZIKV in biological samples. This approach, nonetheless, demands a considerable amount of time and resources such as kits and reagents that, in endemic areas, may result in a substantial financial burden over affected individuals and health services veering away from RT-PCR analysis. This study presents a powerful combination of high-resolution mass spectrometry and a machine-learning prediction model for data analysis to assess the existence of ZIKV infection across a series of patients that bear similar symptomatic conditions, but not necessarily are infected with the disease. By using mass spectrometric data that are inputted with the developed decision-making algorithm, we were able to provide a set of features that work as a "fingerprint" for this specific pathophysiological condition, even after the acute phase of infection. Since both mass spectrometry and machine learning approaches are well-established and have largely utilized tools within their respective fields, this combination of methods emerges as a distinct alternative for clinical applications, providing a diagnostic screening-faster and more accurate-with improved cost-effectiveness when compared to existing technologies.

A distinct role of pectate lyases in the formation of feeding structures induced by cyst and root-knot nematodes.

PubMed

Wieczorek, K; Elashry, A; Quentin, M; Grundler, F M W; Favery, B; Seifert, G J; Bohlmann, H

2014-09-01

Pectin in the primary plant cell wall is thought to be responsible for its porosity, charge density, and microfibril spacing and is the main component of the middle lamella. Plant-parasitic nematodes secrete cell wall-degrading enzymes that macerate the plant tissue, facilitating the penetration and migration within the roots. In sedentary endoparasitic nematodes, these enzymes are released only during the migration of infective juveniles through the root. Later, nematodes manipulate the expression of host plant genes, including various cell wall enzymes, in order to induce specific feeding sites. In this study, we investigated expression of two Arabidopsis pectate lyase-like genes (PLL), PLL18 (At3g27400) and PLL19 (At4g24780), together with pectic epitopes with different degrees of methylesterification in both syncytia induced by the cyst nematode Heterodera schachtii and giant cells induced by the root-knot nematode Meloidogyne incognita. We confirmed upregulation of PLL18 and PLL19 in both types of feeding sites with quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and in situ RT-PCR. Furthermore, the functional analysis of mutants demonstrated the important role of both PLL genes in the development and maintenance of syncytia but not giant cells. Our results show that both enzymes play distinct roles in different infected root tissues as well as during parasitism of different nematodes.

Low-level lasers alter mRNA levels from traditional reference genes used in breast cancer cells

NASA Astrophysics Data System (ADS)

Teixeira, A. F.; Canuto, K. S.; Rodrigues, J. A.; Fonseca, A. S.; Mencalha, A. L.

2017-07-01

Cancer is among the leading causes of mortality worldwide, increasing the importance of treatment development. Low-level lasers are used in several diseases, but some concerns remains on cancers. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a technique used to understand cellular behavior through quantification of mRNA levels. Output data from target genes are commonly relative to a reference that cannot vary according to treatment. This study evaluated reference genes levels from MDA-MB-231 cells exposed to red or infrared lasers at different fluences. Cultures were exposed to red and infrared lasers, incubated (4 h, 37 °C), total RNA was extracted and cDNA synthesis was performed to evaluate mRNA levels from ACTB, GUSB and TRFC genes by RT-qPCR. Specific amplification was verified by melting curves and agarose gel electrophoresis. RefFinder enabled data analysis by geNorm, NormFinder and BestKeeper. Specific amplifications were obtained and, although mRNA levels from ACTB, GUSB or TRFC genes presented no significant variation through traditional statistical analysis, Excel-based tools revealed that the use of these reference genes are dependent of laser characteristics. Our data showed that exposure to low-level red and infrared lasers at different fluences alter the mRNA levels from ACTB, GUSB and TRFC in MDA-MB-231 cells.

Cloning and expression analysis of a novel G-protein-coupled receptor selectively expressed on granulocytes.

PubMed

Yousefi, S; Cooper, P R; Potter, S L; Mueck, B; Jarai, G

2001-06-01

The migration of neutrophils into sites of acute and chronic inflammation is mediated by chemokines. We used degenerate-primer reverse transcriptase-polymerase chain reaction (RT-PCR) to analyze chemokine receptor expression in neutrophils and identify novel receptors. RNA was isolated from human peripheral blood neutrophils and from neutrophils that had been stimulated for 5 h with granulocyte-macrophage colony-stimulating factor or by coculturing with primary human bronchial epithelial cells. Amplification products were cloned, and clone redundancy was determined. Seven known G-protein-coupled receptors were identified among 38 clones-CCR1, CCR4, CXCR1, CXCR2, CXCR4, HM63, and FPR1-as well as a novel gene, EX33. The full-length EX33 clone was obtained, and an in silico approach was used to identify the putative murine homologue. The EX33 gene encodes a 396-amino-acid protein with limited sequence identity to known receptors. Expression studies of several known chemokine receptors and EX33 revealed that resting neutrophils expressed higher levels of CXCRs and EX33 compared with activated neutrophils. Northern blot experiments revealed that EX33 is expressed mainly in bone marrow, lung, and peripheral blood leukocytes. Using RT-PCR analysis, we showed more abundant expression of EX33 in neutrophils and eosinophils, in comparison with that in T- or B-lymphocytes, indicating cell-specific expression among leukocytes.

Isolation and identification of highly pathogenic avian influenza virus subtype H5N1 in peafowl (Pavo cristatus).

PubMed

Ismail, Mahmoud Moussa; Khan, Owais Ahmed; Cattoli, Giovanni; Lu, Huaguang

2010-03-01

An outbreak of highly pathogenic avian influenza (HPAI) virus subtype H5N1 was first diagnosed in a "backyard" flock of peafowl (Pavo cristatus) raised on palace premises in the Kingdom of Saudi Arabia in December 3, 2007. The flock consisted of 40 peafowl, and their ages ranged from 3 to 5 years old. Affected birds suffered from depression, anorexia, and white diarrhea. Four dead birds were submitted for HPAI diagnosis at the Central Veterinary Diagnostic Laboratory in Riyadh. Brain and liver tissues and tracheal and cloacal swabs were taken from the dead birds and processed for a real-time reverse transcriptase (RT)-PCR test and virus isolation in specific-pathogen-free embryonating chicken eggs. The H5N1 subtype of avian influenza virus was isolated from the four dead birds and identified by a real-time RT-PCR before and after egg inoculation. The virus isolates were characterized as HPAI H5N1 virus by sequencing analysis. Phylogenetic comparisons revealed that the H5N1 viruses isolated from peafowl belong to the genetic clade 2.2 according to the World Health Organization nomenclature. The peafowl H5N1 virus falls into 2.2.2 sublineage II and clusters with the H5N1 viruses isolated from poultry in Saudi Arabia in 2007-08.

Respiratory syncytial virus outbreak in the basic military training cAMP of the republic of Korea Air Force.

PubMed

Park, Won-Ju; Yoo, Seok-Ju; Lee, Suk-Ho; Chung, Jae-Woo; Jang, Keun-Ho; Moon, Jai-Dong

2015-01-01

An outbreak of acute febrile illness occurred in the Republic of Korea Air Force boot camp from May to July 2011. An epidemiological investigation of the causative agent, which was of a highly infective nature, was conducted. Throat swabs were carried out and a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) assay was performed to identify possible causative factors. The mean age of patients who had febrile illness during the study period was 20.24 years. The multiplex RT-PCR assay identified respiratory syncytial virus (RSV) as the causative agent. The main symptoms were sore throat (76.0%), sputum (72.8%), cough (72.1%), tonsillar hypertrophy (67.9%), and rhinorrhea (55.9%). The mean temperature was 38.75°C and the attack rate among the recruits was 15.7% (588 out of 3750 recruits), while the mean duration of fever was 2.3 days. The prognosis was generally favorable with supportive care but recurrent fever occurred in 10.1% of the patients within a month. This is the first epidemiological study of an RSV outbreak that developed in a healthy young adult group. In the event of an outbreak of an acute febrile illness of a highly infective nature in facilities used by a young adult group, RSV should be considered among the possible causative agents.

RITA inhibits growth of human hepatocellular carcinoma through induction of apoptosis.

PubMed

Wang, Haihe; Chen, Guofu; Wang, Hongzhi; Liu, Chunbo

2013-01-01

RBP-J-interacting and tubulin-associated (RITA) is a novel RBP-J-interacting protein that downregulates Notch-mediated transcription. The current study focuses on the antitumor effect of RITA in human hepatocellular carcinoma (HCC) and aims to explore its molecular mechanism. Thirty paired HCC and adjacent non-tumoral liver samples were analyzed by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). RITA overexpression was induced by transfection of a pcDNA3.1-Flag-RITA plasmid into HepG2 cells. RITA knockdown was achieved by siRNA transfection. mRNA and protein expression of target genes were quantified by qRT-PCR and Western blotting, respectively. Cell proliferation and apoptosis were measured using MTT assay and flow cytometry. Our results demonstrate that adjacent nontumoral liver samples exhibited increased RITA expression compared to HCC tissues (p < 0.05); RITA levels were associated with tumor differentiation status. Overexpression of RITA suppressed cell proliferation and promoted early apoptosis, while its silencing promoted cell growth dramatically (p < 0.05). RITA overexpression upregulated p53 and reduced cyclin E levels, whereas silencing of RITA had the opposite effect on p53 and cyclin E expression. Our in vitro results represent the first evidence that RITA might suppress tumor growth and induce apoptosis in HCCs, and may be a potent antitumoral agent for HCC treatment that deserves further exploration.

Local Synthesis of Pepsin in Barrett's Esophagus and the Role of Pepsin in Esophageal Adenocarcinoma.

PubMed

Samuels, Tina; Hoekzema, Craig; Gould, Jon; Goldblatt, Matthew; Frelich, Matthew; Bosler, Matthew; Lee, Sang-Hyuk; Johnston, Nikki

2015-11-01

Despite widespread use of proton pump inhibitors (PPIs), the incidence of esophageal adenocarcinoma (EAC) continues to rise. PPIs reduce reflux acidity, but only transiently inactivate gastric enzymes. Nonacid reflux, specifically nonacid pepsin, contributes to carcinogenesis in the larynx. Given the carcinogenic potential of pepsin and inefficacy of PPIs to prevent EAC, the presence and effect of pepsin in the esophagus should be investigated. Normal and Barrett's biopsies from 8 Barrett's esophagus patients were collected for pepsin analysis via Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR). Human esophageal cells cultured from healthy patients were treated with pepsin (0.01-1 mg/mL; 1-20 hours), acid (pH 4)±pepsin (5 minutes); real-time RT-PCR, ELISA, and cell migration were assayed. Pepsin was detected in all 8 Barrett's and 4 of 8 adjacent normal specimens. Pepsinogen mRNA was observed in 22 Barrett's, but not in normal adjacent samples. Pepsin induced PTSG2 (COX-2) and IL-1β expression and cell migration in vitro. Pepsin is synthesized by metaplastic, Barrett's esophageal mucosa. Nonacid pepsin increases metrics of tumorigenicity in esophageal epithelial cells in vitro. These findings implicate refluxed and locally synthesized pepsin in development and progression of EAC and, in part, explain the inefficacy of PPIs in the prevention of EAC. © The Author(s) 2015.

Gene expression profile of human Down syndrome leukocytes.

PubMed

Malagó, Wilson; Sommer, César A; Del Cistia Andrade, Camillo; Soares-Costa, Andrea; Abrao Possik, Patricia; Cassago, Alexandre; Santejo Silveira, Henrique C; Henrique-Silva, Flavio

2005-08-01

Identification of differences in the gene expression patterns of Down syndrome and normal leukocytes. We constructed the first Down syndrome leukocyte serial analysis of gene expression (SAGE) library from a 28 year-old patient. This library was analyzed and compared with a normal leukocyte SAGE library using the eSAGE software. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to validate the results. We found that a large number of unidentified transcripts were overexpressed in Down syndrome leukocytes and some transcripts coding for growth factors (e.g. interleukin 8, IL-8), ribosomaproteins (e.g. L13a, L29, and L37), and transcription factors (e.g., Jun B, Jun D, and C/EBP beta) were underexpressed. The SAGE data were successfully validated for the genes IL-8, CXCR4, BCL2A1, L13a, L29, L37, and GTF3A using RT-PCR. Our analysis identified significant changes in the expression pattern of Down syndrome leukocytes compared with normal ones, including key regulators of growth and proliferation, ribosomal proteins, and a large number of overexpressed transcripts that were not matched in UniGene clusters and that may represent novel genes related to Down syndrome. This study offers a new insight into transcriptional changes in Down syndrome leukocytes and indicates candidate genes for further investigations into the molecular mechanism of Down syndrome pathology.

Etiology of Influenza-Like Illnesses from Sentinel Network Practitioners in Réunion Island, 2011-2012

PubMed Central

Brottet, Elise; Jaffar-Bandjee, Marie-Christine; Li-Pat-Yuen, Ghislaine; Filleul, Laurent

2016-01-01

In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. PMID:27654509

Expression of c-Jun and Bcl-2 family proteins in apoptotic photoreceptors of RCS rats.

PubMed

Katai, Naomichi; Yanagidaira, Tomoko; Senda, Nami; Murata, Toshinori; Yoshimura, Nagahisa

2006-01-01

To determine if c-Jun and Bcl-2 family proteins play a role in photoreceptor apoptosis in Royal College of Surgeons (RCS) rats. RCS and Sprague-Dawley rats were used. Cryosections of retinas harvested at various postnatal periods were immunostained with antibodies against c-Jun, Bcl-2, and Bax. Double staining with TdT-dUTP nick-end labeling (TUNEL) or propidium iodide (PI) and antibodies was also done. To study the time course of gene and protein expression, semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting analyses were carried out. TUNEL-positive photoreceptors of RCS rats were stained strongly with antibodies against c-Jun and Bax. The number of immunoreactive cells increased on days 21 and 28 after birth (P21 and P28) and decreased on P45. Semiquantitative RT-PCR analysis showed that mRNAs for c-Jun and Bax were upregulated at P21 and P28, but those for Bcl-2 were unchanged. On immunoblotting, a 43-kDa band was revealed by the anti-c-Jun antibody and a 21-kDa band, by the anti-Bax antibody. Protein expression of c-Jun and Bax were increased at both P21 and P28. The temporal profiles of immunoreactivity, protein expression, and mRNA expression were similar. c-Jun and Bax may play a role in photoreceptor apoptosis in RCS rats.

F4-related mutation and expression analysis of the aminopeptidase N gene in pigs.

PubMed

Goetstouwers, T; Van Poucke, M; Nguyen, V U; Melkebeek, V; Coddens, A; Deforce, D; Cox, E; Peelman, L J

2014-05-01

Intestinal infections with F4 enterotoxigenic Escherichia coli (ETEC) are worldwide an important cause of diarrhea in neonatal and recently weaned pigs. Adherence of F4 ETEC to the small intestine by binding to specific receptors is mediated by F4 fimbriae. Porcine aminopeptidase N (ANPEP) was recently identified as a new F4 receptor. In this study, 7 coding mutations and 1 mutation in the 3' untranslated region (3' UTR)were identified in ANPEP by reverse transcriptase (RT-) PCR and sequencing using 3 F4 receptor-positive (F4R+) and 2 F4 receptor-negative (F4R-) pigs, which were F4 phenotyped based on the MUC4 TaqMan, oral immunization, and the in vitro villous adhesion assay. Three potential differential mutations (g.2615C > T, g.8214A > G, and g.16875C > G) identified by comparative analysis between the 3 F4R+ and 2 F4R- pigs were genotyped in 41 additional F4 phenotyped pigs. However, none of these 3 mutations could be associated with F4 ETEC susceptibility. In addition, the RT-PCR experiments did not reveal any differential expression or alternative splicing in the small intestine of F4R+ and F4R- pigs. In conclusion, we hypothesize that the difference in F4 binding to ANPEP is due to modifications in its carbohydrate moieties.

Opposing actions of Arx and Pax4 in endocrine pancreas development

PubMed Central

Collombat, Patrick; Mansouri, Ahmed; Hecksher-Sørensen, Jacob; Serup, Palle; Krull, Jens; Gradwohl, Gerard; Gruss, Peter

2003-01-01

Genes encoding homeodomain-containing proteins potentially involved in endocrine pancreas development were isolated by combined in silico and nested-PCR approaches. One such transcription factor, Arx, exhibits Ngn3-dependent expression throughout endocrine pancreas development in α, β-precursor, and δ cells. We have used gene targeting in mouse embryonic stem cells to generate Arx loss-of-function mice. Arx-deficient animals are born at the expected Mendelian frequency, but develop early-onset hypoglycemia, dehydration, and weakness, and die 2 d after birth. Immunohistological analysis of pancreas from Arx mutants reveals an early-onset loss of mature endocrine α cells with a concomitant increase in β-and δ-cell numbers, whereas islet morphology remains intact. Our study indicates a requirement of Arx for α-cell fate acquisition and a repressive action on β-and δ-cell destiny, which is exactly the opposite of the action of Pax4 in endocrine commitment. Using multiplex reverse transcriptase PCR (RT-PCR), we demonstrate an accumulation of Pax4 and Arx transcripts in Arx and Pax4 mutant mice, respectively. We propose that the antagonistic functions of Arx and Pax4 for proper islet cell specification are related to the pancreatic levels of the respective transcripts. PMID:14561778

Opposing actions of Arx and Pax4 in endocrine pancreas development.

PubMed

Collombat, Patrick; Mansouri, Ahmed; Hecksher-Sorensen, Jacob; Serup, Palle; Krull, Jens; Gradwohl, Gerard; Gruss, Peter

2003-10-15

Genes encoding homeodomain-containing proteins potentially involved in endocrine pancreas development were isolated by combined in silico and nested-PCR approaches. One such transcription factor, Arx, exhibits Ngn3-dependent expression throughout endocrine pancreas development in alpha, beta-precursor, and delta cells. We have used gene targeting in mouse embryonic stem cells to generate Arx loss-of-function mice. Arx-deficient animals are born at the expected Mendelian frequency, but develop early-onset hypoglycemia, dehydration, and weakness, and die 2 d after birth. Immunohistological analysis of pancreas from Arx mutants reveals an early-onset loss of mature endocrine alpha cells with a concomitant increase in beta-and delta-cell numbers, whereas islet morphology remains intact. Our study indicates a requirement of Arx for alpha-cell fate acquisition and a repressive action on beta-and delta-cell destiny, which is exactly the opposite of the action of Pax4 in endocrine commitment. Using multiplex reverse transcriptase PCR (RT-PCR), we demonstrate an accumulation of Pax4 and Arx transcripts in Arx and Pax4 mutant mice, respectively. We propose that the antagonistic functions of Arx and Pax4 for proper islet cell specification are related to the pancreatic levels of the respective transcripts.

The Expression and Significance of Feces Cyclooxygensae-2 mRNA in Colorectal Cancer and Colorectal Adenomas

PubMed Central

Li, Xiaofeng; Kong, Lixia; Liao, Suhuan; Lu, Jing; Ma, Lin; Long, Xiaohua

2017-01-01

Background/Aim: This study aims to explore the expression and significance of feces cyclooxygensae-2 (COX-2) mRNA in colorectal cancer and colorectal adenomas. Materials and Methods: The expression of feces COX-2 mRNA in colorectal cancer (n = 28), colorectal adenomas (n = 54), and normal control group (n = 11) were examined by reverse transcriptase polymerase chain reaction (RT-PCR). The positive rate of fecal occult blood test (FOBT) were detected in colorectal cancer (n = 30), colorectal adenomas (n = 56), and normal control group (n = 11); the sensitivity of the two methods was also compared. Results: The positive rate of feces COX-2 mRNA in colorectal cancer was 82.1% (25/28), which was significantly higher than colorectal adenomas 59.3% (32/54), and normal tissues 18.2% (2/11), the difference being significant between the three groups (χ2= 13.842, P = 0.001). The positive rate of FOBT in colorectal cancer was 73.3% (10/30), which was significantly higher than colorectal adenomas 10.7% (6/56) and normal tissues 9.1% (1/11), the difference being significant between these three groups (χ2= 7.525, P = 0.023). There was no significant association between feces COX-2 expression and various clinical pathological features of colorectal cancer and colorectal adenomas (P > 0.05). The sensitivity of the RT-PCR method is higher than FOBT, however, the specificity of FOBT is slightly higher than RT-PCR. Conclusions: High expression of feces COX-2 mRNA in colorectal adenomas and colorectal cancer is a common event; it is an early event in the development of colorectal adenomas to colorectal cancer. Feces COX-2 mRNA has a high sensitivity for detect colorectal cancer; combination with FOBT will be the best alternative. Feces COX-2 can be potentially used in the early diagnosis and screening of colorectal cancer. PMID:28139497

The expression and significance of feces cyclooxygensae-2 mRNA in colorectal cancer and colorectal adenomas.

PubMed

Li, Xiaofeng; Kong, Lixia; Liao, Suhuan; Lu, Jing; Ma, Lin; Long, Xiaohua

2017-01-01

This study aims to explore the expression and significance of feces cyclooxygensae-2 (COX-2) mRNA in colorectal cancer and colorectal adenomas. The expression of feces COX-2 mRNA in colorectal cancer (n = 28), colorectal adenomas (n = 54), and normal control group (n = 11) were examined by reverse transcriptase polymerase chain reaction (RT-PCR). The positive rate of fecal occult blood test (FOBT) were detected in colorectal cancer (n = 30), colorectal adenomas (n = 56), and normal control group (n = 11); the sensitivity of the two methods was also compared. The positive rate of feces COX-2 mRNA in colorectal cancer was 82.1% (25/28), which was significantly higher than colorectal adenomas 59.3% (32/54), and normal tissues 18.2% (2/11), the difference being significant between the three groups (χ2= 13.842,P= 0.001). The positive rate of FOBT in colorectal cancer was 73.3% (10/30), which was significantly higher than colorectal adenomas 10.7% (6/56) and normal tissues 9.1% (1/11), the difference being significant between these three groups (χ2= 7.525,P= 0.023). There was no significant association between feces COX-2 expression and various clinical pathological features of colorectal cancer and colorectal adenomas (P > 0.05). The sensitivity of the RT-PCR method is higher than FOBT, however, the specificity of FOBT is slightly higher than RT-PCR. High expression of feces COX-2 mRNA in colorectal adenomas and colorectal cancer is a common event; it is an early event in the development of colorectal adenomas to colorectal cancer. Feces COX-2 mRNA has a high sensitivity for detect colorectal cancer; combination with FOBT will be the best alternative. Feces COX-2 can be potentially used in the early diagnosis and screening of colorectal cancer.

Zika virus epidemic in Brazil. I. Fatal disease in adults: Clinical andlaboratorial aspects

PubMed Central

Azevedo, Raimunda S.S.; Araujo, Marialva T.; Martins Filho, Arnaldo J.; Oliveira, Consuelo S.; Nunes, Bruno T.D.; Cruz, Ana C.R.; Nascimento, Ana G.P.A.C.; Medeiros, Rita C.; Caldas, Cezar A.M.; Araujo, Fernando C.; Quaresma, Juarez A.S.; Vasconcelos, Barbara C.B.; Queiroz, Maria G.L.; Travassos da Rosa, Elizabeth S.; Henriques, Daniele F.; Silva, Eliana V.P.; Chiang, Jannifer O.; Martins, Lívia C.; Medeiros, Daniele B.A.; Lima, Juliana A.; Nunes, Márcio R.T.; Cardoso, Jedson F.; Silva, Sandro P.; Shi, Pei-Yong; Tesh, Robert B.; Rodrigues, Sueli G.; Vasconcelos, Pedro F.C.

2017-01-01

Background Zika virus (ZIKV) was first detected in Brazil in May 2015 and the country experienced an explosive epidemic. However, recent studies indicate that the introduction of ZIKV occurred in late 2013. Cases of microcephaly and deaths associated with ZIKV infection were identified in Brazil in November, 2015. Objectives To determine the etiology of three fatal adult cases. Study design Here we report three fatal adult cases of ZIKV disease. ZIKV infection in these patients was confirmed by cells culture and/or real-time reverse transcriptase polymerase chain reaction (RT-qPCR) and by antigen detection using immunohistochemical assay. Samples of brain and other selected organs taken at autopsy from three patients were also analyzed by histopathological and immunohistological examination. Results The first patient, a 36-year-old man with lupus and receiving prednisone therapy, developed a fulminant ZIKV infection. At autopsy, RT-qPCR of blood and tissues was positive for ZIKV RNA, and the virus was cultured from an organ homogenate. The second patient, a previously healthy female, 16 years of age, presented classic symptoms of Zika fever, but later developed severe thrombocytopenia, anemia and hemorrhagic manifestations and died. A blood sample taken on the seventh day of her illness was positive RT-PCR for ZIKV RNA and research in the serum was positive for antinuclear factor fine speckled (1/640), suggesting Evans syndrome (hemolytic anemia an autoimmune disorder with immune thrombocytopenic purpura) secondary to ZIKV infection. The third patient was a 20-year-old woman hospitalized with fever, pneumonia and hemorrhages, who died on 13 days after admission. Histopathological changes were observed in all viscera examined. ZIKV antigens were detected by immunohistochemistry in viscera specimens of patients 1 and 3. These three cases demonstrate other potential complications of ZIKV infection, in addition to microcephaly and Guillain-Barre syndrome (GBS), and they suggest that individuals with immune suppression and/or autoimmune disorders may be at higher risk of developing severe disease, if infected with ZIKV. PMID:27835759

A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer.

PubMed

Paik, Soonmyung; Shak, Steven; Tang, Gong; Kim, Chungyeul; Baker, Joffre; Cronin, Maureen; Baehner, Frederick L; Walker, Michael G; Watson, Drew; Park, Taesung; Hiller, William; Fisher, Edwin R; Wickerham, D Lawrence; Bryant, John; Wolmark, Norman

2004-12-30

The likelihood of distant recurrence in patients with breast cancer who have no involved lymph nodes and estrogen-receptor-positive tumors is poorly defined by clinical and histopathological measures. We tested whether the results of a reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assay of 21 prospectively selected genes in paraffin-embedded tumor tissue would correlate with the likelihood of distant recurrence in patients with node-negative, tamoxifen-treated breast cancer who were enrolled in the National Surgical Adjuvant Breast and Bowel Project clinical trial B-14. The levels of expression of 16 cancer-related genes and 5 reference genes were used in a prospectively defined algorithm to calculate a recurrence score and to determine a risk group (low, intermediate, or high) for each patient. Adequate RT-PCR profiles were obtained in 668 of 675 tumor blocks. The proportions of patients categorized as having a low, intermediate, or high risk by the RT-PCR assay were 51, 22, and 27 percent, respectively. The Kaplan-Meier estimates of the rates of distant recurrence at 10 years in the low-risk, intermediate-risk, and high-risk groups were 6.8 percent (95 percent confidence interval, 4.0 to 9.6), 14.3 percent (95 percent confidence interval, 8.3 to 20.3), and 30.5 percent (95 percent confidence interval, 23.6 to 37.4). The rate in the low-risk group was significantly lower than that in the high-risk group (P<0.001). In a multivariate Cox model, the recurrence score provided significant predictive power that was independent of age and tumor size (P<0.001). The recurrence score was also predictive of overall survival (P<0.001) and could be used as a continuous function to predict distant recurrence in individual patients. The recurrence score has been validated as quantifying the likelihood of distant recurrence in tamoxifen-treated patients with node-negative, estrogen-receptor-positive breast cancer. Copyright 2004 Massachusetts Medical Society.

[Investigation of West Nile virus RNA in blood donors by real-time RT-PCR].

PubMed

Sahiner, Fatih; Avcı, Ismail Yaşar; Bedir, Orhan; Koru, Ozgür; Sener, Kenan; Yapar, Mehmet; Kubar, Ayhan

2012-07-01

West Nile virus (WNV), a member of Flaviviridae family, is an enveloped, icosahedral symmetric RNA virus. Primary reservoir hosts of WNV are birds, but the virus can cause various infections in humans and other mammals. The most common and natural transmission way of WNV infections is mosquito bites, however, humans can be infected by different routes. The most important non-mosquito transmission route is contaminated blood and blood products. In this study, we aimed to investigate the risk of WNV transmission through blood and blood products in Ankara, Turkey. The presence of WNV RNA was investigated by in house real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in serum samples obtained from 729 healthy blood donors (mean age: 27.7 years; 711 were male), regardless of the donor's seropositivity status since the virus can be transmitted at the early stages of infection when seroconversion has not yet developed. Serum samples were collected in August-September 2009, the period when these infections are more frequent due to mosquito activity. The vast majority of donors (n= 702, 96.3%) have been inhabiting in Ankara and 569 (78%) of donors have had risk factors for arboviral infections (e.g. outdoor activity, mosquito and tick bites). WNV RNA was not detected by real-time RT-PCR analysis in any serum sample included in this study. According to the results of our study, it can be said that the risk of WNV transmission through blood and blood products is low in Ankara. However, WNV seropositivity was detected within the range of 0.56 to 2.4% among blood donors in previous studies and probable and confirmed WNV infections have been reported in our region. In addition, WNV outbreaks have emerged in some countries neighbouring Turkey recently. Thus, the risk of WNV transmission through blood and blood products should not be ignored and blood donor questionnaires should be evaluated in detail.

Selection of reference genes for transcriptional analysis of edible tubers of potato (Solanum tuberosum L.).

PubMed

Mariot, Roberta Fogliatto; de Oliveira, Luisa Abruzzi; Voorhuijzen, Marleen M; Staats, Martijn; Hutten, Ronald C B; Van Dijk, Jeroen P; Kok, Esther; Frazzon, Jeverson

2015-01-01

Potato (Solanum tuberosum) yield has increased dramatically over the last 50 years and this has been achieved by a combination of improved agronomy and biotechnology efforts. Gene studies are taking place to improve new qualities and develop new cultivars. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a bench-marking analytical tool for gene expression analysis, but its accuracy is highly dependent on a reliable normalization strategy of an invariant reference genes. For this reason, the goal of this work was to select and validate reference genes for transcriptional analysis of edible tubers of potato. To do so, RT-qPCR primers were designed for ten genes with relatively stable expression in potato tubers as observed in RNA-Seq experiments. Primers were designed across exon boundaries to avoid genomic DNA contamination. Differences were observed in the ranking of candidate genes identified by geNorm, NormFinder and BestKeeper algorithms. The ranks determined by geNorm and NormFinder were very similar and for all samples the most stable candidates were C2, exocyst complex component sec3 (SEC3) and ATCUL3/ATCUL3A/CUL3/CUL3A (CUL3A). According to BestKeeper, the importin alpha and ubiquitin-associated/ts-n genes were the most stable. Three genes were selected as reference genes for potato edible tubers in RT-qPCR studies. The first one, called C2, was selected in common by NormFinder and geNorm, the second one is SEC3, selected by NormFinder, and the third one is CUL3A, selected by geNorm. Appropriate reference genes identified in this work will help to improve the accuracy of gene expression quantification analyses by taking into account differences that may be observed in RNA quality or reverse transcription efficiency across the samples.

Aseptic meningitis in children--the Singapore experience.

PubMed

Tee, W S N; Choong, C T; Lin, R V T P; Ling, A E

2002-11-01

To study the incidence, aetiology, clinical characteristics and management of paediatric aseptic meningitis in a paediatric hospital in Singapore. Patients with cerebrospinal fluid (CSF) pleocytosis, a negative Gram stain and bacterial culture were reviewed retrospectively from 1 January to 31 December 2000. Eighty-seven patients who fulfilled the criteria for aseptic meningitis and without neurological deficits were studied. In addition, reverse transcriptase polymerase chain reaction (RT-PCR) using pan enterovirus primers was subsequently performed on 73 of these CSF specimens which were available for storage. The incidence of aseptic meningitis was approximately 37 cases per 10,000 admissions. Non-polio enteroviruses were isolated from 29 of 64 (45.3%) CSF and 38 of 52 (73.1%) stool samples. RT-PCR was positive in 43 (58.9%) of the archived CSF specimens. The aetiologies of the remaining cases were mostly unidentified. Their ages ranged from 5 days to 12 years (median, 2 months). All patients except 1 had fever. Vomiting or poor feeding occurred in 44.7%, cough or running nose in 35.3%, irritability was observed in 35.3%, seizures in 7.1%, a rash in 10.6% and diarrhoea in 5.9%. All patients recovered without sequelae. The median CSF white cell count was 212 cells/mm3 (range, 7 to 12,000). The median glucose concentration was 2.7 mmol/L (range, 1.6 to 4.4). The median CSF/blood glucose ratio was 0.52 (range, 0.23 to 0.73). Median length of stay was 7 days (range, 4 to 17). Eighty-four patients (96.6%) received antibiotics for a median of 5.5 days (range, 2 to 14). Enteroviruses were the most common aetiologic agent identified. A method of early diagnosis using RT-PCR for enteroviruses is necessary to reduce the current duration of antibiotic usage and to decrease the length of hospital stay.

Molecular detection of mixed infections with multiple dengue virus serotypes in suspected dengue samples in Tamaulipas, Mexico.

PubMed

Requena-Castro, Rocío; Reyes-López, Miguel Ángel; Rodríguez-Reyna, Rosa Eminé; Palma-Nicolás, Prisco; Bocanegra-García, Virgilio

2017-07-01

This study aimed to detect dengue virus (DENV) serotypes in serum samples obtained in Matamoros Tamaulipas, Mexico, and to determine the concordance of conventional nested reverse transcriptase polymerase chain reaction (RT-PCR) and a serological test [enzyme-linked immunosorbent assay (ELISA NS1)]. Here, we detected mixed infections consisting of four serotypes of DENV. The most prevalent serotype was DENV-1, followed by DENV-4. This is the first report of DENV-4 in our region. Mixed infections were also detected in 21.5% of samples, and the predominant coinfection consisted of DENV-1 and DENV-2. Therefore, continuous epidemiological surveillance of DENV in this area is required to predict future forms of dengue heterologous infections and the effect of this on health care.

An inducible HSP70 gene from the midge Chironomus dilutus: Characterization and transcription profile under environmental stress

USGS Publications Warehouse

Karouna-Renier, N. K.; Rao, K.R.

2009-01-01

In the present study, we identified and characterized an inducible heat shock protein 70 (HSP70) from the midge Chironomus dilutus and investigated the transcriptional profile of the gene under baseline and environmentally stressful conditions. Using real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), we observed increased expression of CD-HSP70-1 in response to both heat shock and copper stress. We also investigated the expression of this gene during midge development. All C. dilutus developmental stages expressed CD-HSP70-1 under normal conditions, although at extremely low levels. Phylogenetic analysis of the amino acid sequence demonstrated distinct clustering of this gene with inducible HSP70s from other insect species. ?? 2008 The Authors.

Evaluating Fumonisin Gene Expression in Fusarium verticillioides.

PubMed

Scala, Valeria; Visentin, Ivan; Cardinale, Francesca

2017-01-01

Transcript levels of key genes in a biosynthetic pathway are often taken as a proxy for metabolite production. This is the case of FUM1, encoding the first dedicated enzyme in the metabolic pathway leading to the production of the mycotoxins Fumonisins by fungal species belonging to the genus Fusarium. FUM1 expression can be quantified by different methods; here, we detail a protocol based on quantitative reverse transcriptase polymerase chain reaction (RT-qPCR), by which relative or absolute transcript abundance can be estimated in Fusaria grown in vitro or in planta. As very seldom commercial kits for RNA extraction and cDNA synthesis are optimized for fungal samples, we developed a protocol tailored for these organisms, which stands alone but can be also easily integrated with specific reagents and kits commercially available.

Monitoring and improving the sensitivity of dengue nested RT-PCR used in longitudinal surveillance in Thailand.

PubMed

Klungthong, Chonticha; Manasatienkij, Wudtichai; Phonpakobsin, Thipwipha; Chinnawirotpisan, Piyawan; Rodpradit, Prinyada; Hussem, Kittinun; Thaisomboonsuk, Butsaya; Ong-ajchaowlerd, Prapapun; Nisalak, Ananda; Kalayanarooj, Siripen; Buddhari, Darunee; Gibbons, Robert V; Jarman, Richard G; Yoon, In-Kyu; Fernandez, Stefan

2015-02-01

AFRIMS longitudinal dengue surveillance in Thailand depends on the nested RT-PCR and the dengue IgM/IgG ELISA. To examine and improve the sensitivity of the nested RT-PCR using a panel of archived samples collected during dengue surveillance. A retrospective analysis of 16,454 dengue IgM/IgG ELISA positive cases collected between 2000 and 2013 was done to investigate the sensitivity of the nested RT-PCR. From these cases, 318 acute serum specimens or extracted RNA, previously found to be negative by the nested RT-PCR, were tested using TaqMan real-time RT-PCR (TaqMan rRT-PCR). To improve the sensitivity of nested RT-PCR, we designed a new primer based on nucleotide sequences from contemporary strains found to be positive by the TaqMan rRT-PCR. Sensitivity of the new nested PCR was calculated using a panel of 87 samples collected during 2011-2013. The percentage of dengue IgM/IgG ELISA positive cases that were negative by the nested RT-PCR varied from 17% to 42% for all serotypes depending on the year. Using TaqMan rRT-PCR, dengue RNA was detected in 194 (61%) of the 318 acute sera or extracted RNA previously found to be negative by the nested RT-PCR. The newly designed DENV-1 specific primer increased the sensitivity of DENV-1 detection by the nested RT-PCR from 48% to 88%, and of all 4 serotypes from 73% to 87%. These findings demonstrate the impact of genetic diversity and signal erosion on the sensitivity of PCR-based methods. Published by Elsevier B.V.

Protein-mediated antagonism between HIV reverse transcriptase ligands nevirapine and MgATP.

PubMed

Zheng, Xunhai; Mueller, Geoffrey A; DeRose, Eugene F; London, Robert E

2013-06-18

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) play a central role in the treatment of AIDS, but their mechanisms of action are incompletely understood. The interaction of the NNRTI nevirapine (NVP) with HIV-1 reverse transcriptase (RT) is characterized by a preference for the open conformation of the fingers/thumb subdomains, and a reported variation of three orders of magnitude between the binding affinity of NVP for RT in the presence or absence of primer/template DNA. To investigate the relationship between conformation and ligand binding, we evaluated the use of methionine NMR probes positioned near the tip of the fingers or thumb subdomains. Such probes would be expected to be sensitive to changes in the local environment depending on the fractions of open and closed RT. Comparisons of the NMR spectra of three conservative mutations, I63M, L74M, and L289M, indicated that M63 showed the greatest shift sensitivity to the addition of NVP. The exchange kinetics of the M63 resonance are fast on the chemical shift timescale, but become slow in the presence of NVP due to the slow binding of RT with the inhibitor. The simplest model consistent with this behavior involves a rapid open/closed equilibrium coupled with a slow interaction of the inhibitor with the open conformation. Studies of RT in the presence of both NVP and MgATP indicate a strong negative cooperativity. Binding of MgATP reduces the fraction of RT bound to NVP, as indicated by the intensity of the NVP-perturbed M230 resonance, and enhances the dissociation rate constant of the NVP, resulting in an increase of the open/closed interconversion rate, so that the M63 resonance moves into the fast/intermediate-exchange regime. Protein-mediated interactions appear to explain most of the affinity variation of NVP for RT. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Grana Padano cheese whey starters: microbial composition and strain distribution.

PubMed

Rossetti, Lia; Fornasari, Maria Emanuela; Gatti, Monica; Lazzi, Camilla; Neviani, Erasmo; Giraffa, Giorgio

2008-09-30

The aim of this work was to evaluate the species composition and the genotypic strain heterogeneity of dominant lactic acid bacteria (LAB) isolated from whey starter cultures used to manufacture Grana Padano cheese. Twenty-four Grana Padano cheese whey starters collected from dairies located over a wide geographic production area in the north of Italy were analyzed. Total thermophilic LAB streptococci and lactobacilli were quantified by agar plate counting. Population structure of the dominant and metabolically active LAB species present in the starters was profiled by reverse transcriptase, length heterogeneity-PCR (RT-LH-PCR), a culture-independent technique successfully applied to study whey starter ecosystems. The dominant bacterial species were Lactobacillus helveticus, Lactobacillus delbrueckii subsp. lactis, Streptococcus thermophilus, and Lactobacillus fermentum. Diversity in the species composition allowed the whey cultures to be grouped into four main typologies, the one containing L. helveticus, L. delbrueckii subsp. lactis, and S. thermophilus being the most frequent one (45% of the cultures analyzed), followed by that containing only the two lactobacilli (40%). Only a minor fraction of the cultures contained L. helveticus alone (4%) or all the four LAB species (11%). Five hundred and twelve strains were isolated from the 24 cultures and identified by M13-PCR fingerprinting coupled with 16S rRNA gene sequencing. Most of the strains were L. helveticus (190 strains; 37% of the total), L delbrueckii subsp. lactis (90 strains; 18%) and S. thermophilus (215 strains; 42%). This result was in good agreement with the qualitative whey starter composition observed by RT-LH-PCR. M13-PCR fingerprinting indicated a markedly low infra-species diversity, i.e. the same biotypes were often found in more than one culture. The distribution of the biotypes into the different cultures was mainly dairy plant-specific rather than correlated with the different production areas.

Quantification of HTLV-1 reverse transcriptase activity in ATL patients treated with zidovudine and interferon-α.

PubMed

Macchi, Beatrice; Balestrieri, Emanuela; Frezza, Caterina; Grelli, Sandro; Valletta, Elena; Marçais, Ambroise; Marino-Merlo, Francesca; Turpin, Jocelyn; Bangham, Charles R; Hermine, Olivier; Mastino, Antonio; Bazarbachi, Ali

2017-05-09

The therapeutic efficacy of the AZT and IFN combination in ATL presumably reflects the inhibition of RT-related functions.HTLV-1-RT activity from short-term cultured PBMCs may represent a predictive correlate of clinical response to AZT/IFN in ATL patients.

Susceptibility of recombinant porcine endogenous retrovirus reverse transcriptase to nucleoside and non-nucleoside inhibitors.

PubMed

Wilhelm, M; Fishman, J A; Pontikis, R; Aubertin, A M; Wilhelm, F X

2002-12-01

Transplantation of organs, tissues or cells from pigs to humans could be a potential solution to the shortage of human organs for transplantation. Porcine endogenous retroviruses (PERVs) remain a major safety concern for porcine xenotransplantation. Thus, finding drugs that could be used as virological prophylaxis (or therapy) against PERV replication would be desirable. One of the most effective ways to block retroviral multiplication is to inhibit the enzyme reverse transcriptase (RT) which catalyzes the reverse transcription of viral RNA to proviral double-stranded DNA. We report here the cloning and expression of PERV RT and its susceptibility to several inhibitors. Our data demonstrate PERV susceptibility in vitro to the triphosphorylated nucleoside analog of zidovudine (AZT) and to ddGTP and to a lesser extent to ddTTP but almost no susceptibility to the non-nucleoside RT inhibitors tested.

Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus

NASA Astrophysics Data System (ADS)

Huang, Su-Hua; Yang, Tsuey-Ching; Tsai, Ming-Hong; Tsai, I.-Shou; Lu, Huang-Chih; Chuang, Pei-Hsin; Wan, Lei; Lin, Ying-Ju; Lai, Chih-Ho; Lin, Cheng-Wen

2008-10-01

Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples.

A new fluorescence/PET probe for targeting intracellular human telomerase reverse transcriptase (hTERT) using Tat peptide-conjugated IgM

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jung, Kyung oh; Biomedical Sciences, Seoul National University College of Medicine; Cancer Research Institute, Seoul National University College of Medicine

Despite an increasing need for methods to visualize intracellular proteins in vivo, the majority of antibody-based imaging methods available can only detect membrane proteins. The human telomerase reverse transcriptase (hTERT) is an intracellular target of great interest because of its high expression in several types of cancer. In this study, we developed a new probe for hTERT using the Tat peptide. An hTERT antibody (IgG or IgM) was conjugated with the Tat peptide, a fluorescence dye and {sup 64}Cu. HT29 (hTERT+) and U2OS (hTERT−) were used to visualize the intracellular hTERT. The hTERT was detected by RT-PCR and western blot. Fluorescencemore » signals for hTERT were obtained by confocal microscopy, live cell imaging, and analyzed by Tissue-FAXS. In nude mice, tumors were visualized using the fluorescence imaging devices Maestro™ and PETBOX. In RT-PCR and western blot, the expression of hTERT was detected in HT29 cells, but not in U2OS cells. Fluorescence signals were clearly observed in HT29 cells and in U2OS cells after 1 h of treatment, but signals were only detected in HT29 cells after 24 h. Confocal microscopy showed that 9.65% of U2OS and 78.54% of HT29 cells had positive hTERT signals. 3D animation images showed that the probe could target intranuclear hTERT in the nucleus. In mice models, fluorescence and PET imaging showed that hTERT in HT29 tumors could be efficiently visualized. In summary, we developed a new method to visualize intracellular and intranuclear proteins both in vitro and in vivo. - Highlights: • We developed new probes for imaging hTERT using Tat-conjugated IgM antibodies labeled with a fluorescent dye and radioisotope. • This probes could be used to overcome limitation of conventional antibody imaging system in live cell imaging. • This system could be applicable to monitor intracellular and intranuclear proteins in vitro and in vivo.« less

Phylogenetic analysis of HIV-1 reverse transcriptase sequences from 382 patients recruited in JJ Hospital of Mumbai, India, between 2002 and 2008.

PubMed

Deshpande, Alaka; Jauvin, Valerie; Pinson, Patricia; Jeannot, Anne Cecile; Fleury, Herve J

2009-06-01

Analysis of reverse transcriptase (RT) sequences of 382 HIV-1 isolates from untreated and treated patients recruited in JJ Hospital (Mumbai, India) between 2002 and 2008 shows that subtype C is largely predominant (98%) and that non-C sequences cluster with A1, B, CRF01_AE, and CRF06_cpx.

Suicide Inhibitors of Reverse Transcriptase in the Therapy of AIDS and Other Retroviruses

DTIC Science & Technology

1990-07-01

I and 10 nanonolar) and compared to the E . Coli recombinant HIV-RT (Kindly donated by Dr. Steven Hughes Fort Detrick M.D.) and the wild type HIV-RT...Both the wild type and E . Coli HIV-RT’s were resistant to PFA showing essentially no inhibition at the lOnM level. Previous studies have shown that...10 nanomolar PFA. j, Sentivitv of Recombinant HIV-Reverse Transcriotase to Foscarnet. RECOMBINANT HIV RT ( E . COLI) + FOSCARNET 350001R 300000 PFA .001

An integrated molecular dynamics, principal component analysis and residue interaction network approach reveals the impact of M184V mutation on HIV reverse transcriptase resistance to lamivudine.

PubMed

Bhakat, Soumendranath; Martin, Alberto J M; Soliman, Mahmoud E S

2014-08-01

The emergence of different drug resistant strains of HIV-1 reverse transcriptase (HIV RT) remains of prime interest in relation to viral pathogenesis as well as drug development. Amongst those mutations, M184V was found to cause a complete loss of ligand fitness. In this study, we report the first account of the molecular impact of M184V mutation on HIV RT resistance to 3TC (lamivudine) using an integrated computational approach. This involved molecular dynamics simulation, binding free energy analysis, principle component analysis (PCA) and residue interaction networks (RINs). Results clearly confirmed that M184V mutation leads to steric conflict between 3TC and the beta branched side chain of valine, decreases the ligand (3TC) binding affinity by ∼7 kcal mol(-1) when compared to the wild type, changes the overall conformational landscape of the protein and distorts the native enzyme residue-residue interaction network. The comprehensive molecular insight gained from this study should be of great importance in understanding drug resistance against HIV RT as well as assisting in the design of novel reverse transcriptase inhibitors with high ligand efficacy on resistant strains.

Revealing the drug-resistant mechanism for diarylpyrimidine analogue inhibitors of HIV-1 reverse transcriptase.

PubMed

Zhang, Hao; Qin, Fang; Ye, Wei; Li, Zeng; Ma, Songyao; Xia, Yan; Jiang, Yi; Zhu, Jiayi; Li, Yixue; Zhang, Jian; Chen, Hai-Feng

2011-09-01

Diaryltriazine (DATA) and diarylpyrimidine (DAPY) were two category inhibitors with highly potent activity for wild type (wt) and four principal mutant types (L100I, K103N, Y181C and Y188L) of HIV-1 reverse transcriptase (RT). We had revealed the drug-resistant mechanism of DATA analogue inhibitors with molecular dynamics simulation and three-dimensional quantitative structure-activity relationship (3D-QSAR) methods. In this work, we investigated the drug-resistant mechanism of DAPY analogue inhibitors. It was found that DAPY analogue inhibitors form more hydrogen bonds and hydrophobic contacts with wild type and mutants of HIV-1 RT than DATA inhibitors. This could explain that DAPY analogue inhibitors are more potent than DATA for the wild type and mutants of HIV-1 RT. Then, 3D-QSAR models were constructed for these inhibitors of wild type and four principal mutant types HIV-1 RT and evaluated by test set compounds. These combined models can be used to design new chemical entities and make quantitative prediction of the bioactivities for HIV-1 RT inhibitors before resorting to in vitro and in vivo experiment. © 2011 John Wiley & Sons A/S.

Substrate-induced stable enzyme-inhibitor complex formation allows tight binding of novel 2-aminopyrimidin-4(3H)-ones to drug-resistant HIV-1 reverse transcriptase mutants.

PubMed

Samuele, Alberta; Facchini, Marcella; Rotili, Dante; Mai, Antonello; Artico, Marino; Armand-Ugón, Mercedes; Esté, José A; Maga, Giovanni

2008-09-01

We recently reported the synthesis and biological evaluation of a novel series of 5-alkyl-2-(N,N-disubstituted)amino-6-(2,6-difluorophenylalkyl)-3,4-dihydropyrimidin-4(3H)-ones (F(2)-N,N-DABOs). These compounds are highly active against both wild-type HIV-1 and the K103N, Y181C, and Y188L mutant strains. Herein we present novel 6-(2-chloro-6-fluorophenylalkyl)-N,N-DABO (2-Cl-6-F-N,N-DABO) derivatives and investigate the molecular basis for their high-affinity binding to HIV-1 reverse transcriptase (RT). Our results show that the new compounds display higher association rates than the difluoro derivatives toward wild-type HIV-1 RT or drug-resistant RT mutant forms. We also show that they preferentially associate to either the free enzyme or the enzyme-nucleic acid binary complex, and that this binding is stabilized upon formation of the ternary complex between HIV-1 RT and both the nucleic acid and nucleotide substrates. Interestingly, one compound showed dissociation rates from the ternary complex with RT mutants K103N and Y181I 10-20-fold slower than from the corresponding complex with wild-type RT.

A reverse transcriptase-dependent mechanism plays central roles in fundamental biological processes.

PubMed

Spadafora, Corrado

2008-01-01

This review summarizes emerging evidence that LINE-1 (Long Interspersed Nuclear Elements) -encoded reverse transcriptase (RT) regulates fundamental biological processes. Earlier studies showed that sperm cells can be used as vectors of both exogenous DNA and RNA molecules in sperm-mediated gene transfer assays. During these studies, a sperm endogenous RT activity was identified, which can reverse-transcribe exogenous RNA directly, or DNA molecules through sequential transcription and reverse transcription. Resulting cDNA copies generated in sperm cells can be delivered to embryos at fertilization, further propagated in tissues as low-copy extrachromosomal structures and transmitted to the progeny in a non-mendelian fashion. Being transcriptionally competent, they can induce phenotypic variations in positive tissues. An RT activity is also present in preimplantation embryos, and its inhibition causes developmental arrest in early preimplantation stages, paralleled by an extensive reprogramming of gene expression. In analogy with this, drug-mediated inhibition of RT activity, or RNA interference-mediated silencing of human LINE-1, reduce cell proliferation and induce differentiation in a variety of cancer cell lines. Furthermore, RT inhibition in vivo antagonizes the growth of human tumors in animal models. As a whole, these data implicate a RT-dependent machinery in the genesis of new genetic information in spermatozoa and in normal and pathological developmental processes.

Detection of canine distemper virus (CDV) through one step RT-PCR combined with nested PCR.

PubMed

Kim, Y H; Cho, K W; Youn, H Y; Yoo, H S; Han, H R

2001-04-01

A one step reverse transcription PCR (RT-PCR) combined nested PCR was set up to increase efficiency in the diagnosis of canine distemper virus (CDV) infection after developement of nested PCR. Two PCR primer sets were designed based on the sequence of nucleocapsid gene of CDV Onderstepoort strain. One-step RT-PCR with the outer primer pair was revealed to detect 10(2) PFU/ml. The sensitivity was increased hundredfold using the one-step RT-PCR combined with the nested PCR. Specificity of the PCR was also confirmed using other related canine virus and peripheral blood mononuclear cells (PBMC) and body secretes of healthy dogs. Of the 51 blood samples from dogs clinically suspected of CD, 45 samples were revealed as positive by one-step RT-PCR combined with nested PCR. However, only 15 samples were identified as positive with a single one step RT-PCR. Therefore approximately 60% increase in the efficiency of the diagnosis was observed by the combined method. These results suggested that one step RT-PCR combined with nested PCR could be a sensitive, specific, and practical method for diagnosis of CDV infection.

Groundwater microbiological quality in Canadian drinking water municipal wells.

PubMed

Locas, Annie; Barthe, Christine; Margolin, Aaron B; Payment, Pierre

2008-06-01

To verify previous conclusions on the use of bacterial indicators suggested in regulations and to investigate virological quality of groundwater, a 1-year study was undertaken on groundwater used as a source of drinking water in 3 provinces in Canada. Raw water from 25 municipal wells was sampled during a 1-year period for a total of 167 samples. Twenty-three sites were selected on the basis of their excellent historical bacteriological water quality data, and 2 sites with known bacteriological contamination were selected as positive controls. Water samples were analyzed for general water quality indicators (aerobic endospores, total coliforms), fecal indicators (Escherichia coli, enterococci, somatic and male-specific coliphages), total culturable human enteric viruses (determined by cell culture and immunoperoxidase), noroviruses (analyzed by reverse-transcriptase -- polymerase chain reaction (RT-PCR)), adenovirus types 40 and 41 (analyzed by integrated cell culture (ICC) - PCR), and enteroviruses and reoviruses types 1, 2, and 3 (analyzed by ICC-RT-PCR). General water quality indicators were found very occasionally at the clean sites but were frequently present at the 2 contaminated sites. Only one of 129 samples from the 23 clean sites was positive for enterococci. These results confirm the value of raw water quality historical data to detect source water contamination affecting wells that are vulnerable. Samples from the 2 contaminated sites confirmed the frequent presence of fecal indicators: E. coli was found in 20/38 samples and enterococci in 12/38 samples. Human enteric viruses were not detected by cell culture on MA-104 cells nor by immunoperoxidase detection in any sample from the clean sites but were found at one contaminated site. By ICC-RT-PCR and ICC-PCR, viruses were found by cytopathic effect in one sample from a clean site and they were found in 3 samples from contaminated sites. The viruses were not detected by the molecular methods but were confirmed as picornaviruses by electron microscopy. Noroviruses were not detected in any samples. The results obtained reinforce the value of frequent sampling of raw water using simple parameters: sampling for total coliforms and E. coli remains the best approach to detect contamination of source water by fecal pollutants and accompanying pathogens. The absence of total coliforms at a site appears to be a good indication of the absence of human enteric viruses.

Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth disease virus and look-alike disease viruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hindson, B J; Reid, S M; Baker, B R

2007-07-26

A high-throughput multiplexed assay was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspect cases of foot-and-mouthmore » disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRT-PCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.« less

On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires.

PubMed

Silas, Sukrit; Makarova, Kira S; Shmakov, Sergey; Páez-Espino, David; Mohr, Georg; Liu, Yi; Davison, Michelle; Roux, Simon; Krishnamurthy, Siddharth R; Fu, Becky Xu Hua; Hansen, Loren L; Wang, David; Sullivan, Matthew B; Millard, Andrew; Clokie, Martha R; Bhaya, Devaki; Lambowitz, Alan M; Kyrpides, Nikos C; Koonin, Eugene V; Fire, Andrew Z

2017-07-11

Cas1 integrase is the key enzyme of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas adaptation module that mediates acquisition of spacers derived from foreign DNA by CRISPR arrays. In diverse bacteria, the cas1 gene is fused (or adjacent) to a gene encoding a reverse transcriptase (RT) related to group II intron RTs. An RT-Cas1 fusion protein has been recently shown to enable acquisition of CRISPR spacers from RNA. Phylogenetic analysis of the CRISPR-associated RTs demonstrates monophyly of the RT-Cas1 fusion, and coevolution of the RT and Cas1 domains. Nearly all such RTs are present within type III CRISPR-Cas loci, but their phylogeny does not parallel the CRISPR-Cas type classification, indicating that RT-Cas1 is an autonomous functional module that is disseminated by horizontal gene transfer and can function with diverse type III systems. To compare the sequence pools sampled by RT-Cas1-associated and RT-lacking CRISPR-Cas systems, we obtained samples of a commercially grown cyanobacterium- Arthrospira platensis Sequencing of the CRISPR arrays uncovered a highly diverse population of spacers. Spacer diversity was particularly striking for the RT-Cas1-containing type III-B system, where no saturation was evident even with millions of sequences analyzed. In contrast, analysis of the RT-lacking type III-D system yielded a highly diverse pool but reached a point where fewer novel spacers were recovered as sequencing depth was increased. Matches could be identified for a small fraction of the non-RT-Cas1-associated spacers, and for only a single RT-Cas1-associated spacer. Thus, the principal source(s) of the spacers, particularly the hypervariable spacer repertoire of the RT-associated arrays, remains unknown. IMPORTANCE While the majority of CRISPR-Cas immune systems adapt to foreign genetic elements by capturing segments of invasive DNA, some systems carry reverse transcriptases (RTs) that enable adaptation to RNA molecules. From analysis of available bacterial sequence data, we find evidence that RT-based RNA adaptation machinery has been able to join with CRISPR-Cas immune systems in many, diverse bacterial species. To investigate whether the abilities to adapt to DNA and RNA molecules are utilized for defense against distinct classes of invaders in nature, we sequenced CRISPR arrays from samples of commercial-scale open-air cultures of Arthrospira platensis , a cyanobacterium that contains both RT-lacking and RT-containing CRISPR-Cas systems. We uncovered a diverse pool of naturally occurring immune memories, with the RT-lacking locus acquiring a number of segments matching known viral or bacterial genes, while the RT-containing locus has acquired spacers from a distinct sequence pool for which the source remains enigmatic. Copyright © 2017 Silas et al.

Identification and characterization of mobile genetic elements LINEs from Brassica genome.

PubMed

Nouroz, Faisal; Noreen, Shumaila; Khan, Muhammad Fiaz; Ahmed, Shehzad; Heslop-Harrison, J S Pat

2017-09-05

Among transposable elements (TEs), the LTR retrotransposons are abundant followed by non-LTR retrotransposons in plant genomes, the lateral being represented by LINEs and SINEs. Computational and molecular approaches were used for the characterization of Brassica LINEs, their diversity and phylogenetic relationships. Four autonomous and four non-autonomous LINE families were identified and characterized from Brassica. Most of the autonomous LINEs displayed two open reading frames, ORF1 and ORF2, where ORF1 is a gag protein domain, while ORF2 encodes endonuclease (EN) and a reverse transcriptase (RT). Three of four families encoded an additional RNase H (RH) domain in pol gene common to 'R' and 'I' type of LINEs. The PCR analyses based on LINEs RT fragments indicate their high diversity and widespread occurrence in tested 40 Brassica cultivars. Database searches revealed the homology in LINE sequences in closely related genera Arabidopsis indicating their origin from common ancestors predating their separation. The alignment of 58 LINEs RT sequences from Brassica, Arabidopsis and other plants depicted 4 conserved domains (domain II-V) showing similarity to previously detected domains. Based on RT alignment of Brassica and 3 known LINEs from monocots, Brassicaceae LINEs clustered in separate clade, further resolving 4 Brassica-Arabidopsis specific families in 2 sub-clades. High similarities were observed in RT sequences in the members of same family, while low homology was detected in members across the families. The investigation led to the characterization of Brassica specific LINE families and their diversity across Brassica species and their cultivars. Copyright © 2017 Elsevier B.V. All rights reserved.

Rapid colorimetric detection of Zika virus from serum and urine specimens by reverse transcription loop-mediated isothermal amplification (RT-LAMP)

PubMed Central

Biggerstaff, Brad J.; Tanner, Nathan A.; Lauterbach, Molly; Lanciotti, Robert S.

2017-01-01

Zika virus (ZIKV) has emerged as a major global public health concern in the last two years due to its link as a causative agent of human birth defects. Its rapid expansion into the Western Hemisphere as well as the ability to be transmitted from mother to fetus, through sexual transmission and possibly through blood transfusions has increased the need for a rapid and expansive public health response to this unprecedented epidemic. A non-invasive and rapid ZIKV diagnostic screening assay that can be performed in a clinical setting throughout pregnancy is vital for prenatal care of women living in areas of the world where exposure to the virus is possible. To meet this need we have developed a sensitive and specific reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay to detect ZIKV RNA in urine and serum with a simple visual detection. RT-LAMP results were shown to have a limit of detection 10-fold higher than qRT-PCR. As little as 1.2 RNA copies/μl was detected by RT-LAMP from a panel of 178 diagnostic specimens. The assay was shown to be highly specific for ZIKV RNA when tested with diagnostic specimens positive for dengue virus (DENV) and chikungunya virus (CHIKV). The assay described here illustrates the potential for a fast, reliable, sensitive and specific assay for the detection of ZIKV from urine or serum that can be performed in a clinical or field setting with minimal equipment and technological expertise. PMID:28945787

Point-of-care diagnostic assay for the detection of Zika virus using the recombinase polymerase amplification method.

PubMed

Vasileva Wand, Nadina I; Bonney, Laura C; Watson, Robert J; Graham, Victoria; Hewson, Roger

2018-06-13

The sudden and explosive expansion of Zika virus (ZIKV) from the African continent through Oceania and culminating in the outbreak in South America has highlighted the importance of new rapid point-of-care diagnostic tools for the control and prevention of transmission. ZIKV infection has devastating consequences, such as neurological congenital malformations in infants born to infected mothers and Guillain-Barré syndrome in adults. Additionally, its potential for transmission through vector bites, as well as from person to person through blood transfusions and sexual contact, are important considerations for prompt diagnosis. Recombinase polymerase amplification (RPA), an isothermal method, was developed as an alternative field-applicable assay to PCR. Here we report the development of a novel ZIKV real-time reverse transcriptase RPA (RT-RPA) assay capable of detecting a range of different ZIKV strains from a variety of geographical locations. The ZIKV RT-RPA was shown to be highly sensitive, being capable of detecting as few as five copies of target nucleic acid per reaction, and suitable for use with a battery-operated portable device. The ZIKV RT-RPA demonstrated 100 % specificity and 83 % sensitivity in clinical samples. Furthermore, we determined that the ZIKV RT-RPA is a versatile assay that can be applied to crude samples, such as saliva and serum, and can be used as a vector surveillance tool on crude mosquito homogenates. Therefore, the developed ZIKV RT-RPA is a useful diagnostic tool that can be transferred to a resource-limited location, eliminating the need for a specialized and sophisticated laboratory environment and highly trained staff.

Rapid colorimetric detection of Zika virus from serum and urine specimens by reverse transcription loop-mediated isothermal amplification (RT-LAMP).

PubMed

Calvert, Amanda E; Biggerstaff, Brad J; Tanner, Nathan A; Lauterbach, Molly; Lanciotti, Robert S

2017-01-01

Zika virus (ZIKV) has emerged as a major global public health concern in the last two years due to its link as a causative agent of human birth defects. Its rapid expansion into the Western Hemisphere as well as the ability to be transmitted from mother to fetus, through sexual transmission and possibly through blood transfusions has increased the need for a rapid and expansive public health response to this unprecedented epidemic. A non-invasive and rapid ZIKV diagnostic screening assay that can be performed in a clinical setting throughout pregnancy is vital for prenatal care of women living in areas of the world where exposure to the virus is possible. To meet this need we have developed a sensitive and specific reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay to detect ZIKV RNA in urine and serum with a simple visual detection. RT-LAMP results were shown to have a limit of detection 10-fold higher than qRT-PCR. As little as 1.2 RNA copies/μl was detected by RT-LAMP from a panel of 178 diagnostic specimens. The assay was shown to be highly specific for ZIKV RNA when tested with diagnostic specimens positive for dengue virus (DENV) and chikungunya virus (CHIKV). The assay described here illustrates the potential for a fast, reliable, sensitive and specific assay for the detection of ZIKV from urine or serum that can be performed in a clinical or field setting with minimal equipment and technological expertise.

Appearance of drug resistance-associated mutations in human immunodeficiency virus type 1 protease and reverse transcriptase derived from drug-treated Indonesian patients.

PubMed

Khairunisa, Siti Qamariyah; Kotaki, Tomohiro; Witaningrum, Adiana Mutamsari; Yunifiar M, Muhammad Qushai; Sukartiningrum, Septhia Dwi; Nasronudin; Kameoka, Masanori

2015-02-01

Although HIV-1 drug resistance is a major obstacle in Indonesia, information on drug resistance is limited. In this study, the viral subtype and appearance of drug resistance mutations in the HIV-1 protease (PR) and reverse transcriptase (RT) genes were determined among drug-treated, HIV-1-infected patients in Surabaya. HIV-1 patients who received antiretroviral therapy (ART) more than 2 years were randomly recruited regardless of the viral load or ART failure. Fifty-eight HIV-1 PR genes and 53 RT genes were sequenced. CRF01_AE viruses were identified as the predominant strain. Major drug resistance mutations were not detected in the PR genes. In contrast, 37.7% (20/53) of the participants had one or more major drug resistance mutations in the RT genes, predominantly M184V (28.3%), K103N (11.3%), and thymidine analogue mutations (TAMs) (20.8%). The high prevalence of drug resistance mutations in RT genes indicated the necessity of monitoring the effectiveness of ART in Indonesia.

Generation of thermostable Moloney murine leukemia virus reverse transcriptase variants using site saturation mutagenesis library and cell-free protein expression system.

PubMed

Katano, Yuta; Li, Tongyang; Baba, Misato; Nakamura, Miyo; Ito, Masaaki; Kojima, Kenji; Takita, Teisuke; Yasukawa, Kiyoshi

2017-12-01

We attempted to increase the thermostability of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT). The eight-site saturation mutagenesis libraries corresponding to Ala70-Arg469 in the whole MMLV RT (Thr24-Leu671), in each of which 1 out of 50 amino acid residues was replaced with other amino acid residue, were constructed. Seven-hundred and sixty eight MMLV RT clones were expressed using a cell-free protein expression system, and their thermostabilities were assessed by the temperature of thermal treatment at which they retained cDNA synthesis activity. One clone D200C was selected as the most thermostable variant. The highest temperature of thermal treatment at which D200C exhibited cDNA synthesis activity was 57ºC, which was higher than for WT (53ºC). Our results suggest that a combination of site saturation mutagenesis library and cell-free protein expression system might be useful for generation of thermostable MMLV RT in a short period of time for expression and selection.

New Subtypes and Genetic Recombination in HIV Type 1-Infecting Patients with Highly Active Antiretroviral Therapy in Peru (2008–2010)

PubMed Central

Acuña, Maribel; Gazzo, Cecilia; Salinas, Gabriela; Cárdenas, Fanny; Valverde, Ada; Romero, Soledad

2012-01-01

Abstract HIV-1 subtype B is the most frequent strain in Peru. However, there is no available data about the genetic diversity of HIV-infected patients receiving highly active antiretroviral therapy (HAART) here. A group of 267 patients in the Peruvian National Treatment Program with virologic failure were tested for genotypic evidence of HIV drug resistance at the Instituto Nacional de Salud (INS) of Peru between March 2008 and December 2010. Viral RNA was extracted from plasma and the segments of the protease (PR) and reverse transcriptase (RT) genes were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), purified, and fully sequenced. Consensus sequences were submitted to the HIVdb Genotypic Resistance Interpretation Algorithm Database from Stanford University, and then aligned using Clustal X v.2.0 to generate a phylogenetic tree using the maximum likelihood method. Intrasubtype and intersubtype recombination analyses were performed using the SCUEAL program (Subtype Classification by Evolutionary ALgo-rithms). A total of 245 samples (91%) were successfully genotyped. The analysis obtained from the HIVdb program showed 81.5% resistance cases (n=198). The phylogenetic analysis revealed that subtype B was predominant in the population (98.8%), except for new cases of A, C, and H subtypes (n=4). Of these cases, only subtype C was imported. Likewise, recombination analysis revealed nine intersubtype and 20 intrasubtype recombinant cases. This is the first report of the presence of HIV-1 subtypes C and H in Peru. The introduction of new subtypes and circulating recombinants forms can make it difficult to distinguish resistance profiles in patients and consequently affect future treatment strategies against HIV in this country. PMID:22559065

New subtypes and genetic recombination in HIV type 1-infecting patients with highly active antiretroviral therapy in Peru (2008-2010).

PubMed

Yabar, Carlos Augusto; Acuña, Maribel; Gazzo, Cecilia; Salinas, Gabriela; Cárdenas, Fanny; Valverde, Ada; Romero, Soledad

2012-12-01

HIV-1 subtype B is the most frequent strain in Peru. However, there is no available data about the genetic diversity of HIV-infected patients receiving highly active antiretroviral therapy (HAART) here. A group of 267 patients in the Peruvian National Treatment Program with virologic failure were tested for genotypic evidence of HIV drug resistance at the Instituto Nacional de Salud (INS) of Peru between March 2008 and December 2010. Viral RNA was extracted from plasma and the segments of the protease (PR) and reverse transcriptase (RT) genes were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), purified, and fully sequenced. Consensus sequences were submitted to the HIVdb Genotypic Resistance Interpretation Algorithm Database from Stanford University, and then aligned using Clustal X v.2.0 to generate a phylogenetic tree using the maximum likelihood method. Intrasubtype and intersubtype recombination analyses were performed using the SCUEAL program (Subtype Classification by Evolutionary ALgo-rithms). A total of 245 samples (91%) were successfully genotyped. The analysis obtained from the HIVdb program showed 81.5% resistance cases (n=198). The phylogenetic analysis revealed that subtype B was predominant in the population (98.8%), except for new cases of A, C, and H subtypes (n=4). Of these cases, only subtype C was imported. Likewise, recombination analysis revealed nine intersubtype and 20 intrasubtype recombinant cases. This is the first report of the presence of HIV-1 subtypes C and H in Peru. The introduction of new subtypes and circulating recombinants forms can make it difficult to distinguish resistance profiles in patients and consequently affect future treatment strategies against HIV in this country.

Discovery and analysis of an active long terminal repeat-retrotransposable element in Aspergillus oryzae.

PubMed

Jie Jin, Feng; Hara, Seiichi; Sato, Atsushi; Koyama, Yasuji

2014-01-01

Wild-type Aspergillus oryzae RIB40 contains two copies of the AO090005001597 gene. We previously constructed A. oryzae RIB40 strain, RKuAF8B, with multiple chromosomal deletions, in which the AO090005001597 copy number was found to be increased significantly. Sequence analysis indicated that AO090005001597 is part of a putative 6,000-bp retrotransposable element, flanked by two long terminal repeats (LTRs) of 669 bp, with characteristics of retroviruses and retrotransposons, and thus designated AoLTR (A. oryzae LTR-retrotransposable element). AoLTR comprised putative reverse transcriptase, RNase H, and integrase domains. The deduced amino acid sequence alignment of AoLTR showed 94% overall identity with AFLAV, an A. flavus Tf1/sushi retrotransposon. Quantitative real-time RT-PCR showed that AoLTR gene expression was significantly increased in the RKuAF8B, in accordance with the increased copy number. Inverse PCR indicated that the full-length retrotransposable element was randomly integrated into multiple genomic locations. However, no obvious phenotypic changes were associated with the increased AoLTR gene copy number.

A sugar gustatory receptor identified from the foregut of cotton bollworm Helicoverpa armigera.

PubMed

Xu, Wei; Zhang, Hui-Jie; Anderson, Alisha

2012-12-01

Helicoverpa armigera (Hübner) is one of the most polyphagous and cosmopolitan pest species, the larvae of which feed on numerous important crops. The gustatory system is critical in guiding insect feeding behavior. Here, we identified a gustatory receptor from H. armigera, HaGR9, which shows high levels of identity to DmGR43a from Drosophila melanogaster and BmGR9 from Bombyx mori. Reverse transcriptase PCR (RT-PCR) revealed HaGR9 is highly expressed in larval foregut, with little or no expression in other chemosensory tissues. Membrane topology studies indicated that, like two previously studied B. mori GRs, BmGR8 and BmGR53, HaGR9 has an inverted topology relative to G protein-coupled receptors (GPCRs), an intracellular N-terminus and an extracellular C-terminus. Calcium imaging studies confirmed HaGR9 is a sugar receptor showing dose-dependent responses to D-galactose, D-maltose, and D-fructose. This highly-expressed foregut-specific gustatory receptor may contribute to the regulation of larval feeding behavior.

Sequence quality analysis tool for HIV type 1 protease and reverse transcriptase.

PubMed

Delong, Allison K; Wu, Mingham; Bennett, Diane; Parkin, Neil; Wu, Zhijin; Hogan, Joseph W; Kantor, Rami

2012-08-01

Access to antiretroviral therapy is increasing globally and drug resistance evolution is anticipated. Currently, protease (PR) and reverse transcriptase (RT) sequence generation is increasing, including the use of in-house sequencing assays, and quality assessment prior to sequence analysis is essential. We created a computational HIV PR/RT Sequence Quality Analysis Tool (SQUAT) that runs in the R statistical environment. Sequence quality thresholds are calculated from a large dataset (46,802 PR and 44,432 RT sequences) from the published literature ( http://hivdb.Stanford.edu ). Nucleic acid sequences are read into SQUAT, identified, aligned, and translated. Nucleic acid sequences are flagged if with >five 1-2-base insertions; >one 3-base insertion; >one deletion; >six PR or >18 RT ambiguous bases; >three consecutive PR or >four RT nucleic acid mutations; >zero stop codons; >three PR or >six RT ambiguous amino acids; >three consecutive PR or >four RT amino acid mutations; >zero unique amino acids; or <0.5% or >15% genetic distance from another submitted sequence. Thresholds are user modifiable. SQUAT output includes a summary report with detailed comments for troubleshooting of flagged sequences, histograms of pairwise genetic distances, neighbor joining phylogenetic trees, and aligned nucleic and amino acid sequences. SQUAT is a stand-alone, free, web-independent tool to ensure use of high-quality HIV PR/RT sequences in interpretation and reporting of drug resistance, while increasing awareness and expertise and facilitating troubleshooting of potentially problematic sequences.

Validated reverse transcription droplet digital PCR serves as a higher order method for absolute quantification of Potato virus Y strains.

PubMed

Mehle, Nataša; Dobnik, David; Ravnikar, Maja; Pompe Novak, Maruša

2018-05-03

RNA viruses have a great potential for high genetic variability and rapid evolution that is generated by mutation and recombination under selection pressure. This is also the case of Potato virus Y (PVY), which comprises a high diversity of different recombinant and non-recombinant strains. Consequently, it is hard to develop reverse transcription real-time quantitative PCR (RT-qPCR) with the same amplification efficiencies for all PVY strains which would enable their equilibrate quantification; this is specially needed in mixed infections and other studies of pathogenesis. To achieve this, we initially transferred the PVY universal RT-qPCR assay to a reverse transcription droplet digital PCR (RT-ddPCR) format. RT-ddPCR is an absolute quantification method, where a calibration curve is not needed, and it is less prone to inhibitors. The RT-ddPCR developed and validated in this study achieved a dynamic range of quantification over five orders of magnitude, and in terms of its sensitivity, it was comparable to, or even better than, RT-qPCR. RT-ddPCR showed lower measurement variability. We have shown that RT-ddPCR can be used as a reference tool for the evaluation of different RT-qPCR assays. In addition, it can be used for quantification of RNA based on in-house reference materials that can then be used as calibrators in diagnostic laboratories.

Development of a highly sensitive real-time nested RT-PCR assay in a single closed tube for detection of enterovirus 71 in hand, foot, and mouth disease.

PubMed

Niu, Peihua; Qi, Shunxiang; Yu, Benzhang; Zhang, Chen; Wang, Ji; Li, Qi; Ma, Xuejun

2016-11-01

Enterovirus 71 (EV71) is one of the major causative agents of outbreaks of hand, foot, and mouth disease (HFMD). A commercial TaqMan probe-based real-time PCR assay has been widely used for the differential detection of EV71 despite its relatively high cost and failure to detect samples with a low viral load (Ct value > 35). In this study, a highly sensitive real-time nested RT-PCR (RTN RT-PCR) assay in a single closed tube for detection of EV71 in HFMD was developed. The sensitivity and specificity of this assay were evaluated using a reference EV71 stock and a panel of controls consisting of coxsackievirus A16 (CVA16) and common respiratory viruses, respectively. The clinical performance of this assay was evaluated and compared with those of a commercial TaqMan probe-based real-time PCR (qRT-PCR) assay and a traditional two-step nested RT-PCR assay. The limit of detection for the RTN RT-PCR assay was 0.01 TCID50/ml, with a Ct value of 38.3, which was the same as that of the traditional two-step nested RT-PCR assay and approximately tenfold lower than that of the qRT-PCR assay. When testing the reference strain EV71, this assay showed favorable detection reproducibility and no obvious cross-reactivity. The testing results of 100 clinical throat swabs from HFMD-suspected patients revealed that 41 samples were positive for EV71 by both RTN RT-PCR and traditional two-step nested RT-PCR assays, whereas only 29 were EV71 positive by qRT-PCR assay.

Evaluation of approaches to monitor Staphylococcus aureus virulence factor expression during human disease.

PubMed

Rozemeijer, Wouter; Fink, Pamela; Rojas, Eduardo; Jones, C Hal; Pavliakova, Danka; Giardina, Peter; Murphy, Ellen; Liberator, Paul; Jiang, Qin; Girgenti, Douglas; Peters, Remco P H; Savelkoul, Paul H M; Jansen, Kathrin U; Anderson, Annaliesa S; Kluytmans, Jan

2015-01-01

Staphylococcus aureus is a versatile pathogen of medical significance, using multiple virulence factors to cause disease. A prophylactic S. aureus 4-antigen (SA4Ag) vaccine comprising capsular polysaccharide (types 5 and 8) conjugates, clumping factor A (ClfA) and manganese transporter C (MntC) is under development. This study was designed to characterize S. aureus isolates recovered from infected patients and also to investigate approaches for examining expression of S. aureus vaccine candidates and the host response during human infection. Confirmation of antigen expression in different disease states is important to support the inclusion of these antigens in a prophylactic vaccine. Hospitalized patients with diagnosed S. aureus wound (27) or bloodstream (24) infections were enrolled. Invasive and nasal carriage S. aureus isolates were recovered and characterized for genotypic diversity. S. aureus antigen expression was evaluated directly by real-time, quantitative, reverse-transcriptase PCR (qRT-PCR) analysis and indirectly by serology using a competitive Luminex immunoassay. Study isolates were genotypically diverse and all had the genes encoding the antigens present in the SA4Ag vaccine. S. aureus nasal carriage was detected in 55% of patients, and in those subjects 64% of the carriage isolates matched the invasive strain. In swab samples with detectable S. aureus triosephosphate isomerase housekeeping gene expression, RNA transcripts encoding the S. aureus virulence factors ClfA, MntC, and capsule polysaccharide were detected by qRT-PCR. Antigen expression was indirectly confirmed by increases in antibody titer during the course of infection from acute to convalescent phase. Demonstration of bacterial transcript expression together with immunological response to the SA4Ag antigens in a clinically relevant patient population provides support for inclusion of these antigens in a prophylactic vaccine.

Overexpression of MDR-1 and CDR-2 genes in fluconazole resistance of Candida albicans isolated from patients with vulvovaginal candidiasis.

PubMed

Khosravi Rad, K; Falahati, M; Roudbary, M; Farahyar, S; Nami, S

2016-12-01

Candida albicans ( C. albicans ) is an opportunistic fungus that can colonize women's mucosal epithelial cell surfaces, causing vulvovaginitis in specific circumstances. The major genes contributing to drug resistance in C. albicans are the candida drug resistance ( CDR ) and multi drug resistance ( MDR ) genes. The purpose of this study was to evaluate the CDR-2 and MDR-1 gene expression patterns in C. albicans strains isolated from patients with recurrent vulvovaginal candidiasis. In this study, 40 isolates of fluconazole-resistant C. albicans were cultured on Sabouraud dextrose agar. These isolates were collected from women with vulvovaginitis who were referred to a clinic in Tehran, Iran, and transferred to a mycology laboratory. Then, RNA was extracted from the isolates using phenol-chloroform and glass beads, and the complementary DNA (cDNA) was synthetized. To detect the semi-quantitative expression of CDR-2 and MDR-1 genes, the reverse transcriptase-PCR (RT-PCR) technique was performed using specific primers. Our findings indicated that of the 40 C. albicans isolates, 35 (87.5%) strains were positive for mRNA of the CDR-2 gene, 32 (80%) strains expressed mRNA of the MDR-1 gene, and 30 (75%) strains were confirmed to express mRNA of both the CDR-2 and MDR-1 genes simultaneously using the RT-PCR assay. According to the obtained results, the expression rates of CDR-2 and MDR-1 genes were high in fluconazole-resistant C. albicans isolates, which can cause treatments to fail and result in chronic infections. Inhibiting these important genes using novel or natural agents can help with the treatment of chronic and recurrent vaginitis.

A biosensor assay for the detection of Mycobacterium avium subsp. paratuberculosis in fecal samples

PubMed Central

Kumanan, Vijayarani; Nugen, Sam R.; Baeumner, Antje J.

2009-01-01

A simple, membrane-strip-based lateral-flow (LF) biosensor assay and a high-throughput microtiter plate assay have been combined with a reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of a small number (ten) of viable Mycobacterium (M.) avium subsp. paratuberculosis (MAP) cells in fecal samples. The assays are based on the identification of the RNA of the IS900 element of MAP. For the assay, RNA was extracted from fecal samples spiked with a known quantity of (101 to 106) MAP cells and amplified using RT-PCR and identified by the LF biosensor and the microtiter plate assay. While the LF biosensor assay requires only 30 min of assay time, the overall process took 10 h for the detection of 10 viable cells. The assays are based on an oligonucleotide sandwich hybridization assay format and use either a membrane flow through system with an immobilized DNA probe that hybridizes with the target sequence or a microtiter plate well. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye, sulforhodamine B. The dye in the liposomes provides a signal that can be read visually, quantified with a hand-held reflectometer, or with a fluorescence reader. Specificity analysis of the assays revealed no cross reactivity with other mycobacteria, such as M. avium complex, M. ulcerans, M. marium, M. kansasii, M. abscessus, M. asiaticum, M. phlei, M. fortuitum, M. scrofulaceum, M. intracellulare, M. smegmatis, and M. bovis. The overall assay for the detection of live MAP organisms is comparatively less expensive and quick, especially in comparison to standard MAP detection using a culture method requiring 6-8 weeks of incubation time, and is significantly less expensive than real-time PCR. PMID:19255522

Messenger RNA profiling of human platelets by microarray hybridization.

PubMed

Bugert, Peter; Dugrillon, Alex; Günaydin, Ayse; Eichler, Hermann; Klüter, Harald

2003-10-01

Platelets are generally believed to be inactive in terms of de novo protein synthesis. On the other hand, the presence of ribosomes and mRNA molecules is well established. Many studies have used reverse transcriptase (RT) -PCR for detection of gene transcripts in platelets. As RT-PCR is a very sensitive method, any leukocyte contamination of platelet preparations can lead to false results. We performed three filtration procedures to minimize leukocyte contamination of pooled buffy-coat platelet concentrates prior to RNA isolation. Furthermore, by applying a genomic PCR approach with 50 amplification cycles we demonstrated that nucleated cells were not detectable. Microarray hybridization was used to analyze 9,850 individual human genes in RNA from purified platelets. In total we identified 1,526 (15.5%) positive genes. The data were confirmed in six individual experiments each performed on a PC pooled from four individual blood donations. Genes specific for nucleated blood cells such as CD4, CD83 and others were negative and verified the purity of PC. Overrepresentation of positive genes was found in the functional categories of glycoproteins/integrins (22.6% vs. 15.5%, p=0.029) and receptors (20.7% vs. 15.5%, p<0.001). Gene transcripts encoding RANTES, GRO-alpha, MIP-1alpha, MIP-1beta, and others were found at high levels of signal intensity and confirmed literature data. This work provides a mRNA profile of human platelets and a complete list of results can be downloaded from the website of our institute www.ma.uni-heidelberg.de/inst/iti/plt_array.xls. The knowledge about gene transcripts may have an impact on the characterization of novel proteins and their functions in platelets.

Prostacyclin synthase expression and epigenetic regulation in nonsmall cell lung cancer.

PubMed

Cathcart, Mary-Clare; Gray, Steven G; Baird, Anne-Marie; Boyle, Elaine; Gately, Kathy; Kay, Elaine; Cummins, Robert; Pidgeon, Graham P; O'Byrne, Kenneth J

2011-11-15

Prostacyclin synthase (PGIS) metabolizes prostaglandin H(2), into prostacyclin. This study aimed to determine the expression profile of PGIS in nonsmall cell lung cancer (NSCLC) and examine potential mechanisms involved in PGIS regulation. PGIS expression was examined in human NSCLC and matched controls by reverse transcriptase polymerase chain reaction (RT-PCR), Western analysis, and immunohistochemistry. A 204-patient NSCLC tissue microarray was stained for PGIS and cyclooxygenase 2 (COX2) expression. Staining intensity was correlated with clinical parameters. Epigenetic mechanisms underpinning PGIS promoter expression were examined using RT-PCR, methylation-specific PCR, and chromatin immunoprecipitation analysis. PGIS expression was reduced/absent in human NSCLC protein samples (P < .0001), but not mRNA relative to matched controls. PGIS tissue expression was higher in squamous cell carcinoma (P = .004) and in male patients (P < .05). No significant correlation of PGIS or COX2 expression with overall patient survival was observed, although COX2 was prognostic for short-term (2-year) survival (P < .001). PGIS mRNA expression was regulated by DNA CpG methylation and histone acetylation in NSCLC cell lines, with chromatin remodeling taking place directly at the PGIS gene. PGIS mRNA expression was increased by both demethylation agents and histone deacetylase inhibitors. Protein levels were unaffected by demethylation agents, whereas PGIS protein stability was negatively affected by histone deacetylase inhibitors. PGIS protein expression is reduced in NSCLC, and does not correlate with overall patient survival. PGIS expression is regulated through epigenetic mechanisms. Differences in expression patterns between mRNA and protein levels suggest that PGIS expression and protein stability are regulated post-translationally. PGIS protein stability may have an important therapeutic role in NSCLC. Copyright © 2011 American Cancer Society.

Identification of growth stage molecular markers in Trichoderma sp. 'atroviride type B' and their potential application in monitoring fungal growth and development in soil.

PubMed

Mendoza-Mendoza, Artemio; Steyaert, Johanna; Nieto-Jacobo, Maria Fernanda; Holyoake, Andrew; Braithwaite, Mark; Stewart, Alison

2015-11-01

Several members of the genus Trichoderma are biocontrol agents of soil-borne fungal plant pathogens. The effectiveness of biocontrol agents depends heavily on how they perform in the complex field environment. Therefore, the ability to monitor and track Trichoderma within the environment is essential to understanding biocontrol efficacy. The objectives of this work were to: (a) identify key genes involved in Trichoderma sp. 'atroviride type B' morphogenesis; (b) develop a robust RNA isolation method from soil; and (c) develop molecular marker assays for characterizing morphogenesis whilst in the soil environment. Four cDNA libraries corresponding to conidia, germination, vegetative growth and conidiogenesis were created, and the genes identified by sequencing. Stage specificity of the different genes was confirmed by either Northern blot or quantitative reverse-transcriptase PCR (qRT-PCR) analysis using RNA from the four stages. con10, a conidial-specific gene, was observed in conidia, as well as one gene also involved in subsequent stages of germination (L-lactate/malate dehydrogenase encoding gene). The germination stage revealed high expression rates of genes involved in amino acid and protein biosynthesis, while in the vegetative-growth stage, genes involved in differentiation, including the mitogen-activated protein kinase kinase similar to Kpp7 from Ustilago maydis and the orthologue to stuA from Aspergillus nidulans, were preferentially expressed. Genes involved in cell-wall synthesis were expressed during conidiogenesis. We standardized total RNA isolation from Trichoderma sp. 'atroviride type B' growing in soil and then examined the expression profiles of selected genes using qRT-PCR. The results suggested that the relative expression patterns were cyclic and not accumulative.

Novel chromosomal rearrangements and break points at the t(6;9) in salivary adenoid cystic carcinoma: association with MYB-NFIB chimeric fusion, MYB expression, and clinical outcome.

PubMed

Mitani, Yoshitsugu; Rao, Pulivarthi H; Futreal, P Andrew; Roberts, Dianna B; Stephens, Philip J; Zhao, Yi-Jue; Zhang, Li; Mitani, Mutsumi; Weber, Randal S; Lippman, Scott M; Caulin, Carlos; El-Naggar, Adel K

2011-11-15

To investigate the molecular genetic heterogeneity associated with the t(6:9) in adenoid cystic carcinoma (ACC) and correlate the findings with patient clinical outcome. Multimolecular and genetic techniques complemented with massive pair-ended sequencing and single-nucleotide polymorphism array analyses were used on tumor specimens from 30 new and 52 previously analyzed fusion transcript-negative ACCs by reverse transcriptase PCR (RT-PCR). MYB mRNA expression level was determined by quantitative RT-PCR. The results of 102 tumors (30 new and 72 previously reported cases) were correlated with the clinicopathologic factors and patients' survival. The FISH analysis showed 34 of 82 (41.5%) fusion-positive tumors and molecular techniques identified fusion transcripts in 21 of the 82 (25.6%) tumors. Detailed FISH analysis of 11 out the 15 tumors with gene fusion without transcript formation showed translocation of NFIB sequences to proximal or distal sites of the MYB gene. Massive pair-end sequencing of a subset of tumors confirmed the proximal translocation to an NFIB sequence and led to the identification of a new fusion gene (NFIB-AIG1) in one of the tumors. Overall, MYB-NFIB gene fusion rate by FISH was in 52.9% whereas fusion transcript forming incidence was 38.2%. Significant statistical association between the 5' MYB transcript expression and patient survival was found. We conclude that: (i) t(6;9) results in complex genetic and molecular alterations in ACC, (ii) MYB-NFIB gene fusion may not always be associated with chimeric transcript formation, (iii) noncanonical MYB-NFIB gene fusions occur in a subset of tumors, (iv) high MYB expression correlates with worse patient survival.

Association between human papilloma virus/Epstein-Barr virus coinfection and oral carcinogenesis.

PubMed

Jiang, Ru; Ekshyyan, Oleksandr; Moore-Medlin, Tara; Rong, Xiaohua; Nathan, Sean; Gu, Xin; Abreo, Fleurette; Rosenthal, Eben L; Shi, Mingxia; Guidry, Joseph T; Scott, Rona S; Hutt-Fletcher, Lindsey M; Nathan, Cherie-Ann O

2015-01-01

The recent epidemic of head and neck squamous cell carcinomas associated with human papilloma virus (HPV) has not addressed its association with lymphoid tissue in the oropharynx or the potential role of Epstein-Barr virus (EBV)/HPV coinfection. The prevalence of HPV and EBV infection/coinfection and CD21 mRNA expression were determined in normal and cancerous tissues from the oropharynx using in situ hybridization (ISH), p16, and quantitative reverse transcriptase PCR (qRT-PCR). The effects of coinfection on tumorigenicity were evaluated using proliferation and invasion assays. Normal oropharynx, tonsil, non-cancer base of tongue (BOT), and BOT from sleep apnea patients demonstrated EBV positivity ranging from 7% to 36% depending on the site and methods of detection used (qRT-PCR or ISH). Among non-malignant BOT samples, HPV positivity was noted only in 20%. The percent of tonsil and BOT cancers positive for HPV (up to 63% and 80%, respectively) or coinfected with HPV/EBV (up to 25% and 70%, respectively) were both significantly associated with cancer status. Notably, HPV/EBV coinfection was observed only in malignant tissue originating in lymphoid-rich oropharynx sites (tonsil, BOT). CD21 mRNA (the major EBV attachment receptor) was detected in tonsil and BOT epithelium, but not in soft-palate epithelium. Coinfected cell lines showed a significant increase in invasiveness (P < 0.01). There is a high prevalence of HPV/EBV infection and coinfection in BOT and tonsil cancers, possibly reflecting their origins in lymphoid-rich tissue. In vitro, cells modeling coinfection have an increased invasive potential. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Quantitative assessment of hTERT mRNA expression in dysplastic nodules of HBV-related hepatocarcinogenesis.

PubMed

Oh, Bong-Kyeong; Kim, Young-Joo; Park, Young Nyun; Choi, Jinsub; Kim, Kyung Sik; Park, Chanil

2006-04-01

Telomerase reverse transcriptase (hTERT) is the rate-limiting determinant of telomerase, which is critical for carcinogenesis. Dysplastic nodules (DNs) appear to be preneoplastic lesions of hepatocellular carcinomas (HCCs). In this study, in order to characterize DNs, hTERT mRNA, hTERT gene dosage, and mRNA for c-myc, a transcriptional activator of hTERT were studied in human multi-step hepatocarcinogenesis. Fifty four hepatic nodules including 5 large regenerative nodules, 14 low-grade DNs, 7 high-grade DNs, 11 DNs with HCC foci and 17 HCCs, 23 livers with chronic hepatitis/cirrhosis, and 6 normal livers were examined. Transcript levels were measured by real-time quantitative RT-PCR and gene dosages by real-time PCR and Southern blotting. The hTERT mRNA levels increased with the progression of hepatocarcinogenesis, and a significant induction in the transition between low- and high-grade DNs was seen. Most high-grade DNs strongly expressed hTERT mRNA at levels similar to those of HCCs. Twenty-one percent of low-grade DNs had high levels of hTERT mRNA, up to those of high-grade DNs and there was no difference in the pathological features between low-grade DNs with and without increased hTERT mRNA levels. No correlation was found between hTERT mRNA levels, hTERT gene dosage, and c-myc mRNA levels. These results suggest that the induction of hTERT mRNA is an important early event and that its measurement by real-time quantitative RT-PCR is a useful tool to detect premalignant/malignant tendencies in hepatic nodules. However, hTERT gene dosage and c-myc expression are not the main mechanisms regulating hTERT expression in hepatocarcinogenesis.

Prospective surveillance and molecular characterization of seasonal influenza in a university cohort in Singapore.

PubMed

Virk, Ramandeep Kaur; Tambyah, Paul Anantharajah; Inoue, Masafumi; Lim, Elizabeth Ai-Sim; Chan, Ka-Wei; Chua, Catherine; Tan, Boon-Huan

2014-01-01

Southeast Asia is believed to be a potential locus for the emergence of novel influenza strains, and therefore accurate sentinel surveillance in the region is critical. Limited information exists on sentinel surveillance of influenza-like illness (ILI) in young adults in Singapore in a University campus setting. The objective of the present study was to determine the proportion of ILI caused by influenza A and B viruses in a university cohort in Singapore. We conducted a prospective surveillance study from May through October 2007, at the National University of Singapore (NUS). Basic demographic information and nasopharyngeal swabs were collected from students and staff with ILI. Reverse-transcriptase PCR (RT-PCR) and viral isolation were employed to detect influenza viruses. Sequencing of hemagglutinin (HA) and neuraminidase (NA) genes of some representative isolates was also performed. Overall proportions of influenza A and B virus infections were 47/266 (18%) and 9/266 (3%) respectively. The predominant subtype was A/H3N2 (55%) and the rest were A/H1N1 (45%). The overall sensitivity difference for detection of influenza A viruses using RT-PCR and viral isolation was 53%. Phylogenetic analyses of HA and NA gene sequences of Singapore strains showed identities higher than 98% within both the genes. The strains were more similar to strains included in the WHO vaccine recommendation for the following year (2008). Genetic markers of oseltamivir resistance were not detected in any of the sequenced Singapore isolates. HA and NA gene sequences of Singapore strains were similar to vaccine strains for the upcoming influenza season. No drug resistance was found. Sentinel surveillance on university campuses should make use of molecular methods to better detect emerging and re-emerging influenza viral threats.

Modulation of adipocyte biology by δ(9)-tetrahydrocannabinol.

PubMed

Teixeira, Diana; Pestana, Diogo; Faria, Ana; Calhau, Conceição; Azevedo, Isabel; Monteiro, Rosário

2010-11-01

It is recognized that the endocannabinoid system (ECS) plays a crucial role in the modulation of food intake and other aspects of energy metabolism. In this study, we aimed to investigate the effects of Δ(9)-tetrahydrocannabinol (THC) on adipocyte biology. 3T3-L1 cells were used to evaluate proliferation by sulforhodamine B (SRB) staining and methyl-(3)H-thymidine incorporation after 48 or 72 h of treatment with THC (1-500 nmol/l). Cells were differentiated in the presence or absence of the cannabinoid, and adipogenesis was determined by measuring lipid accumulation and peroxisome proliferator-activated receptor γ (PPARγ) transcription through reverse transcriptase-PCR (RT-PCR). Lipolysis was quantified under basal conditions or after isoproterenol (IP, 100 nmol/l) or insulin (INS, 100 nmol/l) treatment. Transforming growth factor β (TGFβ), diacylglycerol lipase α, and N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD) transcriptions were determined by RT-PCR in preadipocytes and adipocytes and adiponectin only in adipocytes. THC treatment increased culture protein content and reduced methyl-(3)H-thymidine incorporation. Cells treated with THC underwent adipogenesis shown by the expression of PPARγ and had increased lipid accumulation. Basal and IP-stimulated lipolyses were inhibited by THC and there was no effect on lipolysis of INS-treated adipocytes. The effects on methyl-(3)H-thymidine incorporation and lipolysis seem to be mediated through CB1- and CB2-dependent pathways. THC decreased NAPE-PLD in preadipocytes and increased adiponectin and TGFβ transcription in adipocytes. These results show that the ECS interferes with adipocyte biology and may contribute to adipose tissue (AT) remodeling. Although these observations point toward increased AT deposition, the stimulation of adiponectin production and inhibition of lipolysis may be in favor of improved INS sensitivity under cannabinoid influence.

Evaluation of Liquid- and Fog-Based Application of Sterilox Hypochlorous Acid Solution for Surface Inactivation of Human Norovirus▿

PubMed Central

Park, Geun Woo; Boston, Deyanna M.; Kase, Julie A.; Sampson, Mark N.; Sobsey, Mark D.

2007-01-01

Noroviruses (NVs) are the most frequent cause of outbreaks of gastroenteritis in common settings, with surface-mediated transfer via contact with fecally contaminated surfaces implicated in exposure. NVs are environmentally stable and persistent and have a low infectious dose. Several disinfectants have been evaluated for efficacy to control viruses on surfaces, but the toxicity and potential damage to treated materials limits their applicability. Sterilox hypochlorous acid (HOCl) solution (HAS) has shown broad-spectrum antimicrobial activity while being suitable for general use. The objectives of this study were to evaluate the efficacy of HAS to reduce NV both in aqueous suspensions and on inanimate carriers. HOCl was further tested as a fog to decontaminate large spaces. HOCl effectiveness was evaluated using nonculturable human NV measured by reverse transcriptase PCR (RT-PCR) and two surrogate viruses, coliphage MS2 and murine NV, that were detected by both infectivity and RT-PCR. Exposing virus-contaminated carriers of ceramic tile (porous) and stainless steel (nonporous) to 20 to 200 ppm of HOCl solution resulted in ≥99.9% (≥3 log10) reductions of both infectivity and RNA titers of tested viruses within 10 min of exposure time. HOCl fogged in a confined space reduced the infectivity and RNA titers of NV, murine NV, and MS2 on these carriers by at least 99.9% (3 log10), regardless of carrier location and orientation. We conclude that HOCl solution as a liquid or fog is likely to be effective in disinfecting common settings to reduce NV exposures and thereby control virus spread via fomites. PMID:17483283

Rapid group-, serotype-, and vaccine strain-specific identification of poliovirus isolates by real-time reverse transcription-PCR using degenerate primers and probes containing deoxyinosine residues.

PubMed

Kilpatrick, David R; Yang, Chen-Fu; Ching, Karen; Vincent, Annelet; Iber, Jane; Campagnoli, Ray; Mandelbaum, Mark; De, Lina; Yang, Su-Ju; Nix, Allan; Kew, Olen M

2009-06-01

We have adapted our previously described poliovirus diagnostic reverse transcription-PCR (RT-PCR) assays to a real-time RT-PCR (rRT-PCR) format. Our highly specific assays and rRT-PCR reagents are designed for use in the WHO Global Polio Laboratory Network for rapid and large-scale identification of poliovirus field isolates.

Microbiota of an Italian Grana-Like Cheese during Manufacture and Ripening, Unraveled by 16S rRNA-Based Approaches.

PubMed

Alessandria, Valentina; Ferrocino, Ilario; De Filippis, Francesca; Fontana, Mauro; Rantsiou, Kalliopi; Ercolini, Danilo; Cocolin, Luca

2016-07-01

The microbial ecology of cheese involves a rich and complex interaction between starter lactic acid bacteria and nonstarter lactic acid bacteria (NSLAB), mainly originating from raw milk and/or from the environment, that can contribute to the final characteristics of cheese. The aim of the present research was the exploration of the active microbiota by RNA-based approaches during the manufacturing and ripening of a Grana-like cheese. Reverse transcriptase PCR (RT-PCR)-denaturing gradient gel electrophoresis (DGGE) and RNA-based high-throughput sequencing were applied to profile microbial populations, while the enumeration of active bacteria was carried out by using quantitative PCR (qPCR). Three different cheese productions (named D, E, and F) collected in the same month from the same dairy plant were analyzed. The application of the qPCR protocol revealed the presence of 7 log CFU/ml of bacterial load in raw milk, while, during ripening, active bacterial populations ranged from <4 to 8 log CFU/ml. The natural whey starters used in the three productions showed the same microbiota composition, characterized by the presence of Lactobacillus helveticus and Lactobacillus delbrueckii Nevertheless, beta-diversity analysis of the 16S rRNA sequencing data and RT-PCR-DGGE showed a clear clustering of the samples according to the three productions, probably driven by the different milks used. Milk samples were found to be characterized by the presence of several contaminants, such as Propionibacterium acnes, Acidovorax, Acinetobacter, Pseudomonas, and NSLAB. The core genera of the starter tended to limit the development of the spoilage bacteria only in two of the three batches. This study underlines the influence of different factors that can affect the final microbiota composition of the artisanal cheese. This study highlights the importance of the quality of the raw milk in the production of a hard cheese. Independent from the use of a starter culture, raw milk with low microbiological quality can negatively affect the populations of lactic acid bacteria and, as a consequence, impact the quality of the final product due to metabolic processes associated with spoilage bacteria. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Course of c-myc mRNA expression in the regenerating mouse testis determined by competitive reverse transcriptase polymerase chain reaction.

PubMed

Amendola, R

1994-11-01

The c-myc proto-oncogene is a reliable marker of the "G0-early G1" transition, and its down-regulation is believed to be necessary to obtain cellular differentiation. In murine spermatogenesis, the level of c-myc transcripts does not correlate with the rate of cellular division. Proliferation of supposed staminal spermatogonia to reproduce themselves is induced with a local 5 Gy X-ray dose in 90-day-old C57Bl/6 mice. c-myc quantification by a newly developed competitive reverse transcriptase polymerase chain reaction (RT-PCR) was carried out to follow the expression course of this proto-oncogene. Damage and restoration of spermatogenesis were analyzed at days 3, 6, 9, 10, 13, 30, and 60 after injury by relative testes/body weight determination and histological examination. Proliferative status was determined by histone H3 Northern blot analysis. c-myc mRNA level was 10 times higher after 3 days in the irradiated animals compared to the controls. An increasing number of copies were noted up to 10 days, but promptly decreased to the base level found for irradiated mice from 13 to 60 days. Interestingly, the expression of histone H3 detected S phase only in testes at 60 days from damage.

Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth and look-alike disease viruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hindson, B J; Baker, B R; Bentley Tammero, L F

2007-09-18

A high-throughput multiplexed assay (Multiplex Version 1.0) was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspectmore » cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRTPCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.« less

Rapid and sensitive detection of canine distemper virus by one-tube reverse transcription-insulated isothermal polymerase chain reaction.

PubMed

Wilkes, Rebecca P; Tsai, Yun-Long; Lee, Pei-Yu; Lee, Fu-Chun; Chang, Hsiao-Fen Grace; Wang, Hwa-Tang Thomas

2014-09-09

Canine distemper virus (CDV) has been associated with outbreaks of canine infectious respiratory disease in shelters and boarding kennel environments. POCKITTM Nucleic Acid Analyzer is a field-deployable device capable of generating automatically interpreted insulated isothermal polymerase chain reaction (iiPCR) results from extracted nucleic acid within one hour. In this study, reverse transcription iiPCR (RT-iiPCR) was developed to facilitate point-of-need diagnosis of CDV infection. Analytical sensitivity (limit of detection 95%) of the established CDV RT-iiPCR was about 11 copies of in vitro transcribed RNA per reaction. CDV RT-iiPCR generated positive signals from CDV, but not Bordetella bronchiseptica, canine parvovirus, canine herpesvirus, canine adenovirus 2, canine influenza virus (subtype H3N8), canine parainfluenza virus, and canine respiratory coronavirus. To evaluate accuracy of the established reaction in canine distemper clinical diagnosis, 110 specimens from dogs, raccoons, and foxes suspected with CDV infection were tested simultaneously by CDV RT-iiPCR and real-time RT-PCR. CDV RT-iiPCR demonstrated excellent sensitivity (100%) and specificity (100%), compared to real-time RT-PCR. The results indicated an excellent correlation between RT-iiPCR and a reference real time RT-PCR method. Working in a lyophilized format, the established method has great potential to be used for point-of-care diagnosis of canine distemper in animals, especially in resource-limited facilities.

Molecular characters of melon (Cucumismelo L. "Tacapa") in response to karst critical land

NASA Astrophysics Data System (ADS)

Rachmawati, Yuanita; Daryono, Budi Setiadi; Aristya, Ganies Riza

2017-06-01

Yogyakarta district has 158.600 ha critical land and spread off in three Agro Ecosystem zones. Two of them are karsts critical land. Critical lands which contain calcium carbonate in high concentration and water dehydration in upper surface give abiotic stress in wide range of plant. Melon cultivar TACAPA has superior characteristic derived from parental crossing, ♀ Action 434 and ♂ PI 371795 and potential to be developed in karsts critical land. Abscicic acid (ABA) is a phytohormone expressed by plant in abiotic stress condition. CmBG1 is a gene which regulate ABA hormone in melon. The purposes of this research were examining the molecular character of melon cultivar TACAPA in response to karsts critical land in order to study molecular characterization of CmBG1 gene. Analysis was done qualitatively by using Reverse Transcriptase-PCR (RT-PCR) and Electrophoresis, while quantitative analysis was conducted by observing absorbance score in spectrophotometer. CmBG1 gene expression is examined by using Real time PCR (qPCR). Molecular characters obtained are CmBG1 detected in size ±1258 bp, CmBG1 gene concentrations in melon which planted in control media are lower than melon in critical lands media. These results are similar with the real time quantitative analysis method. It also be revealed that melon TACAPA is more potential plant compared to another cultivar that can be developed in karst critical land area.

Role of chicken astrovirus as a causative agent of gout in commercial broilers in India.

PubMed

Bulbule, N R; Mandakhalikar, K D; Kapgate, S S; Deshmukh, V V; Schat, K A; Chawak, M M

2013-01-01

Several outbreaks of gout were reported in commercial broilers in India during 2011 and 2012, causing up to 40% mortality in the birds. Gross and histopathological observations confirmed gout. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis from kidney samples of gout-affected birds indicated the presence of chicken astrovirus (CAstV) in 41.7% of cases and a mixed infection of CAstV and avian nephritis virus (ANV) in 36.4% of cases. CAstV isolated from gout-affected kidneys by inoculating embryonated specific pathogen free (SPF) eggs showed dwarfing in embryos and a cytopathic effect in chicken embryo kidney cells. Inoculation of 1-day-old SPF and broiler chicks with CAstVs caused gout and mortality between 4 and 10 days post inoculation. Virus isolation and qRT-PCR analysis showed the presence of only CAstV in inoculated chicks. Sequence analysis of capsid genes indicated a major group of Indian CAstVs that displayed 92.0 to 99.2% intergroup amino acid identity and 83.9 to 90.4% identity with subgroup Bi CAstVs of UK origin. We designated this group Indian Bi. Analysis of the partial polymerase amino acid sequences of our isolates indicated two groups of CAstVs (Indian 1 and 2) that displayed 90.2 to 95.5% amino acid identity between them. We thus report for the first time that, in addition to infectious bronchitis virus and ANV, CAstVs are a causative agent of gout.

Identification of testis-specific male contraceptive targets: insights from transcriptional profiling of the cycle of the rat seminiferous epithelium and purified testicular cells.

PubMed

Johnston, Daniel S; Jelinsky, Scott A; Zhi, Yu; Finger, Joshua N; Kopf, Gregory S; Wright, William W

2007-12-01

In an effort to identify novel targets for the development of nonhormonal male contraceptives, genome-wide transcriptional profiling of the rat testis was performed. Specifically, enzymatically purified spermatogonia plus early spermatocyctes, pachytene spermatocytes, round spermatids, and Sertoli cells was analyzed along with microdissected rat seminiferous tubules at stages I, II-III, IV-V, VI, VIIa,b, VIIc,d, VIII, IX- XI, XII, XIII-XIV of the cycle of the seminiferous epithelium using RAE 230_2.0 microarrays. The combined analysis of these studies identified 16,971 expressed probe sets on the array. How these expression data, combined with additional bioinformatic data analysis and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis, led to the identification of 58 genes that have 1000-fold higher expression transcriptionally in the testis when compared to over 20 other nonreproductive tissues is described. The products of these genes may play important roles in testicular and/or sperm function, and further investigation on their utility as nonhormonal contraceptive targets is warranted. Moreover, these microarray data have been used to expedite the identification of a mutation in RIKEN cDNA 2410004F06 gene as likely being responsible for spermatogenic failure in a line of infertile mice generated by N-ethyl-N-nitrosourea (ENU) mutagenesis. The microarray data and the qRT-PCR data described are available in the Mammalian Reproductive Genetics database (http://mrg.genetics.washington.edu/).

Gene expression profiling analysis of the effects of low-intensity pulsed ultrasound on induced pluripotent stem cell-derived neural crest stem cells.

PubMed

Xia, Bin; Zou, Yang; Xu, Zhiling; Lv, Yonggang

2017-11-01

Low-intensity pulsed ultrasound (LIPUS) is a noninvasive technique that has been shown to affect cell proliferation, migration, and differentiation and promote the regeneration of damaged peripheral nerve. Our previous studies had proved that LIPUS can significantly promote the neural differentiation of induced pluripotent stem cell-derived neural crest stem cells (iPSCs-NCSCs) and enhance the repair of rat-transected sciatic nerve. To further explore the underlying mechanisms of LIPUS treatment of iPSCs-NCSCs, this study reported the gene expression profiling analysis of iPSCs-NCSCs before and after LIPUS treatment using the RNA-sequencing (RNA-Seq) method. It was found that expression of 76 genes of iPSCs-NCSCs cultured in a serum-free neural induction medium and expression of 21 genes of iPSCs-NCSCs cultured in a neuronal differentiation medium were significantly changed by LIPUS treatment. The differentially expressed genes are related to angiogenesis, nervous system activity and functions, cell activities, and so on. The RNA-seq results were further verified by a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). High correlation was observed between the results obtained from qRT-PCR and RNA-Seq. This study presented new information on the global gene expression patterns of iPSCs-NCSCs after LIPUS treatment and may expand the understanding of the complex molecular mechanism of LIPUS treatment of iPSCs-NCSCs. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

Regulation of heat shock protein message in Jurkat cells cultured under serum-starved and gravity-altered conditions

NASA Technical Reports Server (NTRS)

Lewis, M. L.; Hughes-Fulford, M.

2000-01-01

Although our understanding of effects of space flight on human physiology has advanced significantly over the past four decades, the potential contribution of stress at the cellular and gene regulation level is not characterized. The objective of this ground-based study was to evaluate stress gene regulation in cells exposed to altered gravity and environmentally suboptimal conditions. We designed primers to detect message for both the constitutive and inducible forms of the heat shock protein, HSP-70. Applying the reverse transcriptase-polymerase chain reaction (RT-PCR), we probed for HSP-70 message in human acute T-cell leukemia cells, Jurkat, subjected to three types of environmental stressors: (1) altered gravity achieved by centrifugation (hypergravity) and randomization of the gravity vector in rotating bioreactors, (2) serum starvation by culture in medium containing 0.05% serum, and (3) temperature elevation (42 degrees C). Temperature elevation, as the positive control, significantly increased HSP-70 message, while centrifugation and culture in rotating bioreactors did not upregulate heat shock gene expression. We found a fourfold increase in heat shock message in serum-starved cells. Message for the housekeeping genes, actin and cyclophilin, were constant and comparable to unstressed controls for all treatments. We conclude that gravitational perturbations incurred by centrifugal forces, exceeding those characteristic of a Space Shuttle launch (3g), and culture in rotating bioreactors do not upregulate HSP-70 gene expression. In addition, we found RT-PCR useful for evaluating stress in cultured cells. Copyright 2000 Wiley-Liss, Inc.

Laboratory confirmation of measles in elimination settings: experience from the Republic of the Marshall Islands, 2003.

PubMed

Hyde, Terri B; Nandy, Robin; Hickman, Carole J; Langidrik, Justina R; Strebel, Peter M; Papania, Mark J; Seward, Jane F; Bellini, William J

2009-02-01

To highlight the complications involved in interpreting laboratory tests of measles immunoglobulin M (IgM) for confirmation of infection during a measles outbreak in a highly vaccinated population after conducting a mass immunization campaign as a control measure. This case study was undertaken in the Republic of the Marshall Islands during a measles outbreak in 2003, when response immunization was conducted. A measles case was defined as fever and rash and one or more of cough, coryza or conjunctivitis. Between 13 July and 7 November 2003, serum samples were obtained from suspected measles cases for serologic testing and nasopharyngeal swabs were taken for viral isolation by reverse transcriptase polymerase chain reaction (RT-PCR). Specimens were collected from 201 suspected measles cases (19% of total): of the ones that satisfied the clinical case definition, 45% were IgM positive (IgM+) and, of these, 24% had received measles vaccination within the previous 45 days (up to 45 days after vaccination an IgM+ result could be due to either vaccination or wild-type measles infection). The proportion of IgM+ results varied with clinical presentation, the timing of specimen collection and vaccination status. Positive results on RT-PCR occurred in specimens from eight IgM-negative and four IgM+ individuals who had recently been vaccinated. During measles outbreaks, limiting IgM testing to individuals who meet the clinical case definition and have not been recently vaccinated allows for measles to be confirmed while conserving resources.

Susceptibility of zebrafish (Danio rerio) to a model pathogen, spring viremia of carp virus

USGS Publications Warehouse

Sanders, George E.; Batts, William N.; Winton, James R.

2003-01-01

To improve our understanding of the genetic basis of fish disease, we developed a pathogen model, using zebrafish (Danio rerio) and spring virema of carp virus (SVCV). Replicate groups of 10 fish were acclimated to 20 or 24°C, then were exposed to SVCV concentrations of 103 to 105 plaque-forming units per milliliter (PFU/ml) of water and observed daily. In a second trial, fish were acclimated to 15°C, and replicate groups of 10 fish were exposed to SVCV at a concentration of 105 PFU/ml; however, the temperature was raised 1°C/wk. Moribund fish were collected for histologic examination, and dead fish were assayed for virus by use of cell culture and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Mortality exceeded 50% in fish exposed to 105 PFU of SVCV/ml at the lower temperatures. Clinical signs of disease became evident seven days after viral exposure and were observed most consistently in fish of the 105 PFU/ml groups. Affected zebrafish were anorectic and listless, with epidermal petechial hemorrhages followed by death. Use of plaque assays and RT-PCR analysis confirmed presence of SVCV at titers ≥ 104 PFU/g of tissue. Histologic lesions included multifocal brachial necrosis and melanomacrophage proliferation in gills, liver, and kidneys. These results indicate that zebrafish are susceptible to infection by SVCV under conditions that mimic a natural route of exposure.

Characterization of xanthine dehydrogenase and aldehyde oxidase of Marsupenaeus japonicus and their response to microbial pathogen.

PubMed

Okamura, Yo; Inada, Mari; Elshopakey, Gehad Elsaid; Itami, Toshiaki

2018-05-16

Reactive oxygen species (ROS) play key roles in many physiological processes. In particular, the sterilization mechanism of bacteria using ROS in macrophages is a very important function for biological defense. Xanthine dehydrogenase (XDH) and aldehyde oxidase (AOX), members of the molybdo-flavoenzyme subfamily, are known to generate ROS. Although these enzymes occur in many vertebrates, some insects, and plants, little research has been conducted on XDHs and AOXs in crustaceans. Here, we cloned the entire cDNA sequences of XDH (MjXDH: 4328 bp) and AOX (MjAOX: 4425 bp) from Marsupenaeus japonicus (kuruma shrimp) using reverse transcriptase-polymerase chain reaction (RT-PCR) and random amplification of cDNA ends (RACE). Quantitative real-time RT-PCR transcriptional analysis revealed that MjXDH mRNA is highly expressed in heart and stomach tissues, whereas MjAOX mRNA is highly expressed in the lymphoid organ and intestinal tissues. Furthermore, expression of MjAOX was determined to be up-regulated in the lymphoid organ in response to Vibrio penaeicida at 48 and 72 h after injection; in contrast, hydrogen peroxide (H 2 O 2 ) concentrations increased significantly at 6, 12, 48, and 72 h after injection with white spot syndrome virus (WSSV) and at 72 h after injection with V. penaeicida. To the best of our knowledge, this study is the first to have identified and cloned XDH and AOX from a crustacean species.

Low-level lasers and mRNA levels of reference genes used in Escherichia coli

NASA Astrophysics Data System (ADS)

Teixeira, A. F.; Machado, Y. L. R. C.; Fonseca, A. S.; Mencalha, A. L.

2016-11-01

Low-level lasers are widely used for the treatment of diseases and antimicrobial photodynamic therapy. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is widely used to evaluate mRNA levels and output data from a target gene are commonly relative to a reference mRNA that cannot vary according to treatment. In this study, the level of reference genes from Escherichia coli exposed to red or infrared lasers at different fluences was evaluated. E. coli AB1157 cultures were exposed to red (660 nm) and infrared (808 nm) lasers, incubated (20 min, 37 °C), the total RNA was extracted, and cDNA synthesis was performed to evaluate mRNA levels from arcA, gyrA and rpoA genes by RT-qPCR. Melting curves and agarose gel electrophoresis were carried out to evaluate specific amplification. Data were analyzed by geNorm, NormFinder and BestKeeper. The melting curve and agarose gel electrophoresis showed specific amplification. Although mRNA levels from arcA, gyrA or rpoA genes presented no significant variations trough a traditional statistical analysis, Excel-based tools revealed that these reference genes are not suitable for E. coli cultures exposed to lasers. Our data showed that exposure to low-level red and infrared lasers at different fluences alter the mRNA levels from arcA, gyrA and rpoA in E. coli cells.

Selection of Reference Genes for RT-qPCR Analysis in Coccinella septempunctata to Assess Un-intended Effects of RNAi Transgenic Plants.

PubMed

Yang, Chunxiao; Preisser, Evan L; Zhang, Hongjun; Liu, Yong; Dai, Liangying; Pan, Huipeng; Zhou, Xuguo

2016-01-01

The development of genetically engineered plants that employ RNA interference (RNAi) to suppress invertebrate pests opens up new avenues for insect control. While this biotechnology shows tremendous promise, the potential for both non-target and off-target impacts, which likely manifest via altered mRNA expression in the exposed organisms, remains a major concern. One powerful tool for the analysis of these un-intended effects is reverse transcriptase-quantitative polymerase chain reaction, a technique for quantifying gene expression using a suite of reference genes for normalization. The seven-spotted ladybeetle Coccinella septempunctata , a commonly used predator in both classical and augmentative biological controls, is a model surrogate species used in the environmental risk assessment (ERA) of plant incorporated protectants (PIPs). Here, we assessed the suitability of eight reference gene candidates for the normalization and analysis of C. septempunctata v-ATPase A gene expression under both biotic and abiotic conditions. Five computational tools with distinct algorisms, geNorm, Normfinder, BestKeeper , the Δ C t method, and RefFinder , were used to evaluate the stability of these candidates. As a result, unique sets of reference genes were recommended, respectively, for experiments involving different developmental stages, tissues, and ingested dsRNAs. By providing a foundation for standardized RT-qPCR analysis in C. septempunctata , our work improves the accuracy and replicability of the ERA of PIPs involving RNAi transgenic plants.

Altered serum microRNAs as biomarkers for the early diagnosis of pulmonary tuberculosis infection

PubMed Central

2012-01-01

Background Pulmonary tuberculosis (TB) is a highly lethal infectious disease and early diagnosis of TB is critical for the control of disease progression. The objective of this study was to profile a panel of serum microRNAs (miRNAs) as potential biomarkers for the early diagnosis of pulmonary TB infection. Methods Using TaqMan Low-Density Array (TLDA) analysis followed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) validation, expression levels of miRNAs in serum samples from 30 patients with active tuberculosis and 60 patients with Bordetella pertussis (BP), varicella-zoster virus (VZV) and enterovirus (EV) were analyzed. Results The Low-Density Array data showed that 97 miRNAs were differentially expressed in pulmonary TB patient sera compared with healthy controls (90 up-regulated and 7 down-regulated). Following qRT-PCR confirmation and receiver operational curve (ROC) analysis, three miRNAs (miR-361-5p, miR-889 and miR-576-3p) were shown to distinguish TB infected patients from healthy controls and other microbial infections with moderate sensitivity and specificity (area under curve (AUC) value range, 0.711-0.848). Multiple logistic regression analysis of a combination of these three miRNAs showed an enhanced ability to discriminate between these two groups with an AUC value of 0.863. Conclusions Our study suggests that altered levels of serum miRNAs have great potential to serve as non-invasive biomarkers for early detection of pulmonary TB infection. PMID:23272999

Expectoration of Flaviviruses during sugar feeding by mosquitoes (Diptera: Culicidae).

PubMed

van den Hurk, Andrew F; Johnson, Petrina H; Hall-Mendelin, Sonja; Northill, Judy A; Simmons, Russell J; Jansen, Cassie C; Frances, Stephen P; Smith, Greg A; Ritchie, Scott A

2007-09-01

Biological transmission of arboviruses to a vertebrate host occurs when virions are expelled along with saliva during blood feeding by a hematophagous arthropod. We undertook experiments to determine whether mosquitoes expectorate flaviviruses in their saliva while sugar feeding. Batches of Culex annulirostris Skuse and Culex gelidus Theobald (Diptera: Culicidae) were orally infected with Japanese encephalitis (family Flaviviridae, genus Flavivirus, JEV), Kunjin (family Flaviviridae, genus Flavivirus, KUNV; a subtype of West Nile virus), and Murray Valley encephalitis (family Flaviviridae, genus Flavivirus, MVEV) viruses. After a 7-d extrinsic incubation, these mosquitoes were offered sucrose meals via cotton pledgets, which were removed daily and processed for viral RNA by using real-time TaqMan reverse transcriptase-polymerase chain reaction (RT-PCR) assays. JEV, MVEV, and KUNV RNA was detected in all pledgets removed from batches of Cx. gelidus on days 7-14 postexposure. In contrast, detection rates were variable for Cx. annulirostris, with KUNV detected in 0.3 M sucrose pledgets on all days postexposure, and JEV and MVEV detected on 57 and 50% of days postexposure, respectively. Higher concentrations of sucrose in the pledget did not increase virus detection rates. When individual JEV-infected Cx. gelidus were exposed to the sucrose pledget, 73% of mosquitoes expectorated virus with titers that were detectable by TaqMan RT-PCR. These results clearly show that flaviviruses are expectorated by infected mosquitoes during the process of sugar feeding on artificial pledgets. Potential applications of the method for arboviral bioassays and field surveillance are discussed.

The role of phenylalanine-119 of the reverse transcriptase of mouse mammary tumour virus in DNA synthesis, ribose selection and drug resistance.

PubMed

Entin-Meer, Michal; Sevilya, Ziv; Hizi, Amnon

2002-10-15

Phe-119 in the reverse transcriptase (RT) of mouse mammary tumour virus (MMTV) is homologous with Tyr-115 in HIV type 1 (HIV-1) RT and to Phe-155 in murine leukaemia virus (MLV) RT. By mutating these residues in HIV-1 and MLV RTs (which are strict DNA polymerases) the enzymes were shown to function also as RNA polymerases. Owing to the uniqueness of MMTV as a type B retrovirus, we have generated a Phe-119-Val mutant of MMTV RT to study the involvement of this residue in affecting the catalytic features of this RT. The data presented here show that the mutant MMTV RT can incorporate both deoxyribonucleosides and ribonucleosides while copying either RNA or DNA. In addition, this mutant RT shows resistance to nucleoside analogues and an enhanced fidelity of DNA synthesis; all relative to the wild-type enzyme. The Phe-119-Val mutant is also different from the wild-type enzyme in its preference for most template primers tested and in its ability to synthesize DNA under non-processive and processive conditions. Overall, it is likely that the aromatic side chain of Phe-119 is located at the dNTP-binding site of MMTV RT and thus might be part of a putative "steric gate" that prevents the incorporation of nucleoside triphosphates. Since the only three-dimensional structures of RTs published so far are those of HIV-1 and MLV, it is likely that MMTV RT folds quite similarly to these RTs.

The reverse transcriptase encoded by LINE-1 retrotransposons in the genesis, progression and therapy of cancer

NASA Astrophysics Data System (ADS)

Sciamanna, Ilaria; De Luca, Chiara; Spadafora, Corrado

2016-02-01

In higher eukaryotic genomes, Long Interspersed Nuclear Element 1 (LINE-1) retrotransposons represent a large family of repeated genomic elements. They transpose using a reverse transcriptase (RT), which they encode as part of the ORF2p product. RT inhibition in cancer cells, either via RNA interference-dependent silencing of active LINE-1 elements, or using RT inhibitory drugs, reduces cancer cell proliferation, promotes their differentiation and antagonizes tumor progression in animal models. Indeed, the nonnucleoside RT inhibitor efavirenz has recently been tested in a phase II clinical trial with metastatic prostate cancer patients. An in-depth analysis of ORF2p in a mouse model of breast cancer showed ORF2p to be precociously expressed in precancerous lesions and highly abundant in advanced cancer stages, while being barely detectable in normal breast tissue, providing a rationale for the finding that RT-expressing tumours are therapeutically sensitive to RT inhibitors. We summarise mechanistic and gene profiling studies indicating that highly abundant LINE-1-derived RT can “sequester” RNA substrates for reverse transcription in tumor cells, entailing the formation of RNA:DNA hybrid molecules and impairing the overall production of regulatory miRNAs, with a global impact on the cell transcriptome. Based on these data, LINE-1-ORF2 encoded RT has a tumor-promoting potential that is exerted at an epigenetic level. We propose a model whereby LINE1-RT drives a previously unrecognized global regulatory process, the deregulation of which drives cell transformation and tumorigenesis and possibly implicated in cancer cell heterogeneity.

Quantitation of TGF-beta1 mRNA in porcine mesangial cells by comparative kinetic RT/PCR: comparison with ribonuclease protection assay and in situ hybridization.

PubMed

Ceol, M; Forino, M; Gambaro, G; Sauer, U; Schleicher, E D; D'Angelo, A; Anglani, F

2001-01-01

Gene expression can be examined with different techniques including ribonuclease protection assay (RPA), in situ hybridisation (ISH), and quantitative reverse transcription-polymerase chain reaction (RT/PCR). These methods differ considerably in their sensitivity and precision in detecting and quantifying low abundance mRNA. Although there is evidence that RT/PCR can be performed in a quantitative manner, the quantitative capacity of this method is generally underestimated. To demonstrate that the comparative kinetic RT/PCR strategy-which uses a housekeeping gene as internal standard-is a quantitative method to detect significant differences in mRNA levels between different samples, the inhibitory effect of heparin on phorbol 12-myristate 13-acetate (PMA)-induced-TGF-beta1 mRNA expression was evaluated by RT/PCR and RPA, the standard method of mRNA quantification, and the results were compared. The reproducibility of RT/PCR amplification was calculated by comparing the quantity of G3PDH and TGF-beta1 PCR products, generated during the exponential phases, estimated from two different RT/PCR (G3PDH, r = 0.968, P = 0.0000; TGF-beta1, r = 0.966, P = 0.0000). The quantitative capacity of comparative kinetic RT/PCR was demonstrated by comparing the results obtained from RPA and RT/PCR using linear regression analysis. Starting from the same RNA extraction, but using only 1% of the RNA for the RT/PCR compared to RPA, significant correlation was observed (r = 0.984, P = 0.0004). Moreover the morphometric analysis of ISH signal was applied for the semi-quantitative evaluation of the expression and localisation of TGF-beta1 mRNA in the entire cell population. Our results demonstrate the close similarity of the RT/PCR and RPA methods in giving quantitative information on mRNA expression and indicate the possibility to adopt the comparative kinetic RT/PCR as reliable quantitative method of mRNA analysis. Copyright 2001 Wiley-Liss, Inc.

Abilities of the mCP Agar Method and CRENAME Alpha Toxin-Specific Real-Time PCR Assay To Detect Clostridium perfringens Spores in Drinking Water

PubMed Central

Maheux, Andrée F.; Bérubé, Ève; Boudreau, Dominique K.; Villéger, Romain; Cantin, Philippe; Boissinot, Maurice; Bissonnette, Luc

2013-01-01

We first determined the analytical specificity and ubiquity (i.e., the ability to detect all or most strains) of a Clostridium perfringens-specific real-time PCR (rtPCR) assay based on the cpa gene (cpa rtPCR) by using a bacterial strain panel composed of C. perfringens and non-C. perfringens Clostridium strains. All non-C. perfringens Clostridium strains tested negative, whereas all C. perfringens strains tested positive with the cpa rtPCR, for an analytical specificity and ubiquity of 100%. The cpa rtPCR assay was then used to confirm the identity of 116 putative C. perfringens isolates recovered after filtration of water samples and culture on mCP agar. Colonies presenting discordant results between the phenotype on mCP agar and cpa rtPCR were identified by sequencing the 16S rRNA and cpa genes. Four mCP−/rtPCR+ colonies were identified as C. perfringens, whereas 3 mCP+/rtPCR− colonies were identified as non-C. perfringens. The cpa rtPCR was negative with all 51 non-C. perfringens strains and positive with 64 of 65 C. perfringens strains. Finally, we compared mCP agar and a CRENAME (concentration and recovery of microbial particles, extraction of nucleic acids, and molecular enrichment) procedure plus cpa rtPCR (CRENAME + cpa rtPCR) for their abilities to detect C. perfringens spores in drinking water. CRENAME + cpa rtPCR detected as few as one C. perfringens CFU per 100 ml of drinking water sample in less than 5 h, whereas mCP agar took at least 25 h to deliver results. CRENAME + cpa rtPCR also allows the simultaneous and sensitive detection of Escherichia coli and C. perfringens from the same potable water sample. In itself, it could be used to assess the public health risk posed by drinking water potentially contaminated with pathogens more resistant to disinfection. PMID:24077714

Simultaneous detection of papaya ringspot virus, papaya leaf distortion mosaic virus, and papaya mosaic virus by multiplex real-time reverse transcription PCR.

PubMed

Huo, P; Shen, W T; Yan, P; Tuo, D C; Li, X Y; Zhou, P

2015-12-01

Both the single infection of papaya ringspot virus (PRSV), papaya leaf distortion mosaic virus (PLDMV) or papaya mosaic virus (PapMV) and double infection of PRSV and PLDMV or PapMV which cause indistinguishable symptoms, threaten the papaya industry in Hainan Island, China. In this study, a multiplex real-time reverse transcription PCR (RT-PCR) was developed to detect simultaneously the three viruses based on their distinctive melting temperatures (Tms): 81.0±0.8°C for PRSV, 84.7±0.6°C for PLDMV, and 88.7±0.4°C for PapMV. The multiplex real-time RT-PCR method was specific and sensitive in detecting the three viruses, with a detection limit of 1.0×10(1), 1.0×10(2), and 1.0×10(2) copies for PRSV, PLDMV, and PapMV, respectively. Indeed, the reaction was 100 times more sensitive than the multiplex RT-PCR for PRSV, and 10 times more sensitive than multiplex RT-PCR for PLDMV. Field application of the multiplex real-time RT-PCR demonstrated that some non-symptomatic samples were positive for PLDMV by multiplex real-time RT-PCR but negative by multiplex RT-PCR, whereas some samples were positive for both PRSV and PLDMV by multiplex real-time RT-PCR assay but only positive for PLDMV by multiplex RT-PCR. Therefore, this multiplex real-time RT-PCR assay provides a more rapid, sensitive and reliable method for simultaneous detection of PRSV, PLDMV, PapMV and their mixed infections in papaya.

Citrus and Prunuscopia-like retrotransposons.

PubMed

Asíns, M J; Monforte, A J; Mestre, P F; Carbonell, E A

1999-08-01

Many of the world's most important citrus cultivars ("Washington Navel", satsumas, clementines) have arisen through somatic mutation. This phenomenon occurs fairly often in the various species and varieties of the genus.The presence of copia-like retrotransposons has been investigated in fruit trees, especially citrus, by using a PCR assay designed to detect copia-like reverse transcriptase (RT) sequences. Amplification products from a genotype of each the following species Citrus sinensis, Citrus grandis, Citrus clementina, Prunus armeniaca and Prunus amygdalus, were cloned and some of them sequenced. Southern-blot hybridization using RT clones as probes showed that multiple copies are integrated throughout the citrus genome, while only 1-3 copies are detected in the P. armeniaca genome, which is in accordance with the Citrus and Prunus genome sizes. Sequence analysis of RT clones allowed a search for homologous sequences within three gene banks. The most similar ones correspond to RT domains of copia-like retrotransposons from unrelated plant species. Cluster analysis of these sequences has shown a great heterogeneity among RT domains cloned from the same genotype. This finding supports the hypothesis that horizontal transmission of retrotransposons has occurred in the past. The species presenting a RT sequence most similar to citrus RT clones is Gnetum montanum, a gymnosperm whose distribution area coincides with two of the main centers of origin of Citrus spp. A new C-methylated restriction DNA fragment containing a RT sequence is present in navel sweet oranges, but not in Valencia oranges from which the former originated suggesting, that retrotransposon activity might be, at least in part, involved in the genetic variability among sweet orange cultivars. Given that retrotransposons are quite abundant throughout the citrus genome, their activity should be investigated thoroughly before commercializing any transgenic citrus plant where the transgene(s) is part of a viral genome in order to avoid its possible recombination with an active retroelement. Focusing on other strategies to control virus diseases is recommended in citrus.

Avian influenza virus detection and quantitation by real-time RT-PCR

USDA-ARS?s Scientific Manuscript database

Real-time RT-PCR (rRT-PCR) has been used for avian influenza virus (AIV) detection since the early 2000’s for routine surveillance, during outbreaks and for research. Some of the advantages of rRT-PCR are: high sensitivity, high specificity, rapid time-to-result, scalability, cost, and its inherentl...

Real-time RT-PCR, a necessary tool to support the diagnosis and surveillance of rotavirus in Mexico.

PubMed

De La Cruz Hernández, Sergio Isaac; Anaya Molina, Yazmin; Gómez Santiago, Fabián; Terán Vega, Heidi Lizbeth; Monroy Leyva, Elda; Méndez Pérez, Héctor; García Lozano, Herlinda

2018-04-01

Rotavirus produces diarrhea in children under 5 years old. Most of those conventional methods such as polyacrylamide gel electrophoresis (PAGE) and reverse transcription-polymerase chain reaction (RT-PCR) have been used for rotavirus detection. However, these techniques need a multi-step process to get the results. In comparison with conventional methods, the real-time RT-PCR is a highly sensitive method, which allows getting the results in only one day. In this study a real-time RT-PCR assay was tested using a panel of 440 samples from patients with acute gastroenteritis, and characterized by PAGE and RT-PCR. The results show that the real-time RT-PCR detected rotavirus from 73% of rotavirus-negative samples analyzed by PAGE and RT-PCR; thus, the percentage of rotavirus-positive samples increased to 81%. The results indicate that this real-time RT-PCR should be part of a routine analysis, and as a support of the diagnosis of rotavirus in Mexico. Copyright © 2017 Elsevier Inc. All rights reserved.

Multicenter Clinical Evaluation of the Alere i Respiratory Syncytial Virus Isothermal Nucleic Acid Amplification Assay.

PubMed

Hassan, Ferdaus; Hays, Lindsay M; Bonner, Aleta; Bradford, Bradley J; Franklin, Ruffin; Hendry, Phyllis; Kaminetsky, Jed; Vaughn, Michael; Cieslak, Kristin; Moffatt, Mary E; Selvarangan, Rangaraj

2018-03-01

The Alere i respiratory syncytial virus (RSV) assay is an isothermal nucleic acid amplification test capable of detecting RSV directly from respiratory specimens, with results being available in ≤13 min after test initiation. The objective of this study was to evaluate the performance characteristics of the Alere i RSV assay in a point-of-care setting by using direct nasopharyngeal (NP) swab specimens (direct NP) and nasopharyngeal swab specimens eluted and transported in viral transport medium (VTM NP). The study was a prospective, multicenter, clinical trial conducted at 9 sites across the United States to evaluate the clinical performance of the Alere i RSV assay with respiratory specimens obtained from both children (age, <18 years) and older adults (age, >60 years). The performance of the Alere i RSV assay was compared with that of the reference method, the Prodesse ProFlu+ real-time reverse transcriptase PCR (RT-PCR) assay. All specimens with discrepant test results were tested further by a second FDA-cleared PCR assay (the Verigene respiratory virus plus nucleic acid test; Luminex Inc., TX). A total of 554 subjects with signs and symptoms of respiratory infections were enrolled, and respiratory samples were collected in this study. In comparison with the ProFlu+ real-time RT-PCR, the overall sensitivity and specificity of Alere i RSV assay for the detection of RSV were 98.6% (95% confidence interval [CI], 94.4 to 99.7%) and 98.0% (95% CI, 95.8 to 99.1%), respectively, for direct NP and 98.6% (95% CI, 94.4 to 99.7%) and 97.8% (95% CI, 95.5 to 98.9%), respectively, for VTM NP. The Alere i RSV is a highly sensitive and specific molecular assay ideal for rapid RSV detection in patients in the point-of-care setting due to its minimal hands-on time and rapid result availability. Copyright © 2018 American Society for Microbiology.

Nitrate reduction mechanisms and rates in an unconfined eogenetic karst aquifer in two sites with different redox potential

USGS Publications Warehouse

Henson, Wesley; Huang, Laibin; Graham, Wendy D.; Ogram, Andrew

2017-01-01

This study integrates push-pull tracer tests (PPTT) with microbial characterization of extracted water via quantitative polymerase chain reaction (qPCR) and reverse transcriptase qPCR (RT-qPCR) of selected functional N transformation genes to quantify nitrate reduction mechanisms and rates in sites with different redox potential in a karst aquifer. PPTT treatments with nitrate (AN) and nitrate-fumarate (ANC) were executed in two wells representing anoxic and oxic geochemical end-members. Oxic aquifer zero-order nitrate loss rates (mmol L−1 h−1) were similar for AN and ANC treatment, ranging from 0.03 ± 0.01 to 0.05 ± 0.01. Anoxic aquifer zero-order nitrate loss rates ranged from 0.03 ± 0.02 (AN) to 0.13 ± 0.02 (ANC). Microbial characterization indicates mechanisms influencing these rates were dissimilatory nitrate reduction to ammonium (DNRA) at the anoxic site with AN treatment, assimilatory reduction of nitrate to ammonium (ANRA) with ANC treatment in the water column at both sites, and additional documented nitrate reduction that occurred in unsampled biofilms. With carbon treatment, total numbers of microbes (16S rRNA genes) significantly increased (fourteenfold to thirtyfold), supporting stimulated growth with resulting ANRA. Decreased DNRA gene concentrations (nrfA DNA) and increased DNRA activity ratio (nrfA-cDNA/DNA) supported the assertion that DNRA occurred in the anoxic zone with AN and ANC treatment. Furthermore, decreased DNRA gene copy numbers at the anoxic site with ANC treatment suggests that DNRA microbes in the anoxic site are chemolithoautotrophic. Increased RT-qPCR denitrification gene expression (nirK and nirS) was not observed in water samples, supporting that any observed NO3-N loss due to denitrification may be occurring in unsampled microbial biofilms.

Rapid diagnosis of avian influenza virus in wild birds: Use of a portable rRT-PCR and freeze-dried reagents in the field

USGS Publications Warehouse

Takekawa, John Y.; Hill, N.J.; Schultz, A.K.; Iverson, S.A.; Cardona, C.J.; Boyce, W.M.; Dudley, J.P.

2011-01-01

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal. Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene. The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.

Interpretation of diagnostic laboratory tests for severe acute respiratory syndrome: the Toronto experience

PubMed Central

Tang, Patrick; Louie, Marie; Richardson, Susan E.; Smieja, Marek; Simor, Andrew E.; Jamieson, Frances; Fearon, Margaret; Poutanen, Susan M.; Mazzulli, Tony; Tellier, Raymond; Mahony, James; Loeb, Mark; Petrich, Astrid; Chernesky, Max; McGeer, Allison; Low, Donald E.; Phillips, Elizabeth; Jones, Steven; Bastien, Nathalie; Li, Yan; Dick, Daryl; Grolla, Allen; Fernando, Lisa; Booth, Timothy F.; Henry, Bonnie; Rachlis, Anita R.; Matukas, Larissa M.; Rose, David B.; Lovinsky, Reena; Walmsley, Sharon; Gold, Wayne L.; Krajden, Sigmund

2004-01-01

Background An outbreak of severe acute respiratory syndrome (SARS) began in Canada in February 2003. The initial diagnosis of SARS was based on clinical and epidemiological criteria. During the outbreak, molecular and serologic tests for the SARS-associated coronavirus (SARS-CoV) became available. However, without a “gold standard,” it was impossible to determine the usefulness of these tests. We describe how these tests were used during the first phase of the SARS outbreak in Toronto and offer some recommendations that may be useful if SARS returns. Methods We examined the results of all diagnostic laboratory tests used in 117 patients admitted to hospitals in Toronto who met the Health Canada criteria for suspect or probable SARS. Focusing on tests for SARS-CoV, we attempted to determine the optimal specimen types and timing of specimen collection. Results Diagnostic test results for SARS-CoV were available for 110 of the 117 patients. SARS-CoV was detected by means of reverse-transcriptase polymerase chain reaction (RT-PCR) in at least one specimen in 59 (54.1%) of 109 patients. Serologic test results of convalescent samples were positive in 50 (96.2%) of 52 patients for whom paired serum samples were collected during the acute and convalescent phases of the illness. Of the 110 patients, 78 (70.9%) had specimens that tested positive by means of RT-PCR, serologic testing or both methods. The proportion of RT-PCR test results that were positive was similar between patients who met the criteria for suspect SARS (50.8%, 95% confidence interval [CI] 38.4%–63.2%) and those who met the criteria for probable SARS (58.0%, 95% CI 44.2%–70.7%). SARS-CoV was detected in nasopharyngeal swabs in 33 (32.4%) of 102 patients, in stool specimens in 19 (63.3%) of 30 patients, and in specimens from the lower respiratory tract in 10 (58.8%) of 17 patients. Interpretation These findings suggest that the rapid diagnostic tests in use at the time of the initial outbreak lack sufficient sensitivity to be used clinically to rule out SARS. As tests for SARS-CoV continue to be optimized, evaluation of the clinical presentation and elucidation of a contact history must remain the cornerstone of SARS diagnosis. In patients with SARS, specimens taken from the lower respiratory tract and stool samples test positive by means of RT-PCR more often than do samples taken from other areas. PMID:14707219

Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

PubMed Central

Takekawa, John Y.; Hill, Nichola J.; Schultz, Annie K.; Iverson, Samuel A.; Cardona, Carol J.; Boyce, Walter M.; Dudley, Joseph P.

2011-01-01

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal. Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene. The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances. PMID:21847073

Development of a rapid and sensitive one-step reverse transcription-nested polymerase chain reaction in a single tube using the droplet-polymerase chain reaction machine.

PubMed

Yamaguchi, Akemi; Matsuda, Kazuyuki; Sueki, Akane; Taira, Chiaki; Uehara, Masayuki; Saito, Yasunori; Honda, Takayuki

2015-08-25

Reverse transcription (RT)-nested polymerase chain reaction (PCR) is a time-consuming procedure because it has several handling steps and is associated with the risk of cross-contamination during each step. Therefore, a rapid and sensitive one-step RT-nested PCR was developed that could be performed in a single tube using a droplet-PCR machine. The K562 BCR-ABL mRNA-positive cell line as well as bone marrow aspirates from 5 patients with chronic myelogenous leukemia (CML) and 5 controls without CML were used. We evaluated one-step RT-nested PCR using the droplet-PCR machine. One-step RT-nested PCR performed in a single tube using the droplet-PCR machine enabled the detection of BCR-ABL mRNA within 40min, which was 10(3)-fold superior to conventional RT nested PCR using three steps in separate tubes. The sensitivity of the one-step RT-nested PCR was 0.001%, with sample reactivity comparable to that of the conventional assay. One-step RT-nested PCR was developed using the droplet-PCR machine, which enabled all reactions to be performed in a single tube accurately and rapidly and with high sensitivity. This one-step RT-nested PCR may be applicable to a wide spectrum of genetic tests in clinical laboratories. Copyright © 2015 Elsevier B.V. All rights reserved.

Development of mRNA-specific RT-PCR for the detection of koi herpesvirus (KHV) replication stage.

PubMed

Yuasa, Kei; Kurita, Jun; Kawana, Morihiko; Kiryu, Ikunari; Oseko, Norihisa; Sano, Motohiko

2012-08-13

An mRNA-specific reverse transcription (RT)-PCR primer set spanning the exon junction of a spliced putative terminase gene in the koi herpesvirus (KHV) was developed to detect the replicating stage of the virus. The proposed RT-PCR amplified a target gene from the RNA template, but not from a DNA template extracted from common carp brain (CCB) cells infected with KHV. In addition, the RT-PCR did not amplify the target gene of templates extracted from specific cell lines infected with either CyHV-1 or CyHV-2. RT-PCR detected mRNA from the scales of koi experimentally infected with KHV at 24 h post exposure (hpe). However, unlike conventional PCR, RT-PCR could not detect KHV DNA in fish at 0 hpe. The results indicate that the RT-PCR developed in this study is mRNA-specific and that the assay can detect the replicating stage of KHV from both fish and cultured cells infected with the virus.

Elimination of endogenous aberrant kappa chain transcripts from sp2/0-derived hybridoma cells by specific ribozyme cleavage: utility in genetic therapy of HIV-1 infections.

PubMed Central

Duan, L; Pomerantz, R J

1994-01-01

The pooled degenerate-primer polymerase chain reaction (PCR) technology is now widely used in the amplification and cloning of murine hybridoma-specific immunoglobulin gene cDNAs. The design of primers is mainly based on the highly conserved 5' terminus of immunoglobulin gene variable regions and the constant region in the 3' terminus. Of note, most murine hybridoma cell lines are derived from the Sp2/0 cell line, which is demonstrated to express endogenous aberrant kappa chains (abV kappa). This high-level endogenous abV kappa mixes with specific kappa chains in the hybridomas and interferes with the efficiency of the reverse transcriptase (RT)-PCR cloning strategy. In this report, during the cloning of murine anti-human immunodeficiency virus type I (HIV-1) hybridoma immunoglobulin cDNAs, a specific primer-PCR screening system was developed, based on the abV kappa complementarity-defining region (CDR), to eliminate abV kappa-carrying plasmids. Furthermore, an abV kappa sequence-specific derived ribozyme was developed and packaged in a retroviral expression vector system. This abV kappa ribozyme can be transduced into different murine hybridomas, and expressed intracellularly to potently eliminate endogenous abV kappa RNA. Images PMID:7816635

Identification and validation of reference genes for qRT-PCR studies of the obligate aphid pathogenic fungus Pandora neoaphidis during different developmental stages.

PubMed

Zhang, Shutao; Chen, Chun; Xie, Tingna; Ye, Sudan

2017-01-01

The selection of stable reference genes is a critical step for the accurate quantification of gene expression. To identify and validate the reference genes in Pandora neoaphidis-an obligate aphid pathogenic fungus-the expression of 13classical candidate reference genes were evaluated by quantitative real-time reverse transcriptase polymerase chain reaction(qPCR) at four developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae). Four statistical algorithms, including geNorm, NormFinder, BestKeeper and Delta Ct method were used to rank putative reference genes according to their expression stability and indicate the best reference gene or combination of reference genes for accurate normalization. The analysis of comprehensive ranking revealed that ACT1and 18Swas the most stably expressed genes throughout the developmental stages. To further validate the suitability of the reference genes identified in this study, the expression of cell division control protein 25 (CDC25) and Chitinase 1(CHI1) genes were used to further confirm the validated candidate reference genes. Our study presented the first systematic study of reference gene(s) selection for P. neoaphidis study and provided guidelines to obtain more accurate qPCR results for future developmental efforts.

Pausing kinetics dominates strand-displacement polymerization by reverse transcriptase

PubMed Central

Malik, Omri; Khamis, Hadeel; Rudnizky, Sergei; Marx, Ailie

2017-01-01

Abstract Reverse transcriptase (RT) catalyzes the conversion of the viral RNA into an integration-competent double-stranded DNA, with a variety of enzymatic activities that include the ability to displace a non-template strand concomitantly with polymerization. Here, using high-resolution optical tweezers to follow the activity of the murine leukemia Virus RT, we show that strand-displacement polymerization is frequently interrupted. Abundant pauses are modulated by the strength of the DNA duplex ∼8 bp ahead, indicating the existence of uncharacterized RT/DNA interactions, and correspond to backtracking of the enzyme, whose recovery is also modulated by the duplex strength. Dissociation and reinitiation events, which induce long periods of inactivity and are likely the rate-limiting step in the synthesis of the genome in vivo, are modulated by the template structure and the viral nucleocapsid protein. Our results emphasize the potential regulatory role of conserved structural motifs, and may provide useful information for the development of potent and specific inhibitors. PMID:28973474

Novel Structure of Ty3 Reverse Transcriptase | Center for Cancer Research

Cancer.gov

Retrotransposons are mobile genetic elements that self amplify via a single-stranded RNA intermediate, which is converted to double-stranded DNA by an encoded reverse transcriptase (RT) with both DNA polymerase (pol) and ribonuclease H (RNase) activities. Categorized by whether they contain flanking long terminal repeat (LTR) sequences, retrotransposons play a critical role in the architecture of eukaryotic genomes and are the evolutionary origin of retroviruses, including human immunodeficiency virus (HIV).

Lower genetic variability of HIV-1 and antiretroviral drug resistance in pregnant women from the state of Pará, Brazil.

PubMed

Machado, Luiz Fernando Almeida; Costa, Iran Barros; Folha, Maria Nazaré; da Luz, Anderson Levy Bessa; Vallinoto, Antonio Carlos Rosário; Ishak, Ricardo; Ishak, Marluisa Oliveira Guimarães

2017-04-12

The present study aimed to describe the genetic diversity of HIV-1, as well as the resistance profile of the viruses identified in HIV-1 infected pregnant women under antiretroviral therapy in the state of Pará, Northern Brazil. Blood samples were collected from 45 HIV-1 infected pregnant to determine the virus subtypes according to the HIV-1 protease (PR) gene and part of the HIV-1 reverse transcriptase (RT) gene by sequencing the nucleotides of these regions. Drug resistance mutations and susceptibility to antiretroviral drugs were analyzed by the Stanford HIV Drug Resistance Database. Out of 45 samples, only 34 could be amplified for PR and 30 for RT. Regarding the PR gene, subtypes B (97.1%) and C (2.9%) were identified; for the RT gene, subtypes B (90.0%), F (6.7%), and C (3.3%) were detected. Resistance to protease inhibitors (PI) was identified in 5.8% of the pregnant, and mutations conferring resistance to nucleoside reverse transcriptase inhibitors were found in 3.3%, while mutations conferring resistance to non-nucleoside reverse transcriptase inhibitors were found in 3.3%. These results showed a low frequency of strains resistant to antiretroviral drugs, the prevalence of subtypes B and F, and the persistent low transmission of subtype C in pregnant of the state of Pará, Brazil.

Clinical utility of RT-PCR in assessing HER 2 gene expression versus traditional IHC and FISH in breast cancer patients.

PubMed

Suryavanshi, Moushumi; Mehta, Anurag; Jaipuria, Jiten; Kumar, Dushyant; Vishwakarma, Gayatri; Panigrahi, Manoj Kumar; Verma, Haristuti; Saifi, Mumtaz; Sharma, Sanjeev; Tandon, Simran; Doval, D C; Das, Bhudev C

2018-02-09

IHC and FISH are used for categorizing HER 2 status in breast cancer at the protein and DNA level, respectively. HER 2 expression at the RNA level is quantitative, cheaper, easier to standardize and free from interobserver variation. 115 consecutive patients were tested by IHC, FISH and RT-PCR (test cohort). Assuming FISH result to be the response variable, ROC curves for RT-PCR ratio were analyzed to label HER 2 negative, equivocal and positive cases as RT-PCR score 1, 2 and 3, respectively. Inter-relationships between RT-PCR, IHC and FISH were defined. 'Clinical benefit' of a test was defined as proportion of patients labeled unequivocally as HER 2 positive or negative. Population for 1 year was simulated constraint to previous reports of HER 2 positivity and IHC category distribution by a meta-analysis of previous studies that evaluated concordance between IHC and FISH to determine HER 2 status (simulation cohort). Four diagnostic pathways in the simulation cohort were defined-(1) initial IHC, followed by FISH (conventional pathway); (2) initial RT-PCR, followed by FISH; (3) initial IHC, followed by RT-PCR and then by FISH; (4) initial RT-PCR, followed by IHC and then by FISH. The clinical benefit of IHC and RT-PCR in the four pathways was analyzed and sensitivity analysis for incremental cost-effectiveness ratio and cost-benefit comapring RT-PCR against IHC, both as first-line tests and among those with IHC score 2 as a reflex second-line test was performed by the Monte Carlo technique. 115 patients comprised the study population. While none with IHC score of 0 or 1 was FISH positive for HER 2, all cases with IHC score of 3 were FISH positive. 43 cases were assigned IHC score of 2. Thus, 72 patients benefited from the initial IHC testing [clinical benefit 62.6%], with the overall concordance between IHC and FISH being 100% for those with IHC score of 0, 1 and 3 (conclusive IHC categories). For RT-PCR with 100% concordance, 15.7% (115-97 = 18) patients would have benefited from RT-PCR testing if it was used as a first-line test. If RT-PCR would have been used as a second-line test among those with IHC score 2 (n = 43), then only 6 patients would have been assigned a conclusive RT-PCR category (category 1 or 3) translating to a clinical benefit of 14% (6/43) as a second-line test. As a second-line test it had 51% probability to prove more cost-effective than the conventional pathway, provided the cost of RT-PCR was 0.4 times the cost of IHC. Also in a three-step pathway, RT-PCR upfront would have 56% probability of higher cost-benefit provided the cost of RT-PCR was 0.1 times the cost of IHC. RT-PCR results were found to be suboptimal to IHC in terms of discriminative ability and clinical benefit; thus, it is unlikely to replace IHC as a first-line test in the near future.

High Potency of Indolyl Aryl Sulfone Nonnucleoside Inhibitors towards Drug-Resistant Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutants Is Due to Selective Targeting of Different Mechanistic Forms of the Enzyme

PubMed Central

Cancio, Reynel; Silvestri, Romano; Ragno, Rino; Artico, Marino; De Martino, Gabriella; La Regina, Giuseppe; Crespan, Emmanuele; Zanoli, Samantha; Hübscher, Ulrich; Spadari, Silvio; Maga, Giovanni

2005-01-01

Indolyl aryl sulfone (IAS) nonnucleoside inhibitors have been shown to potently inhibit the growth of wild-type and drug-resistant human immunodeficiency virus type 1 (HIV-1), but their exact mechanism of action has not been elucidated yet. Here, we describe the mechanism of inhibition of HIV-1 reverse transcriptase (RT) by selected IAS derivatives. Our results showed that, depending on the substitutions introduced in the IAS common pharmacophore, these compounds can be made selective for different enzyme-substrate complexes. Moreover, we showed that the molecular basis for this selectivity was a different association rate of the drug to a particular enzymatic form along the reaction pathway. By comparing the activities of the different compounds against wild-type RT and the nonnucleoside reverse transcriptase inhibitor-resistant mutant Lys103Asn, it was possible to hypothesize, on the basis of their mechanism of action, a rationale for the design of drugs which could overcome the steric barrier imposed by the Lys103Asn mutation. PMID:16251294

Synthesis, Activity and Structural Analysis of Novel α-Hydroxytropolone Inhibitors of Human Immunodeficiency Virus Reverse Transcriptase-Associated Ribonuclease H

PubMed Central

Chung, Suhman; Himmel, Daniel M.; Jiang, Jian-Kang; Wojtak, Krzysztof; Bauman, Joseph D.; Rausch, Jason W.; Wilson, Jennifer A.; Beutler, John A.; Thomas, Craig J.; Arnold, Eddy; Le Grice, Stuart F.J.

2011-01-01

The α-hydroxytroplone, manicol (5,7-dihydroxy-2-isopropenyl-9-methyl-1,2,3,4-tetrahydro-benzocyclohepten-6-one) potently and specifically inhibits ribonuclease H (RNase H) activity of human immunodeficiency virus reverse transcriptase (HIV RT) in vitro. However, manicol was ineffective in reducing virus replication in culture. Ongoing efforts to improve the potency and specificity over the lead compound led us to synthesize 14 manicol derivatives that retain the divalent metal-chelating α-hydroxytropolone pharmacophore. These efforts were augmented by a high resolution structure of p66/p51 HIV-1 RT containing the nonnucleoside reverse transcriptase inhibitor (NNRTI), TMC278 and manicol in the DNA polymerase and RNase H active sites, respectively. We demonstrate here that several modified α-hydroxytropolones exhibit antiviral activity at non-cytotoxic concentrations. Inclusion of RNase H active site mutants indicated that manicol analogs can occupy an additional site in or around the DNA polymerase catalytic center. Collectively, our studies will promote future structure-based design of improved α-hydroxytropolones to complement the NRTI and NNRTI currently in clinical use. PMID:21568335

Indolylarylsulfones carrying a heterocyclic tail as very potent and broad spectrum HIV-1 non-nucleoside reverse transcriptase inhibitors.

PubMed

Famiglini, Valeria; La Regina, Giuseppe; Coluccia, Antonio; Pelliccia, Sveva; Brancale, Andrea; Maga, Giovanni; Crespan, Emmanuele; Badia, Roger; Riveira-Muñoz, Eva; Esté, José A; Ferretti, Rosella; Cirilli, Roberto; Zamperini, Claudio; Botta, Maurizio; Schols, Dominique; Limongelli, Vittorio; Agostino, Bruno; Novellino, Ettore; Silvestri, Romano

2014-12-11

We synthesized new indolylarylsulfone (IAS) derivatives carrying a heterocyclic tail at the indole-2-carboxamide nitrogen as potential anti-HIV/AIDS agents. Several new IASs yielded EC50 values <1.0 nM against HIV-1 WT and mutant strains in MT-4 cells. The (R)-11 enantiomer proved to be exceptionally potent against the whole viral panel; in the reverse transcriptase (RT) screening assay, it was remarkably superior to NVP and EFV and comparable to ETV. The binding poses were consistent with the one previously described for the IAS non-nucleoside reverse transcriptase inhibitors. Docking studies showed that the methyl group of (R)-11 points toward the cleft created by the K103N mutation, different from the corresponding group of (S)-11. By calculating the solvent-accessible surface, we observed that the exposed area of RT in complex with (S)-11 was larger than the area of the (R)-11 complex. Compounds 6 and 16 and enantiomer (R)-11 represent novel robust lead compounds of the IAS class.

Evaluation of digital PCR for absolute RNA quantification.

PubMed

Sanders, Rebecca; Mason, Deborah J; Foy, Carole A; Huggett, Jim F

2013-01-01

Gene expression measurements detailing mRNA quantities are widely employed in molecular biology and are increasingly important in diagnostic fields. Reverse transcription (RT), necessary for generating complementary DNA, can be both inefficient and imprecise, but remains a quintessential RNA analysis tool using qPCR. This study developed a Transcriptomic Calibration Material and assessed the RT reaction using digital (d)PCR for RNA measurement. While many studies characterise dPCR capabilities for DNA quantification, less work has been performed investigating similar parameters using RT-dPCR for RNA analysis. RT-dPCR measurement using three, one-step RT-qPCR kits was evaluated using single and multiplex formats when measuring endogenous and synthetic RNAs. The best performing kit was compared to UV quantification and sensitivity and technical reproducibility investigated. Our results demonstrate assay and kit dependent RT-dPCR measurements differed significantly compared to UV quantification. Different values were reported by different kits for each target, despite evaluation of identical samples using the same instrument. RT-dPCR did not display the strong inter-assay agreement previously described when analysing DNA. This study demonstrates that, as with DNA measurement, RT-dPCR is capable of accurate quantification of low copy RNA targets, but the results are both kit and target dependent supporting the need for calibration controls.

Sensitive Deep-Sequencing-Based HIV-1 Genotyping Assay To Simultaneously Determine Susceptibility to Protease, Reverse Transcriptase, Integrase, and Maturation Inhibitors, as Well as HIV-1 Coreceptor Tropism

PubMed Central

Gibson, Richard M.; Meyer, Ashley M.; Winner, Dane; Archer, John; Feyertag, Felix; Ruiz-Mateos, Ezequiel; Leal, Manuel; Robertson, David L.; Schmotzer, Christine L.

2014-01-01

With 29 individual antiretroviral drugs available from six classes that are approved for the treatment of HIV-1 infection, a combination of different phenotypic and genotypic tests is currently needed to monitor HIV-infected individuals. In this study, we developed a novel HIV-1 genotypic assay based on deep sequencing (DeepGen HIV) to simultaneously assess HIV-1 susceptibilities to all drugs targeting the three viral enzymes and to predict HIV-1 coreceptor tropism. Patient-derived gag-p2/NCp7/p1/p6/pol-PR/RT/IN- and env-C2V3 PCR products were sequenced using the Ion Torrent Personal Genome Machine. Reads spanning the 3′ end of the Gag, protease (PR), reverse transcriptase (RT), integrase (IN), and V3 regions were extracted, truncated, translated, and assembled for genotype and HIV-1 coreceptor tropism determination. DeepGen HIV consistently detected both minority drug-resistant viruses and non-R5 HIV-1 variants from clinical specimens with viral loads of ≥1,000 copies/ml and from B and non-B subtypes. Additional mutations associated with resistance to PR, RT, and IN inhibitors, previously undetected by standard (Sanger) population sequencing, were reliably identified at frequencies as low as 1%. DeepGen HIV results correlated with phenotypic (original Trofile, 92%; enhanced-sensitivity Trofile assay [ESTA], 80%; TROCAI, 81%; and VeriTrop, 80%) and genotypic (population sequencing/Geno2Pheno with a 10% false-positive rate [FPR], 84%) HIV-1 tropism test results. DeepGen HIV (83%) and Trofile (85%) showed similar concordances with the clinical response following an 8-day course of maraviroc monotherapy (MCT). In summary, this novel all-inclusive HIV-1 genotypic and coreceptor tropism assay, based on deep sequencing of the PR, RT, IN, and V3 regions, permits simultaneous multiplex detection of low-level drug-resistant and/or non-R5 viruses in up to 96 clinical samples. This comprehensive test, the first of its class, will be instrumental in the development of new antiretroviral drugs and, more importantly, will aid in the treatment and management of HIV-infected individuals. PMID:24468782

KCC isoforms in a human lens epithelial cell line (B3) and lens tissue extracts.

PubMed

Misri, Sandeep; Chimote, Ameet A; Adragna, Norma C; Warwar, Ronald; Brown, Thomas L; Lauf, Peter K

2006-11-01

We recently reported potassium-chloride cotransporter activity in human lens epithelial B3 (HLE-B3) cells. The purpose of the present study was to demonstrate in these cells as well as in human lens tissue the potassium-chloride cotransport (KCC) isoforms by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunofluorescence microscopy. Of the four KCC genes known to encode the respective proteins and their spliced variants, RT-PCR with both rat and human primers revealed the predicted cDNA fragments of KCC1, KCC3a, KCC3b, and KCC4 but not KCC2 in both HLE-B3 cells and in human lens tissue extracts from cataractous patients. Polyclonal rabbit (rb) anti-rat (rt) and anti-human (hm) antibodies against rtKCC1 and hmKCC3, respectively, and a commercially available rb-anti-mouse (ms) KCC4 antibody were used. Rb anti-rtKCC1-ECL3 [against epitopes within the large extracellular loop 3 (ECL3)] revealed a 150kDa band in HLE-B3 cells consistent with the known molecular weight of KCC1. Rb anti-hmKCC3-ECL3 yielded three bands of 150, 122 and 105kDa, evidence for the presence of KCC3a, KCC3b and possibly KCC3c isoforms. The 122 and 112kDa bands were also demonstrated by rb anti-hmKCC3-CTD [the C-terminal domain (CTD)]. Rb anti-msKCC4 antibody only showed a 100kDa band in HLE-B3 cells. In the human lens tissues, a 115kDa protein was detected with rb anti-rtKCC1-ECL3 and a 100kDa band with rb anti-msKCC4, however, no bands with rb anti-hmKCC3-ECL3 or rb anti-hmKCC3-CTD. Fluorescence microscopy revealed immunocytochemical cytoplasmic and membrane labeling of HLE-B3 cells with anti-KCC1, -KCC3 (laser confocal microscopy) and -KCC4 antibodies and a Cy3-tagged secondary antibody. Hence HLE-B3 cells expressed proteins of the KCC1, KCC3a, b, and KCC4 isoforms, whereas surgically removed cataractous lens tissue expressed only those of KCC1 and KCC4.

Highly Sensitive Assay for Detection of Enterovirus in Clinical Specimens by Reverse Transcription-PCR with an Armored RNA Internal Control

PubMed Central

Beld, Marcel; Minnaar, René; Weel, Jan; Sol, Cees; Damen, Marjolein; van der Avoort, Harry; Wertheim-van Dillen, Pauline; Breda, Alex van; Boom, René

2004-01-01

The objective of the present study was the development of a diagnostic reverse transcription (RT)-PCR for the specific detection of enterovirus (EV) RNA in clinical specimens controlled by an internal control (IC) RNA. The IC RNA contains the same primer binding sites as EV RNA but has a different probe region. The IC RNA was packaged into an MS2 phage core particle (armored) and was added to the clinical sample to allow monitoring of both extraction efficiency and RT-PCR efficiency. Serial dilutions of the IC RNA were made, and the detection limit of the RT-PCR was tested in a background of EV RNA-negative cerebrospinal fluid. The sensitivity and specificity of the RT-PCR assay were tested by using all 64 known EV serotypes, several non-EV serotypes, and two Quality Control for Molecular Diagnostics (QCMD) Program EV proficiency panels from 2001 and 2002. In total, 322 clinical specimens were tested by RT-PCR, and to establish the clinical utility of the RT-PCR, a comparison of the results of viral culture and RT-PCR was done with 87 clinical specimens. The lower limit of sensitivity was reached at about 150 copies of IC RNA/ml. All 64 EV serotypes were positive, while all non-EV serotypes were negative. All culture-positive samples of the 2001 QCMD proficiency panel (according to the 50% tissue culture infective doses per milliliter) were positive by RT-PCR. Invalid results, i.e., negativity for both EV RNA and IC RNA, due to inhibition of RT-PCR were observed for 33.3% of the members of the 2002 QCMD proficiency panel and 3.1% of the clinical specimens. Inhibition of RT-PCR could be relieved by the addition of 400 ng of bovine α-casein per μl to both the RT reaction mixture and the PCR mixture. With this optimized protocol, the results for all samples of the 2002 QCMD proficiency panel and all clinical specimens except one fecal sample (0.3%) were valid. Evaluation of the clinical samples demonstrated that EV infection could be detected in 12 of 87 samples (13.8%) by RT-PCR, while viral culture was negative. Our data show that the RT-PCR with armored IC RNA offers a very reliable and rapid diagnostic tool for the detection of EV in clinical specimens and that the addition of bovine α-casein relieved inhibition of the RT-PCR for 99.7% of clinical specimens. PMID:15243060

Detection of Helicobacter pylori by Real-Time PCR for 16s rRNA in Stools of NonInfected Healthy Children, Using ELISA Antigen Stool Test as the Gold Standard.

PubMed

George, Sergio; Mamani, Nora; Lucero, Yalda; Torres, Juan Pablo; Farfán, Mauricio; Lagomarcino, Anne J; Orellana, Andrea; O'Ryan, Miguel

2016-12-01

We previously detected Helicobacter pylori infection by stool antigen ELISA assay in 33-41% of asymptomatic Chilean children between 2-3 years of age, of which 11-20% had a transient infection and 21-22% a persistent infection. A total of 88% of ELISA-positive samples were also rtPCR positive, while 37/133 (33%) of ELISA-negative stool samples were rtPCR positive. The significance of a ELISA-negative/rtPCR-positive sample requires clarification. We aimed to determine whether rtPCR is able to detect persistent infections not detected by ELISA. We selected 36 children with an ELISA-negative/rtPCR-positive stool sample, of which 25 were never H. pylori infected according to ELISA, and 11 had a transient infection with an ELISA-positive sample before or after the discordant sample. At least two additional consecutive ELISA-negative samples per child were tested in duplicate by rtPCR for the 16s rRNA gene. A total of 14 of 78 (17.9%) rtPCR reactions were positive, but only 4/78 (5.1%) were positive in both duplicates, representing a total of 3/36 (8.3%) children with an additional rtPCR-positive sample, only one of whom was persistently negative by ELISA. One child with a transient infection had two positive rtPCR reactions despite negative ELISA samples. In H. pylori noninfected or transiently infected children, as determined by stool ELISA, additional ELISA-negative/rtPCR-positive stool samples were found in 8.3% of children, but a possible persistent infection was only identified in 2.7% of children. Thus, the characterization of infection dynamics in children is not being misrepresented by application of stool ELISA. Furthermore, rtPCR does not significantly improve dynamic characterization. © 2016 John Wiley & Sons Ltd.

Massively parallel single-molecule and single-cell emulsion reverse transcription polymerase chain reaction using agarose droplet microfluidics.

PubMed

Zhang, Huifa; Jenkins, Gareth; Zou, Yuan; Zhu, Zhi; Yang, Chaoyong James

2012-04-17

A microfluidic device for performing single copy, emulsion Reverse Transcription Polymerase Chain Reaction (RT-PCR) within agarose droplets is presented. A two-aqueous-inlet emulsion droplet generator was designed and fabricated to produce highly uniform monodisperse picoliter agarose emulsion droplets with RT-PCR reagents in carrier oil. Template RNA or cells were delivered from one inlet with RT-PCR reagents/cell lysis buffer delivered separately from the other. Efficient RNA/cell encapsulation and RT-PCR at the single copy level was achieved in agarose-in-oil droplets, which, after amplification, can be solidified into agarose beads for further analysis. A simple and efficient method to graft primer to the polymer matrix using 5'-acrydite primer was developed to ensure highly efficient trapping of RT-PCR products in agarose. High-throughput single RNA molecule/cell RT-PCR was demonstrated in stochastically diluted solutions. Our results indicate that single-molecule RT-PCR can be efficiently carried out in agarose matrix. Single-cell RT-PCR was successfully performed which showed a clear difference in gene expression level of EpCAM, a cancer biomarker gene, at the single-cell level between different types of cancer cells. This work clearly demonstrates for the first time, single-copy RT-PCR in agarose droplets. We believe this will open up new possibilities for viral RNA detection and single-cell transcription analysis.

Simultaneous detection of three lily viruses using Triplex IC-RT-PCR.

PubMed

Zhang, Yubao; Wang, Yajun; Xie, Zhongkui; Yang, Guo; Guo, Zhihong; Wang, Le

2017-11-01

Viruses commonly infecting lily (Lilium spp.) include: Lily symptomless virus (LSV), Cucumber mosaic virus (CMV) and Lily mottle virus (LMoV). These viruses usually co-infect lilies causing severe economic losses in terms of quantity and quality of flower and bulb production around the world. Reliable and precise detection systems need to be developed for virus identification. We describe the development of a triplex immunocapture (IC) reverse transcription (RT) polymerase chain reaction (PCR) assay for the simultaneous detection of LSV, CMV and LMoV. The triplex IC-RT-PCR was compared with a quadruplex RT-PCR assay. Relative to the quadruplex RT-PCR, the specificity of the triplex IC-RT-PCR system for LSV, CMV and LMoV was 100% for field samples. The sensitivity of the triplex IC-RT-PCR system was 99.4%, 81.4% and 98.7% for LSV, CMV and LMoV, respectively. Agreement (κ) between the results obtained from the two tests was 0.968, 0.844 and 0.984 for LSV, CMV and LMoV, respectively. This is the first report of the simultaneous detection of LSV, CMV and LMoV in a triplex IC-RT-PCR assay. In particular we believe this convenient and reliable triplex IC-RT-PCR method could be used routinely for large-scale field surveys or crop health monitoring of lily. Copyright © 2017. Published by Elsevier B.V.

Enzymatic and viability RT-qPCR assays for evaluation of enterovirus, hepatitis A virus and norovirus inactivation: Implications for public health risk assessment.

PubMed

Monteiro, S; Santos, R

2018-04-01

To assess the potential of a viability dye and an enzymatic reverse transcription quantitative PCR (RT-qPCR) pretreatment to discriminate between infectious and noninfectious enteric viruses. Enterovirus (EntV), norovirus (NoV) GII.4 and hepatitis A virus (HAV) were inactivated at 95°C for 10 min, and four methods were used to compare the efficiency of inactivation: (i) cell culture plaque assay for HAV and EntV, (ii) RT-qPCR alone, (iii) RT-qPCR assay preceded by RNase treatment, and (iv) pretreatment with a viability dye (reagent D (RD)) followed by RT-qPCR. In addition, heat-inactivated NoV was treated with RD coupled with surfactants to increase the efficiency of the viability dye. No treatment was able to completely discriminate infectious from noninfectious viruses. RD-RT-qPCR reduced more efficiently the detection of noninfectious viruses with little to no removal observed with RNase. RD-RT-qPCR method was the closest to cell culture assay. The combination of surfactants and RD did not show relevant improvements on the removal of inactivated viruses signal compared with viability RT-qPCR, with the exception of Triton X-100. The use of surfactant/RD-RT-qPCR, although not being able to completely remove the signal from noninfectious viral particles, yielded a better estimation of viral infectivity. Surfactant/RD-RT-qPCR may be an advantageous tool for a better detection of infectious viruses with potential significant impact in the risk assessment of the presence of enteric viruses. © 2017 The Society for Applied Microbiology.

Inactivation conditions for human Norovirus measured by an in situ capture-qRT-PCR Method

USDA-ARS?s Scientific Manuscript database

Human noroviruses (HuNoVs) are the major cause of epidemic non-bacterial gastroenteritis. Due to the inability to cultivate HuNoVs, it has been a challenge to determine their infectivity. Quantitative real-time RT-PCR (qRT-PCR) is widely used in detecting HuNoVs. However, qRT-PCR only detects the...

Expression of an Mg2+-Dependent HIV-1 RNase H Construct for Drug Screening▿†

PubMed Central

Farias, Richard V.; Vargas, Deborah A.; Castillo, Andres E.; Valenzuela, Beatriz; Coté, Marie L.; Roth, Monica J.; Leon, Oscar

2011-01-01

A single polypeptide of the HIV-1 reverse transcriptase that reconstituted Mg2+-dependent RNase H activity has been made. Using molecular modeling, the construct was designed to encode the p51 subunit joined by a linker to the thumb (T), connection (C), and RNase H (R) domains of p66. This p51-G-TCR construct was purified from the soluble fraction of an Escherichia coli strain, MIC2067(DE3), lacking endogenous RNase HI and HII. The p51-G-TCR RNase H construct displayed Mg2+-dependent activity using a fluorescent nonspecific assay and showed the same cleavage pattern as HIV-1 reverse transcriptase (RT) on substrates that mimic the tRNA removal required for second-strand transfer reactions. The mutant E706Q (E478Q in RT) was purified under similar conditions and was not active. The RNase H of the p51-G-TCR RNase H construct and wild type HIV-1 RT had similar Kms for an RNA-DNA hybrid substrate and showed similar inhibition kinetics to two known inhibitors of the HIV-1 RT RNase H. PMID:21768506

Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by real-time RT-PCR.

PubMed

Kamau, Everlyn; Agoti, Charles N; Lewa, Clement S; Oketch, John; Owor, Betty E; Otieno, Grieven P; Bett, Anne; Cane, Patricia A; Nokes, D James

2017-03-01

Direct immuno-fluorescence test (IFAT) and multiplex real-time RT-PCR have been central to RSV diagnosis in Kilifi, Kenya. Recently, these two methods showed discrepancies with an increasing number of PCR undetectable RSV-B viruses. Establish if mismatches in the primer and probe binding sites could have reduced real-time RT-PCR sensitivity. Nucleoprotein (N) and glycoprotein (G) genes were sequenced for real-time RT-PCR positive and negative samples. Primer and probe binding regions in N gene were checked for mismatches and phylogenetic analyses done to determine molecular epidemiology of these viruses. New primers and probe were designed and tested on the previously real-time RT-PCR negative samples. N gene sequences revealed 3 different mismatches in the probe target site of PCR negative, IFAT positive viruses. The primers target sites had no mismatches. Phylogenetic analysis of N and G genes showed that real-time RT-PCR positive and negative samples fell into distinct clades. Newly designed primers-probe pair improved detection and recovered previous PCR undetectable viruses. An emerging RSV-B variant is undetectable by a quite widely used real-time RT-PCR assay due to polymorphisms that influence probe hybridization affecting PCR accuracy. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

HIV/AIDS Medicines

MedlinePlus

... few years. But today, there are many effective medicines to fight the infection, and people with HIV ... healthier lives. There are five major types of medicines: Reverse transcriptase (RT) inhibitors - interfere with a critical ...

Immunocapture reverse transcription-polymerase chain reaction combined with nested PCR greatly increases the detection of Prunus necrotic ring spot virus in the peach.

PubMed

Helguera, P R; Taborda, R; Docampo, D M; Ducasse, D A

2001-06-01

A detection system based on nested PCR after IC-RT-PCR (IC-RT-PCR-Nested PCR) was developed to improve indexing of Prunus necrotic ringspot virus in peach trees. Inhibitory effects and inconsistencies of the standard IC-RT-PCR were overcome by this approach. IC-RT-PCR-Nested PCR improved detection by three orders of magnitude compared with DAS-ELISA for the detection of PNRSV in leaves. Several different tissues were evaluated and equally consistent results were observed. The main advantages of the method are its consistency, high sensitivity and easy application in quarantine programs.

Evaluation of a sensitive reverse transcription PCR-enzymelinked immunosorbent assay for detection of hepatitis A virus in oysters (Saccostrea glomerata) on the east coast of the Gulf of Thailand.

PubMed

Intamaso, Uraiwan; Ketkhunthod, Sitthisak

2014-05-01

Hepatitis A virus (HAV) contamination in food can lead to major health problems. We developed a combination reverse transcription (RT) PCR method plus enzyme-linked immunosorbent assay (ELISA) to detect HAV in fresh oysters harvested along the east coast of the Gulf of Thailand. Viral nucleic acid was extracted via the glycine-arginine-polyethylene glycol method followed by RT-PCR amplification with specifically designed primers against HAV and an ELISA to detect the digoxigenin-labeled RT-PCR products. The ELISA in concert with the RT-PCR protocol further increased the detection sensitivity by 100-fold for the HAV genome and 10-fold in artificially contaminated oysters. The overall sensitivity of the RT-PCR in combination with the ELISA was 31.88 pg and 16 PFU/g, respectively. The ELISA increases the specificity of the RT-PCR assay for detecting naturally occurring HAV in oysters. This combined RT-PCR-ELISA approach is a practical and sensitive method for HAV detection and can be utilized in routine screening for HAV in shellfish.

Comparison of 2 Assays for Diagnosing Rotavirus and Evaluating Vaccine Effectiveness in Children with Gastroenteritis

PubMed Central

Mijatovic-Rustempasic, Slavica; Tam, Ka Ian; Lyde, Freda C.; Payne, Daniel C.; Szilagyi, Peter; Edwards, Kathryn; Staat, Mary Allen; Weinberg, Geoffrey A.; Hall, Caroline B.; Chappell, James; McNeal, Monica; Gentsch, Jon R.; Bowen, Michael D.; Parashar, Umesh D.

2013-01-01

We compared rotavirus detection rates in children with acute gastroenteritis (AGE) and in healthy controls using enzyme immunoassays (EIAs) and semiquantitative real-time reverse transcription PCR (qRT-PCR). We calculated rotavirus vaccine effectiveness using different laboratory-based case definitions to determine which best identified the proportion of disease that was vaccine preventable. Of 648 AGE patients, 158 (24%) were EIA positive, and 157 were also qRT-PCR positive. An additional 65 (10%) were qRT-PCR positive but EIA negative. Of 500 healthy controls, 1 was EIA positive and 24 (5%) were qRT-PCR positive. Rotavirus vaccine was highly effective (84% [95% CI 71%–91%]) in EIA-positive children but offered no significant protection (14% [95% CI −105% to 64%]) in EIA-negative children for whom virus was detected by qRT-PCR alone. Children with rotavirus detected by qRT-PCR but not by EIA were not protected by vaccination, suggesting that rotavirus detected by qRT-PCR alone might not be causally associated with AGE in all patients. PMID:23876518

Evaluation of a duplex reverse-transcription real-time PCR assay for the detection of encephalomyocarditis virus.

PubMed

Qin, Shaomin; Underwood, Darren; Driver, Luke; Kistler, Carol; Diallo, Ibrahim; Kirkland, Peter D

2018-06-01

We evaluated a fluorogenic probe-based assay for the detection of encephalomyocarditis virus (EMCV) by comparing a set of published primers and probe to a new set of primers and probe. The published reagents failed to amplify a range of Australian isolates and an Italian reference strain of EMCV. In contrast, an assay based on 2 new sets of primers and probes that were run in a duplex reverse-transcription real-time PCR (RT-rtPCR) worked well, with high amplification efficiency. The analytical sensitivity was ~100-fold higher than virus isolation in cell culture. The intra-assay variation was 0.21-4.90%. No cross-reactivity was observed with a range of other porcine viruses. One hundred and twenty-two clinical specimens were tested simultaneously by RT-rtPCR and virus isolation in cell culture; 72 specimens gave positive results by RT-rtPCR, and 63 of these were also positive by virus isolation. Of 245 archived cell culture isolates of EMCV that were tested in the RT-rtPCR, 242 samples were positive. The new duplex RT-rtPCR assay is a reliable tool for the detection of EMCV in clinical specimens and for use in epidemiologic investigations.

Interactions between HIV-1 Reverse Transcriptase and the Downstream Template Strand in Stable Complexes with Primer-Template

PubMed Central

Rutvisuttinunt, Wiriya; Meyer, Peter R.; Scott, Walter A.

2008-01-01

Background Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) forms stable ternary complexes in which RT is bound tightly at fixed positions on the primer-template (P/T). We have probed downstream interactions between RT and the template strand in the complex containing the incoming dNTP (+1 dNTP•RT•P/T complex) and in the complex containing the pyrophosphate analog, foscarnet (foscarnet•RT•P/T complex). Methods and Results UV-induced cross-linking between RT and the DNA template strand was most efficient when a bromodeoxyuridine residue was placed in the +2 position (the first template position downstream from the incoming dNTP). Furthermore, formation of the +1 dNTP•RT•P/T complex on a biotin-containing template inhibited binding of streptavidin when biotin was in the +2 position on the template but not when the biotin was in the +3 position. Streptavidin pre-bound to a biotin residue in the template caused RT to stall two to three nucleotides upstream from the biotin residue. The downstream border of the complex formed by the stalled RT was mapped by digestion with exonuclease RecJF. UV-induced cross-linking of the complex formed by the pyrophosphate analog, foscarnet, with RT and P/T occurred preferentially with bromodeoxyuridine in the +1 position on the template in keeping with the location of RT one base upstream in the foscarnet•RT•P/T complex (i.e., in the pre-translocation position). Conclusions For +1 dNTP•RT•P/T and foscarnet•RT•P/T stable complexes, tight interactions were observed between RT and the first unpaired template nucleotide following the bound dNTP or the primer terminus, respectively. PMID:18974785

A Novel Microbicide/Contraceptive Intravaginal Ring Protects Macaque Genital Mucosa against SHIV-RT Infection Ex Vivo.

PubMed

Villegas, Guillermo; Calenda, Giulia; Ugaonkar, Shweta; Zhang, Shimin; Kizima, Larisa; Mizenina, Olga; Gettie, Agegnehu; Blanchard, James; Cooney, Michael L; Robbiani, Melissa; Fernández-Romero, José A; Zydowsky, Thomas M; Teleshova, Natalia

2016-01-01

Women need multipurpose prevention products (MPTs) that protect against sexually transmitted infections (STIs) and provide contraception. The Population Council has developed a prototype intravaginal ring (IVR) releasing the non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 (M), zinc acetate (ZA), carrageenan (CG) and levonorgestrel (LNG) (MZCL IVR) to protect against HIV, HSV-2, HPV and unintended pregnancy. Our objective was to evaluate the anti-SHIV-RT activity of MZCL IVR in genital mucosa. First, macaque vaginal tissues were challenged with SHIV-RT in the presence of (i) MIV-150 ± LNG or (ii) vaginal fluids (VF); available from studies completed earlier) collected at various time points post insertion of MZCL and MZC IVRs. Then, (iii) MZCL IVRs (vs. LNG IVRs) were inserted in non-Depo Provera-treated macaques for 24h and VF, genital biopsies, and blood were collected and tissues were challenged with SHIV-RT. Infection was monitored with one step SIV gag qRT-PCR or p27 ELISA. MIV-150 (LCMS/MS, RIA), LNG (RIA) and CG (ELISA) were measured in different compartments. Log-normal generalized mixed linear models were used for analysis. LNG did not affect the anti-SHIV-RT activity of MIV-150 in vitro. MIV-150 in VF from MZC/MZCL IVR-treated macaques inhibited SHIV-RT in vaginal mucosa in a dose-dependent manner (p<0.05). MIV-150 in vaginal tissue from MZCL IVR-treated animals inhibited ex vivo infection relative to baseline (96%; p<0.0001) and post LNG IVR group (90%, p<0.001). No MIV-150 dose-dependent protection was observed, likely because of high MIV-150 concentrations in all vaginal tissue samples. In cervical tissue, MIV-150 inhibited infection vs. baseline (99%; p<0.05). No cervical tissue was available for MIV-150 measurement. Exposure to LNG IVR did not change tissue infection level. These observations support further development of MZCL IVR as a multipurpose prevention technology to improve women's sexual and reproductive health.

Characterization of glial cell K-Cl cotransport.

PubMed

Gagnon, Kenneth B E; Adragna, Norma C; Fyffe, Robert E W; Lauf, Peter K

2007-01-01

The molecular mechanism of K-Cl cotransport (KCC) consists of at least 4 isoforms, KCC 1, 2, 3, and 4 which, in multiple combinations, exist in most cells, including erythrocytes and neuronal cells. We utilized reverse-transcriptase-polymerase chain reaction (RT-PCR) and ion flux studies to characterize KCC activity in an immortalized in vitro cell model for fibrous astrocytes, the rat C6 glioblastoma cell. Isoform-specific sets of oligonucleotide primers were synthesized for NKCC1, KCC1, KCC2, KCC3, KCC4, and also for NKCC1 and actin. K-Cl cotransport activity was determined by measuring either the furosemide-sensitive, or the Cl(-)-dependent bumetanide-insensitive Rb(+) (a K(+) congener) influx in the presence of the Na/K pump inhibitor ouabain. Rb(+) influx was measured at a fixed external Cl concentrations, [Cl(-)](e), as a function of varying external Rb concentrations, [Rb(+)](e), and at a fixed [Rb(+)](e) as a function of varying [Cl(-)](e), and with equimolar Cl replacement by anions of the chaotropic series. RT-PCR of C6 glioblastoma (C6) cells identified mRNA for three KCC isoforms (1, 3, and 4). NKCC1 mRNA was also detected. The apparent K(m) for KCC-mediated Rb(+) influx was 15 mM [Rb(+)](e), and V(max) 12.5 nmol Rb(+) * mg protein(-1) * minute(-1). The calculated apparent K(m) for external Cl(-) was 13 mM and V(max) 14.4 nmol Rb(+) * mg protein(-1) * minute(-1). The anion selectivity sequence of the furosemide-sensitive Rb(+) influx was Cl(-)>Br-=NO(3)(-)>I(-)=SCN(-)>Sfm(-) (sulfamate). Established activators of K-Cl cotransport, hyposmotic shock and N-ethylmaleimide (NEM) pretreatment, stimulated furosemide-sensitive Rb(+) influx. A ñ50% NEM-induced loss of intracellular K(+) was prevented by furosemide. We have identified by RT-PCR the presence of three distinct KCC isoforms (1, 3, and 4) in rat C6 glioblastoma cells, and functionally characterized the anion selectivity and kinetics of their collective sodium-independent cation-chloride cotransport activity.

Simultaneous VENTANA IHC and RT-PCR testing of ALK status in Chinese non-small cell lung cancer patients and response to crizotinib.

PubMed

Xu, Chun-Wei; Wang, Wen-Xian; Chen, Yan-Ping; Chen, Yu; Liu, Wei; Zhong, Li-Hua; Chen, Fang-Fang; Zhuang, Wu; Song, Zheng-Bo; Chen, Xiao-Hui; Huang, Yun-Jian; Guan, Yan-Fang; Yi, Xin; Lv, Tang-Feng; Zhu, Wei-Feng; Lu, Jian-Ping; Wang, Xiao-Jiang; Shi, Yi; Lin, Xian-Dong; Chen, Gang; Song, Yong

2018-04-11

ALK rearrangement-advanced NSCLC patients respond to crizotinib. ALK rearrangement is currently determined with RT-PCR. VENTANA IHC is a standard method to identify ALK protein overexpression in NSCLC; however, VENTANA IHC has rarely been used to determine the response to crizotinib in Chinese patients with NSCLC and ALK overexpression. To better clarify the clinical implication of VENTANA IHC to detect ALK rearrangements, we conducted this study to analyze VENTANA IHC and RT-PCR in a large cohort of Chinese patients with NSCLC undergoing screening for ALK rearrangements. A total of 1720 patients with NSCLC who had ALK rearrangements detected by VENTANA IHC and/or RT-PCR were included in this analysis. We compared the efficacy and survival of ALK-positive patients detected by VENTANA IHC and RT-PCR. We used NGS to identify patients in whom the two methods were inconsistent. Among 1720 patients, 187 (10.87%) were shown to be ALK-positive by VENTANA IHC and/or RT-PCR, and 66 received crizotinib treatment. We identified 10.27% (172/1674) of patients as ALK-positive by the VENTANA IHC method, and 12.73% (41/322) of patients had ALK rearrangements by the RT-PCR method. Twenty-nine of 276 (10.51%) ALK-positive patients were simultaneously analyzed using VENTANA IHC and RT-PCR. The overall response rates were 65.90% (29/44) by VENTANA IHC and 55.88% (19/34) by RT-PCR. The disease control rates were 86.36% (38/44) by VENTANA IHC and 76.47% (26/34) by RT-PCR. In contrast, the median progression-free survival for VENTANA IHC and RT-PCR was 8.5 and 9.2 months, respectively. The VENTANA IHC and RT-PCR results obtained for 6 of 17 ALK-positive patients were inconsistent based on NGS; specifically, 4 patients had EML4-ALK fusions, 2 patients had non EML4-ALK fusions, 1 patient had a KCL1-ALK fusion, and one patient had a FBXO36-ALK fusion. VENTANA IHC is a reliable and rapid screening tool used in routine pathologic laboratories for the identification of suitable candidates for ALK-targeted therapy. VENTANA IHC has moderate sensitivity and a slightly higher association with response to therapy with ALK inhibitors, and some VENTANA IHC-positive, but RT-PCR-negative cases may benefit from crizotinib.

Dengue Virus Serotype 2 Established in Northern Mozambique (2015-2016).

PubMed

Oludele, John; Lesko, Birgitta; Mahumane Gundane, Isabel; de Bruycker-Nogueira, Fernanda; Muianga, Argentina; Ali, Sadia; Mula, Flora; Chelene, Imelda; Falk, Kerstin I; Barreto Dos Santos, Flávia; Gudo, Eduardo Samo

2017-11-01

After the report of an outbreak of dengue virus serotype 2 in 2014 in Nampula and Pemba cities, northern Mozambique, a surveillance system was established by the National Institute of Health. A study was performed during 2015-2016 to monitor the trend of the outbreak and confirm the circulating serotype of dengue virus (DENV). After the inclusion of consenting patients who met the case definition, samples from 192 patients were tested for the presence of nonstructural protein 1 antigen, and 60/192 (31%) samples were positive. Further analysis included DENV IgM antibodies, with 39 (20%) IgM positive cases. Reverse transcriptase (RT) PCR was performed for identification of the prevailing DENV serotype; 21/23 tested samples were DENV-2 positive, with DENV-2 present in both affected cities. When sequencing DENV, phenotype Cosmopolitan was identified. The surveillance indicates ongoing spread of DENV-2 in northern Mozambique 2 years after the first report of the outbreak.

Failure to Detect Borna Disease Virus Antibody and RNA from Peripheral Blood Mononuclear Cells of Psychiatric Patients

PubMed Central

Na, Kyoung-Sae; Tae, Seong-Ho; Song, Jin-won

2009-01-01

Objective Borna disease virus (BDV) is a highly neurotropic agent causing various neuropsychiatric symptoms in animals. Over the past two decades, it has been suggested that BDV might be associated with human psychiatric diseases. We aimed to investigate whether BDV is associated with psychiatric patients in Korea. Methods We recruited 60 normal controls and 198 psychiatric patients (98 patients with depressive disorder, 60 with schizophrenia, and 40 with bipolar disorder). We used an indirect immunofluorescence antibody (IFA) test for the BDV antibody and a real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assay for p24 and p40 RNA from peripheral blood mononuclear cells (PBMCs). Results Neither the BDV antibody nor p24, p40 RNA was detected in controls and patients groups. Conclusion Our results suggest that BDV might not be associated with psychiatric patients in Korea. PMID:20140130

Stable prevalence of Powassan virus in Ixodes scapularis in a northern Wisconsin focus.

PubMed

Brackney, Doug E; Nofchissey, Robert A; Fitzpatrick, Kelly A; Brown, Ivy K; Ebel, Gregory D

2008-12-01

Deer tick virus (DTV), a variant of Powassan virus (POWV), appears to be maintained in nature in an enzootic cycle between Ixodes scapularis ticks and small mammals. Although POWV infection of human beings is rare, a recent report suggests increasing incidence and the possibility that POWV may be an emerging tick-borne zoonosis. Therefore, we assessed the long-term stability of the POWV transmission cycle in northwestern Wisconsin. Adult I. scapularis and Dermacentor variabilis were collected from Hayward and Spooner, Wisconsin, screened for infection by reverse transcriptase polymerase chain reaction (RT-PCR), and virus was isolated. Seventeen of 1,335 (1.3%) of I. scapularis and 0 of 222 (0%) of D. variabilis ticks were infected. All isolated virus belonged to the DTV genotype of POWV. These findings suggest stable transmission of POWV in this focus over ten years and highlight the potential for this agent to emerge as a public health concern.

Scarabaecin, a novel cysteine-containing antifungal peptide from the rhinoceros beetle, Oryctes rhinoceros.

PubMed

Tomie, Tetsuya; Ishibashi, Jun; Furukawa, Seiichi; Kobayashi, Satoe; Sawahata, Ryoko; Asaoka, Ai; Tagawa, Michito; Yamakawa, Minoru

2003-07-25

A novel antifungal peptide, scarabaecin (4080Da), was isolated from the coconut rhinoceros beetle, Oryctes rhinoceros. Scarabaecin cDNA was cloned by reverse transcriptase-polymerase chain reactions (RT-PCR) using a primer based on the N-terminal amino acid sequence. The amino acid sequence deduced from scarabaecin cDNA showed no significant similarity to those of reported proteins. Chemically synthesized scarabaecin indicated antifungal activity against phytopathogenic fungi such as Pyricularia oryzae, Rhizoctonia solani, and Botrytis cinerea, but not against phytopathogenic bacteria. It showed weak activity against Bauberia bassiana, an insect pathogenic fungus, and Staphylococcus aureus, a pathogenic bacterium. Scarabaecin showed chitin binding property and its K(d) was 1.315 microM. A comparison of putative chitin-binding domains among scarabaecin, invertebrate, and plant chitin-binding proteins suggests that scarabaecin is a new member of chitin-binding antimicrobial proteins.

Isolation, sequence identification and tissue expression profiles of 3 novel porcine genes: ASPA, NAGA, and HEXA.

PubMed

Shu, Xianghua; Liu, Yonggang; Yang, Liangyu; Song, Chunlian; Hou, Jiafa

2008-01-01

The complete coding sequences of 3 porcine genes - ASPA, NAGA, and HEXA - were amplified by the reverse transcriptase polymerase chain reaction (RT-PCR) based on the conserved sequence information of the mouse or other mammals and referenced pig ESTs. These 3 novel porcine genes were then deposited in the NCBI database and assigned GeneIDs: 100142661, 100142664 and 100142667. The phylogenetic tree analysis revealed that the porcine ASPA, NAGA, and HEXA all have closer genetic relationships with the ASPA, NAGA, and HEXA of cattle. Tissue expression profile analysis was also carried out and results revealed that swine ASPA, NAGA, and HEXA genes were differentially expressed in various organs, including skeletal muscle, the heart, liver, fat, kidney, lung, and small and large intestines. Our experiment is the first one to establish the foundation for further research on these 3 swine genes.

PALATAL DYSMORPHOGENESIS: QUANTITATIVE RT-PCR

EPA Science Inventory

ABSTRACT

Palatal Dysmorphogenesis : Quantitative RT-PCR

Gary A. Held and Barbara D. Abbott

Reverse transcription PCR (RT-PCR) is a very sensitive method for detecting mRNA in tissue samples. However, as it is usually performed it is does not yield quantitativ...

Enterovirus D68 detection in respiratory specimens: Association with severe disease.

PubMed

Engelmann, Ilka; Fatoux, Marie; Lazrek, Mouna; Alidjinou, Enagnon K; Mirand, Audrey; Henquell, Cécile; Dewilde, Anny; Hober, Didier

2017-07-01

Molecular techniques increased the number of documented respiratory infections. In a substantial number of cases the causative agent remains undetected. Since August 2014, an increase in Enterovirus(EV)-D68 infections was reported. We aimed to investigate epidemiology and clinical relevance of EV-D68. From June to December 2014 and from September to December 2015, 803 and 847 respiratory specimens, respectively, were tested for respiratory viruses with a multiplex RT-PCR. This multiplex RT-PCR does not detect EV-D68. Therefore, 457 (2014) and 343 (2015) specimens with negative results were submitted to an EV-specific-RT-PCR. EV-positive specimens were tested with an EV-D68-specific-RT-PCR and genotyped. Eleven specimens of 2014 tested positive in the EV-specific-RT-PCR and of these seven were positive in the EV-D68-specific-RT-PCR. Typing confirmed these as EV-D68. Median age of EV-D68-positive patients was 3 years (1 month-91 years). Common symptoms included fever (n = 6, 86%), respiratory distress (n = 5, 71%), and cough (n = 4, 57%). All EV-D68-positive patients were admitted to hospital, 4 (57%) were admitted to intensive care units and 6 (86%) received oxygen. One patient suffered from acute flaccid paralysis. Seven specimens of 2015 were positive in the EV-specific-RT-PCR but negative in the EV-D68-specific-RT-PCR. In conclusion, use of an EV-specific-RT-PCR allowed us to detect EV-D68 circulation in autumn 2014 that was not detected by the multiplex RT-PCR and was associated with severe disease. © 2017 Wiley Periodicals, Inc.

Development of duplex RT-PCR-ELISA for the simultaneous detection of hepatitis A virus and hepatitis E virus.

PubMed

Tahk, Hongmin; Lee, Min Hwa; Lee, Kang Bum; Cheon, Doo-Sung; Choi, Changsun

2011-07-01

This study aimed to develop a specific and sensitive duplex reverse transcription polymerase chain reaction enzyme-linked immunosorbent assay (duplex RT-PCR-ELISA) for hepatitis A virus (HAV) and hepatitis E virus (HEV). Duplex RT-PCR-ELISA could detect and differentiate HAV and HEV with specific probes. When ELISA technique was used to detect probe-bound RT-PCR products, duplex RT-PCR-ELISA could detect as little as 0.1 ng/μL HAV and HEV from clinical samples. Human norovirus, enterovirus, poliovirus, murine norovirus and feline calicivirus were used for the specificity test; all were negative. Therefore duplex RT-PCR-ELISA can be used for the simultaneous detection of HAV and HEV in contaminated fecal samples. Copyright © 2011 Elsevier B.V. All rights reserved.

Performance of nested RT-PCR on CSF for tuberculous meningitis diagnosis in HIV-infected patients.

PubMed

Gualberto, F A S; Gonçalves, M G; Fukasawa, L O; Santos, A M Ramos Dos; Sacchi, C T; Harrison, L H; Boulware, D R; Vidal, J E

2017-10-01

Timely diagnosis of tuberculous meningitis (TBM) in patients with human immunodeficiency virus (HIV) infection remains a challenge. Despite the current scale-up of the Xpert® MTB/RIF assay, other molecular diagnostic tools are necessary, particularly in referral centres in low- and middle-income countries without Xpert testing. To determine the diagnostic performance of nested real-time polymerase chain reaction (nRT-PCR) in HIV-infected TBM patients categorised according to standardised clinical case definitions. Based on clinical, laboratory and imaging data, HIV-infected patients with suspected TBM were prospectively categorised as 'definite TBM', 'probable TBM', 'possible TBM' or 'not TBM'. We evaluated nRT-PCR sensitivity and specificity in diagnosing TBM among definite TBM cases, and among definite + probable TBM cases. Ninety-two participants were enrolled in the study. nRT-PCR sensitivity for definite TBM (n = 8) was 100% (95%CI 67-100) and 86% (95%CI 60-96) for both definite and probable TBM (n = 6). Assuming that 'not TBM' patients (n = 74) were true-negatives, nRT-PCR specificity was 100% (95%CI 95-100). The possible TBM group (n = 4) had no nRT-PCR positives. The nRT-PCR is a useful rule-in test for HIV-infected patients with TBM according to international consensus case definitions. As nRT-PCR cannot exclude TBM, studies comparing and combining nRT-PCR with other assays are necessary for a rule-out test.

A Guideline to Family-Wide Comparative State-of-the-Art Quantitative RT-PCR Analysis Exemplified with a Brassicaceae Cross-Species Seed Germination Case Study[W][OA

PubMed Central

Graeber, Kai; Linkies, Ada; Wood, Andrew T.A.; Leubner-Metzger, Gerhard

2011-01-01

Comparative biology includes the comparison of transcriptome and quantitative real-time RT-PCR (qRT-PCR) data sets in a range of species to detect evolutionarily conserved and divergent processes. Transcript abundance analysis of target genes by qRT-PCR requires a highly accurate and robust workflow. This includes reference genes with high expression stability (i.e., low intersample transcript abundance variation) for correct target gene normalization. Cross-species qRT-PCR for proper comparative transcript quantification requires reference genes suitable for different species. We addressed this issue using tissue-specific transcriptome data sets of germinating Lepidium sativum seeds to identify new candidate reference genes. We investigated their expression stability in germinating seeds of L. sativum and Arabidopsis thaliana by qRT-PCR, combined with in silico analysis of Arabidopsis and Brassica napus microarray data sets. This revealed that reference gene expression stability is higher for a given developmental process between distinct species than for distinct developmental processes within a given single species. The identified superior cross-species reference genes may be used for family-wide comparative qRT-PCR analysis of Brassicaceae seed germination. Furthermore, using germinating seeds, we exemplify optimization of the qRT-PCR workflow for challenging tissues regarding RNA quality, transcript stability, and tissue abundance. Our work therefore can serve as a guideline for moving beyond Arabidopsis by establishing high-quality cross-species qRT-PCR. PMID:21666000

RT-qPCR Demonstrates Light-Dependent AtRBCS1A and AtRBCS3B mRNA Expressions in "Arabidopsis thaliana" Leaves

ERIC Educational Resources Information Center

Chang, Ming-Mei; Li, Anna; Feissner, Robert; Ahmad, Talal

2016-01-01

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is widely used in diagnosis and research to determine specific mRNA expressions in cells. As RT-qPCR applications increase, it is necessary to provide undergraduates hands-on experience of this modern technique. Here, we report a 3-week laboratory exercise using RT-qPCR to…

One-step cross-genogroup multiplex RT-qPCR with an internal control system for the detection of infectious pancreatic necrosis virus (IPNV).

PubMed

Hoferer, Marc; Braun, Anne; Skrypski, Julia; Bock, Sabine; Thalheim, Sabine; Sting, Reinhard

2017-09-01

Infectious pancreatic necrosis virus (IPNV) causes great losses in fish hatcheries world-wide. The detection of IPNV can be challenging in certain circumstances, particularly due to low viral load and the genetic variability of this RNA virus. For the first time, this project created a quantitative triplex real-time reverse transcription PCR (RT-qPCR), including an endogenous control system, for specific, sensitive and rapid detection of IPNV in routine diagnostics. Multiple sequence alignment of 46 nucleotide sequences of the segment A genome obtained from the NCBI database allowed the design of two RT-qPCR systems covering the IPNV genogroup 1 and genogroups 2-5, respectively. The completed triplex RT-qPCR including a salmonid-specific endogenous control showed high specificity and an analytical sensitivity of 20-40 oligonucleotide copies. Testing of dilution series of virus-loaded cell culture suspensions proved equality of the triplex RT-qPCR with virus detection in cell culture and a higher sensitivity than conventional RT-PCR in field samples. In comparative studies of a total of 77 field samples tested, 51 showed identical positive and 19 identical negative results in cell culture and the triplex RT-qPCR. However, seven other samples yielded positive results in the triplex RT-qPCR, but negative results in cell culture. Copyright © 2017 Elsevier B.V. All rights reserved.

Silent mutations at codons 65 and 66 in reverse transcriptase alleviate indel formation and restore fitness in subtype B HIV-1 containing D67N and K70R drug resistance mutations

PubMed Central

Telwatte, Sushama; Hearps, Anna C.; Johnson, Adam; Latham, Catherine F.; Moore, Katie; Agius, Paul; Tachedjian, Mary; Sonza, Secondo; Sluis-Cremer, Nicolas; Harrigan, P. Richard; Tachedjian, Gilda

2015-01-01

Resistance to combined antiretroviral therapy (cART) in HIV-1-infected individuals is typically due to nonsynonymous mutations that change the protein sequence; however, the selection of synonymous or ‘silent’ mutations in the HIV-1 genome with cART has been reported. These silent K65K and K66K mutations in the HIV-1 reverse transcriptase (RT) occur in over 35% of drug-experienced individuals and are highly associated with the thymidine analog mutations D67N and K70R, which confer decreased susceptibility to most nucleoside and nucleotide RT inhibitors. However, the basis for selection of these silent mutations under selective drug pressure is unknown. Using Illumina next-generation sequencing, we demonstrate that the D67N/K70R substitutions in HIV-1 RT increase indel frequency by 100-fold at RT codons 65–67, consequently impairing viral fitness. Introduction of either K65K or K66K into HIV-1 containing D67N/K70R reversed the error-prone DNA synthesis at codons 65–67 in RT and improved viral replication fitness, but did not impact RT inhibitor drug susceptibility. These data provide new mechanistic insights into the role of silent mutations selected during antiretroviral therapy and have broader implications for the relevance of silent mutations in the evolution and fitness of RNA viruses. PMID:25765644

Novel nonnucleoside inhibitors that select nucleoside inhibitor resistance mutations in human immunodeficiency virus type 1 reverse transcriptase.

PubMed

Zhang, Zhijun; Walker, Michelle; Xu, Wen; Shim, Jae Hoon; Girardet, Jean-Luc; Hamatake, Robert K; Hong, Zhi

2006-08-01

Mutations in and around the catalytic site of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) are associated with resistance to nucleoside RT inhibitors (NRTIs), whereas changes in the hydrophobic pocket of the RT are attributed to nonnucleoside RT inhibitor (NNRTI) resistance. In this study, we report a novel series of nonnucleoside inhibitors of HIV-1, exemplified by VRX-329747 and VRX-413638, which inhibit both NNRTI- and NRTI-resistant HIV-1 isolates. Enzymatic studies indicated that these compounds are HIV-1 RT inhibitors. Surprisingly, however, following prolonged (6 months) tissue culture selection, this series of nonnucleoside inhibitors did not select NNRTI-resistant mutations in HIV-1 RT. Rather, four mutations (M41L, A62T/V, V118I, and M184V) known to cause resistance to NRTIs and two additional novel mutations (S68N and G112S) adjacent to the catalytic site of the enzyme were selected. Although the M184V mutation appears to be the initial mutation to establish resistance, this mutation alone confers only a two- to fourfold decrease in susceptibility to VRX-329747 and VRX-413638. At least two additional mutations must accumulate for significant resistance. Moreover, while VRX-329747-selected viruses are resistant to lamivudine and emtricitabine due to the M184V mutation, they remain susceptible to zidovudine, stavudine, dideoxyinosine, abacavir, tenofovir, and efavirenz. These results directly demonstrate that VRX-329747 and VRX-413638 are novel nonnucleoside inhibitors of HIV-1 RT with the potential to augment current therapies.

Novel Nonnucleoside Inhibitors That Select Nucleoside Inhibitor Resistance Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase

PubMed Central

Zhang, Zhijun; Walker, Michelle; Xu, Wen; Shim, Jae Hoon; Girardet, Jean-Luc; Hamatake, Robert K.; Hong, Zhi

2006-01-01

Mutations in and around the catalytic site of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) are associated with resistance to nucleoside RT inhibitors (NRTIs), whereas changes in the hydrophobic pocket of the RT are attributed to nonnucleoside RT inhibitor (NNRTI) resistance. In this study, we report a novel series of nonnucleoside inhibitors of HIV-1, exemplified by VRX-329747 and VRX-413638, which inhibit both NNRTI- and NRTI-resistant HIV-1 isolates. Enzymatic studies indicated that these compounds are HIV-1 RT inhibitors. Surprisingly, however, following prolonged (6 months) tissue culture selection, this series of nonnucleoside inhibitors did not select NNRTI-resistant mutations in HIV-1 RT. Rather, four mutations (M41L, A62T/V, V118I, and M184V) known to cause resistance to NRTIs and two additional novel mutations (S68N and G112S) adjacent to the catalytic site of the enzyme were selected. Although the M184V mutation appears to be the initial mutation to establish resistance, this mutation alone confers only a two- to fourfold decrease in susceptibility to VRX-329747 and VRX-413638. At least two additional mutations must accumulate for significant resistance. Moreover, while VRX-329747-selected viruses are resistant to lamivudine and emtricitabine due to the M184V mutation, they remain susceptible to zidovudine, stavudine, dideoxyinosine, abacavir, tenofovir, and efavirenz. These results directly demonstrate that VRX-329747 and VRX-413638 are novel nonnucleoside inhibitors of HIV-1 RT with the potential to augment current therapies. PMID:16870771

Reverse transcription and polymerase chain reaction: principles and applications in dentistry.

PubMed

Santos, Carlos Ferreira Dos; Sakai, Vivien Thiemy; Machado, Maria Aparecida de Andrade Moreira; Schippers, Daniela Nicole; Greene, Andrew Seth

2004-03-01

Various molecular biology techniques have become available in the last few years. One of the most revolutionary of these techniques regarding nucleic acid analysis is the polymerase chain reaction (PCR), which was first described in 1985. This method relies on the exponential amplification of specific DNA fragments, resulting in millions of copies that can serve as templates for different kinds of analyses. PCR can be preceded by a reverse transcription (RT) reaction in order to produce cDNA from RNA (RT-PCR). RT-PCR provides the possibility to assess gene transcription in cells or tissues. PCR and RT-PCR techniques have been instrumental in dental research, and show potential to be used for diagnosis as well as for treatment and prevention of many diseases (dental caries, periodontal disease, endodontic infections and oral cancer). Compared to other traditional methodologies, PCR and RT-PCR show many advantages including high specificity, sensitivity, and speed. Since PCR and RT-PCR are relatively new techniques and are not available to most students and professionals involved with dentistry, the aim of this work is to present the details of these techniques as well as dental literature reports in which they were used.

Complete inactivation of HIV-1 using photo-labeled non-nucleoside reverse transcriptase inhibitors.

PubMed

Rios, Adan; Quesada, Jorge; Anderson, Dallas; Goldstein, Allan; Fossum, Theresa; Colby-Germinario, Susan; Wainberg, Mark A

2011-01-01

We demonstrate that a photo-labeled derivative of the non-nucleoside reverse transcriptase inhibitor (NNRTI) dapivirine termed DAPY, when used together with exposure to ultraviolet light, was able to completely and irreversibly inactivate both HIV-1 RT activity as well as infectiousness in each of a T cell line and peripheral blood mononuclear cells. Control experiments using various concentrations of DAPY revealed that a combination of exposure to ultraviolet light together with use of the specific, high affinity photo-labeled compound was necessary for complete inactivation to occur. This method of HIV RT inactivation may have applicability toward preservation of an intact viral structure and warrants further investigation in regard to the potential of this approach to elicit a durable, broad protective immune response. Copyright © 2010 Elsevier B.V. All rights reserved.

Detection of 22 common leukemic fusion genes using a single-step multiplex qRT-PCR-based assay.

PubMed

Lyu, Xiaodong; Wang, Xianwei; Zhang, Lina; Chen, Zhenzhu; Zhao, Yu; Hu, Jieying; Fan, Ruihua; Song, Yongping

2017-07-25

Fusion genes generated from chromosomal translocation play an important role in hematological malignancies. Detection of fusion genes currently employ use of either conventional RT-PCR methods or fluorescent in situ hybridization (FISH), where both methods involve tedious methodologies and require prior characterization of chromosomal translocation events as determined by cytogenetic analysis. In this study, we describe a real-time quantitative reverse transcription PCR (qRT-PCR)-based multi-fusion gene screening method with the capacity to detect 22 fusion genes commonly found in leukemia. This method does not require pre-characterization of gene translocation events, thereby facilitating immediate diagnosis and therapeutic management. We performed fluorescent qRT-PCR (F-qRT-PCR) using a commercially-available multi-fusion gene detection kit on a patient cohort of 345 individuals comprising 108 cases diagnosed with acute myeloid leukemia (AML) for initial evaluation; remaining patients within the cohort were assayed for confirmatory diagnosis. Results obtained by F-qRT-PCR were compared alongside patient analysis by cytogenetic characterization. Gene translocations detected by F-qRT-PCR in AML cases were diagnosed in 69.4% of the patient cohort, which was comparatively similar to 68.5% as diagnosed by cytogenetic analysis, thereby demonstrating 99.1% concordance. Overall gene fusion was detected in 53.7% of the overall patient population by F-qRT-PCR, 52.9% by cytogenetic prediction in leukemia, and 9.1% in non-leukemia patients by both methods. The overall concordance rate was calculated to be 99.0%. Fusion genes were detected by F-qRT-PCR in 97.3% of patients with CML, followed by 69.4% with AML, 33.3% with acute lymphoblastic leukemia (ALL), 9.1% with myelodysplastic syndromes (MDS), and 0% with chronic lymphocytic leukemia (CLL). We describe the use of a F-qRT-PCR-based multi-fusion gene screening method as an efficient one-step diagnostic procedure as an effective alternative to lengthy conventional diagnostic procedures requiring both cytogenetic analysis followed by targeted quantitative reverse transcription (qRT-PCR) methods, thus allowing timely patient management.

Synthesis and evaluation of "AZT-HEPT", "AZT-pyridinone", and "ddC-HEPT" conjugates as inhibitors of HIV reverse transcriptase.

PubMed

Pontikis, R; Dollé, V; Guillaumel, J; Dechaux, E; Note, R; Nguyen, C H; Legraverend, M; Bisagni, E; Aubertin, A M; Grierson, D S; Monneret, C

2000-05-18

To test the concept that HIV reverse transcriptase could be effectively inhibited by "mixed site inhibitors", a series of seven conjugates containing both a nucleoside analogue component (AZT 1, ddC 2) and a nonnucleoside type inhibitor (HEPT analogue 12, pyridinone 27) were synthesized and evaluated for their ability to block HIV replication. The (N-3 and C-5)AZT-HEPT conjugates 15, 22, and 23 displayed 2-5 microM anti-HIV activity, but they had no effect on the replication of HIV-2 or the HIV-1 strain with the Y181C mutation. The (C-5)AZT-pyridinone conjugates 34-37 were found to be inactive. In marked contrast, the ddC-HEPT molecule 26 displayed the same potency (EC(50) = 0.45 microM) against HIV-1 (wild type and the Y181C nevirapine-resistant strain) and HIV-2 in cell culture. No synergistic effect was observed for these bis-substrate inhibitors, suggesting that the two individual inhibitor components in these molecules do not bind simultaneously in their respective sites. Interestingly, however, the results indicate that the AZT-HEPT conjugates and the ddC-HEPT derivative 26 inhibit reverse transcriptase (RT) in an opposite manner. One explanation for this difference is that the former compounds interact preferentially with the hydrophobic pocket in RT, whereas 26 (after supposed triphosphorylation) inhibits RT through binding in the catalytic site.

The removal of RNA primers from DNA synthesized by the reverse transcriptase of the retrotransposon Tf1 is stimulated by Tf1 integrase.

PubMed

Herzig, Eytan; Voronin, Nickolay; Hizi, Amnon

2012-06-01

The Tf1 retrotransposon represents a group of long terminal repeat retroelements that use an RNA self-primer for initiating reverse transcription while synthesizing the minus-sense DNA strand. Tf1 reverse transcriptase (RT) was found earlier to generate the self-primer in vitro. Here, we show that this RT can remove from the synthesized cDNA the entire self-primer as well as the complete polypurine tract (PPT) sequence (serving as a second primer for cDNA synthesis). However, these primer removals, mediated by the RNase H activity of Tf1 RT, are quite inefficient. Interestingly, the integrase of Tf1 stimulated the specific Tf1 RT-directed cleavage of both the self-primer and PPT, although there was no general enhancement of the RT's RNase H activity (and the integrase by itself is devoid of any primer cleavage). The RTs of two prototype retroviruses, murine leukemia virus and human immunodeficiency virus, showed only a partial and nonspecific cleavage of both Tf1-associated primers with no stimulation by Tf1 integrase. Mutagenesis of Tf1 integrase revealed that the complete Tf1 integrase protein (excluding its chromodomain) is required for stimulating the Tf1 RT primer removal activity. Nonetheless, a double mutant integrase that has lost its integration functions can still stimulate the RT's activity, though heat-inactivated integrase cannot enhance primer removals. These findings suggest that the enzymatic activity of Tf1 integrase is not essential for stimulating the RT-mediated primer removal, while the proper folding of this protein is obligatory for this function. These results highlight possible new functions of Tf1 integrase in the retrotransposon's reverse transcription process.

The Removal of RNA Primers from DNA Synthesized by the Reverse Transcriptase of the Retrotransposon Tf1 Is Stimulated by Tf1 Integrase

PubMed Central

Herzig, Eytan; Voronin, Nickolay

2012-01-01

The Tf1 retrotransposon represents a group of long terminal repeat retroelements that use an RNA self-primer for initiating reverse transcription while synthesizing the minus-sense DNA strand. Tf1 reverse transcriptase (RT) was found earlier to generate the self-primer in vitro. Here, we show that this RT can remove from the synthesized cDNA the entire self-primer as well as the complete polypurine tract (PPT) sequence (serving as a second primer for cDNA synthesis). However, these primer removals, mediated by the RNase H activity of Tf1 RT, are quite inefficient. Interestingly, the integrase of Tf1 stimulated the specific Tf1 RT-directed cleavage of both the self-primer and PPT, although there was no general enhancement of the RT's RNase H activity (and the integrase by itself is devoid of any primer cleavage). The RTs of two prototype retroviruses, murine leukemia virus and human immunodeficiency virus, showed only a partial and nonspecific cleavage of both Tf1-associated primers with no stimulation by Tf1 integrase. Mutagenesis of Tf1 integrase revealed that the complete Tf1 integrase protein (excluding its chromodomain) is required for stimulating the Tf1 RT primer removal activity. Nonetheless, a double mutant integrase that has lost its integration functions can still stimulate the RT's activity, though heat-inactivated integrase cannot enhance primer removals. These findings suggest that the enzymatic activity of Tf1 integrase is not essential for stimulating the RT-mediated primer removal, while the proper folding of this protein is obligatory for this function. These results highlight possible new functions of Tf1 integrase in the retrotransposon's reverse transcription process. PMID:22491446

Cloning of gene-encoded stem bromelain on system coming from Pichia pastoris as therapeutic protein candidate

NASA Astrophysics Data System (ADS)

Yusuf, Y.; Hidayati, W.

2018-01-01

The process of identifying bacterial recombination using PCR, and restriction, and then sequencing process was done after identifying the bacteria. This research aimed to get a yeast cell of Pichia pastoris which has an encoder gene of stem bromelain enzyme. The production of recombinant stem bromelain enzymes using yeast cells of P. pastoris can produce pure bromelain rod enzymes and have the same conformation with the enzyme’s conformation in pineapple plants. This recombinant stem bromelain enzyme can be used as a therapeutic protein in inflammatory, cancer and degenerative diseases. This study was an early stage of a step series to obtain bromelain rod protein derived from pineapple made with genetic engineering techniques. This research was started by isolating the RNA of pineapple stem which was continued with constructing cDNA using reserve transcriptase-PCR technique (RT-PCR), doing the amplification of bromelain enzyme encoder gene with PCR technique using a specific premiere couple which was designed. The process was continued by cloning into bacterium cells of Escherichia coli. A vector which brought the encoder gene of stem bromelain enzyme was inserted into the yeast cell of P. pastoris and was continued by identifying the yeast cell of P. pastoris which brought the encoder gene of stem bromelain enzyme. The research has not found enzyme gene of stem bromelain in yeast cell of P. pastoris yet. The next step is repeating the process by buying new reagent; RNase inhibitor, and buying liquid nitrogen.

Identification of sexually dimorphic gene expression in brain tissue of the fish Leporinus macrocephalus through mRNA differential display and real time PCR analyses.

PubMed

Alves-Costa, Fernanda A; Wasko, A P

2010-03-01

Differentially expressed genes in males and females of vertebrate species generally have been investigated in gonads and, to a lesser extent, in other tissues. Therefore, we attempted to identify sexually dimorphic gene expression in the brains of adult males and females of Leporinus macrocephalus, a gonochoristic fish species that presents a ZZ/ZW sex determination system, throughout a comparative analysis using differential display reverse transcriptase-PCR and real-time PCR. Four cDNA fragments were characterized, representing candidate genes with differential expression between the samples. Two of these fragments presented no significant identity with previously reported gene sequences. The other two fragments, isolated from male specimens, were associated to the gene that codes for the protein APBA2 (amyloid beta (A4) precursor protein-binding, family A, member 2) and to the Rab 37 gene, a member of the Ras oncogene family. The overexpression of these genes has been associated to a greater production of the beta-amyloid protein which, in turns, is the major factor that leads to Alzheimer's disease, and to the development of brain-tumors, respectively. Quantitative RT-PCR analyses revealed a higher Apba2 gene expression in males, thus validating the previous data on differential display. L. macrocephalus may represent an interesting animal model to the understanding of the function of several vertebrate genes, including those involved in neurodegenerative and cancer diseases.

A tumor-promoting mechanism mediated by retrotransposon-encoded reverse transcriptase is active in human transformed cell lines

PubMed Central

Sciamanna, Ilaria; Gualtieri, Alberto; Cossetti, Cristina; Osimo, Emanuele Felice; Ferracin, Manuela; Macchia, Gianfranco; Aricò, Eleonora; Prosseda, Gianni; Vitullo, Patrizia; Misteli, Tom; Spadafora, Corrado

2013-01-01

LINE-1 elements make up the most abundant retrotransposon family in the human genome. Full-length LINE-1 elements encode a reverse transcriptase (RT) activity required for their own retrotranpsosition as well as that of non-autonomous Alu elements. LINE-1 are poorly expressed in normal cells and abundantly in cancer cells. Decreasing RT activity in cancer cells, by either LINE-1-specific RNA interference, or by RT inhibitory drugs, was previously found to reduce proliferation and promote differentiation and to antagonize tumor growth in animal models. Here we have investigated how RT exerts these global regulatory functions. We report that the RT inhibitor efavirenz (EFV) selectively downregulates proliferation of transformed cell lines, while exerting only mild effects on non-transformed cells; this differential sensitivity matches a differential RT abundance, which is high in the former and undetectable in the latter. Using CsCl density gradients, we selectively identify Alu and LINE-1 containing DNA:RNA hybrid molecules in cancer but not in normal cells. Remarkably, hybrid molecules fail to form in tumor cells treated with EFV under the same conditions that repress proliferation and induce the reprogramming of expression profiles of coding genes, microRNAs (miRNAs) and ultraconserved regions (UCRs). The RT-sensitive miRNAs and UCRs are significantly associated with Alu sequences. The results suggest that LINE-1-encoded RT governs the balance between single-stranded and double-stranded RNA production. In cancer cells the abundant RT reverse-transcribes retroelement-derived mRNAs forming RNA:DNA hybrids. We propose that this impairs the formation of double-stranded RNAs and the ensuing production of small regulatory RNAs, with a direct impact on gene expression. RT inhibition restores the ‘normal’ small RNA profile and the regulatory networks that depend on them. Thus, the retrotransposon-encoded RT drives a previously unrecognized mechanism crucial to the transformed state in tumor cells. PMID:24345856

Template-independent DNA synthesis activity associated with the reverse transcriptase of the long terminal repeat retrotransposon Tf1.

PubMed

Oz-Gleenberg, Iris; Herzig, Eytan; Hizi, Amnon

2012-01-01

Reverse transcriptases (RTs) possess a non-templated addition (NTA) activity while synthesizing DNA with blunt-ended DNA primer/templates. Interestingly, the RT of the long terminal repeat retrotransposon Tf1 has an NTA activity that is substantially higher than that of HIV-1 or murine leukemia virus RTs. By performing steady state kinetics, we found that the differences between the NTA activities of Tf1 and HIV-1 RTs can be explained by the substantially lower K(M) value for the incoming dNTP of Tf1 RT (while the differences between the apparent k(cat) values of these two RTs are relatively small). Furthermore, the K(M) values, calculated for both RTs with the same dNTP, are much lower for the template-dependent synthesis (TDS) than those of NTA. However, TDS of HIV-1 RT is higher than that of Tf1 RT. The overall relative order of the apparent k(cat)/K(M) values for dATP is: HIV-1 RT (TDS) > Tf1 RT (TDS) > Tf1 RT (NTA) > HIV-1 RT (NTA). Under the employed conditions, Tf1 RT can add up to seven nucleotides to the blunt-ended substrate, while the other RTs add mostly a single nucleotide. The NTA activity of Tf1 RT is restricted to DNA primers. Furthermore, the NTA activity of Tf1 and HIV-1 RTs is suppressed by ATP, as it competes with the incoming dATP (although ATP is not incorporated by the NTA activity of the RTs). The unusually high NTA activity of Tf1 RT can explain why, after completing cDNA synthesis, the in vivo generated Tf1 cDNA has relatively long extra sequences beyond the highly conserved CA at its 3'-ends. © 2011 The Authors Journal compilation © 2011 FEBS.

Downregulation of telomerase activity by diclofenac and curcumin is associated with cell cycle arrest and induction of apoptosis in colon cancer.

PubMed

Rana, Chandan; Piplani, Honit; Vaish, Vivek; Nehru, Bimla; Sanyal, S N

2015-08-01

Uncontrolled cell proliferation is the hallmark of cancer, and cancer cells have typically acquired damage to genes that directly regulate their cell cycles. The synthesis of DNA onto the end of chromosome during the replicative phase of cell cycle by telomerase may be necessary for unlimited proliferation of cells. Telomerase, a ribonucleoprotein enzyme is considered as a universal therapeutic target of cancer because of its preferential expression in cancer cells and its presence in 90 % of tumors. We studied the regulation of telomerase and telomerase reverse transcriptase catalytic subunit (TERT) by diclofenac and curcumin, alone and also in combination, in 1, 2-dimethylhydrazine dihydrochloride-induced colorectal cancer in rats. The relationship of telomerase activity with tumors suppressor proteins (p51, Rb, p21), cell cycle machinery, and apoptosis was also studied. Telomerase is highly expressed in DMH group and its high activity is associated with increased TERT expression. However, telomerase is absent or is present at lower levels in normal tissue. CDK4, CDK2, cyclin D1, and cyclin E are highly expressed in DMH as assessed by RT-PCR, qRT-PCR, Western blot, and immunofluorescence analysis. Diclofenac and curcumin overcome these carcinogenic effects by downregulating telomerase activity, diminishing the expression of TERT, CDK4, CDK2, cyclin D1, and cyclin E. The anticarcinogenic effects shown after the inhibition of telomerase activity by diclofenac and curcumin may be associated with upregulation of tumor suppressor proteins p51, Rb, and p21, whose activation induces the cells cycle arrest and apoptosis.

Anti-chikungunya activity of luteolin and apigenin rich fraction from Cynodon dactylon.

PubMed

Murali, Krishnan Saravana; Sivasubramanian, Srinivasan; Vincent, Savariar; Murugan, Shanmugaraj Bala; Giridaran, Bupesh; Dinesh, Sundaram; Gunasekaran, Palani; Krishnasamy, Kaveri; Sathishkumar, Ramalingam

2015-05-01

To obtain luteolin and apigenin rich fraction from the ethanolic extract of Cynodon dactylon (L.) (C. dactylon) Pers and evaluate the fraction's cytotoxicity and anti-Chikungunya potential using Vero cells. The ethanolic extract of C. dactylon was subjected to silica gel column chromatography to obtain anti-chikungunya virus (CHIKV) fraction. Reverse phase-HPLC and GC-MS studies were carried out to identify the major phytochemicals in the fraction using phytochemical standards. Cytotoxicity and the potential of the fraction against CHIKV were evaluated in vitro using Vero cells. Reduction in viral replication was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) after treating the viral infected Vero cells with the fraction. Reverse Phase-HPLC and GC-MS studies confirmed the presence of flavonoids, luteolin and apigenin as major phytochemicals in the anti-CHIKV ethanolic fraction of C. dactylon. The fraction was found to exhibit potent viral inhibitory activity (about 98%) at the concentration of 50 µg/mL as observed by reduction in cytopathic effect, and the cytotoxic concentration of the fraction was found to be 250 µg/mL. RT-PCR analyses indicated that the reduction in viral mRNA synthesis in fraction treated infected cells was much higher than the viral infected control cells. Luteolin and apigenin rich ethanolic fraction from C. dactylon can be utilized as a potential therapeutic agent against CHIKV infection as the fraction does not show cytotoxicity while inhibiting the virus. Copyright © 2015 Hainan Medical College. Production and hosting by Elsevier B.V. All rights reserved.

Peroxisome proliferator-activated receptor delta (PPARdelta) activation protects H9c2 cardiomyoblasts from oxidative stress-induced apoptosis.

PubMed

Pesant, Matthieu; Sueur, Stéphanie; Dutartre, Patrick; Tallandier, Mireille; Grimaldi, Paul A; Rochette, Luc; Connat, Jean-Louis

2006-02-01

Activation of peroxisome proliferator-activated receptor alpha (PPARalpha) and PPARgamma plays beneficial roles in cardiovascular disorders such as atherosclerosis and heart reperfusion. Although PPARalpha and gamma have been documented to reduce oxidative stress in the vasculature and the heart, the role of PPARdelta remains poorly studied. We focused on PPARdelta function in the regulation of oxidative stress-induced apoptosis in the rat cardiomyoblast cell line H9c2. Using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we showed that PPARdelta is the predominantly expressed isotype whereas PPARalpha was weakly detected. By performing cell viability assays, we also showed that the selective PPARdelta agonist GW501516 protected cells from H(2)O(2)-induced cell death. The protective effect of GW501516 was due to an inhibition of H(2)O(2)-triggered apoptosis as shown by annexin-V labeling, DNA fragmentation analysis, and caspase-3 activity measurement. We demonstrated by transient transfection of a dominant negative mutant of PPARdelta that the protection induced by GW501516 was totally dependent on PPARdelta. Semi-quantitative RT-PCR and Western blotting analysis demonstrated that GW501516 treatment upregulated catalase. Moreover, forced overexpression of catalase inhibited H(2)O(2)-triggered apoptosis, as evidenced by annexin-V labeling. Taken together, our results account for an important role of PPARdelta in inhibiting the onset of oxidative stress-induced apoptosis in H9c2 cells. PPARdelta appears to be a new therapeutic target for the regulation of heart reperfusion-associated oxidative stress and stimulation of enzymatic antioxidative defences.

Correlations between A/H1N1 influenza and acute cellular rejection in liver transplantation patients.

PubMed

Stucchi, R S B; Boin, I F S F; Angerami, R Nogueira; Sinckoc, V; Sa, F Cesar; Seva-Pereira, T; Escanhoela, C A Fazzio

2010-12-01

Influenza is a common cause of respiratory infection in transplant recipients. It is expected that A/H1N1 influenza virus causes more severe disease in solid-organ recipients. Our goal was to describe two A/H1N1 infections that occurred after Orthotopic liver transplantation followed by acute allograft rejection episodes. From March 2009 to March 2010 we observe two liver transplant patients with symptoms suggestive of A/H1N1 infection. The diagnosis was out based on a temperature of 37.8°C (100°F) or higher and the presence of a cough or using materials from anasopharyngeal and oropharyngeal swabs a sore throat. The diagnosis was confirmed by viral RNA detection by real-time reverse-transcriptase-polymerase-chain-reaction assay (RT-PCR) using materials from nasopharyngeal and oropharyngeal swabs. We performed the RT-PCR assay for A/H1N1 detection in a liver biopsy from one patient. Both patients were treated with usual doses of oseltamivir (75 mg twice daily for 5 days). One patient developed acute bacterial sinusitis requiring antibiotic therapy. Thereafter the liver enzymes increased and transplant biopsies showed moderate-to-severe acute cellular rejection. They were treated with corticosteroids. The liver enzymes normalized after 3 months. A/H1N1 influenza can lead to a severe acute cellular rejection episode with corticosteroid resistant treatment in liver transplant patients. Transplant centers should be aware of a possible relationship between A/H1N1 infections and acute allograft rejection episodes. Copyright © 2010 Elsevier Inc. All rights reserved.

Fisetin as a caloric restriction mimetic protects rat brain against aging induced oxidative stress, apoptosis and neurodegeneration.

PubMed

Singh, Sandeep; Singh, Abhishek Kumar; Garg, Geetika; Rizvi, Syed Ibrahim

2018-01-15

In the present study, attempts have been made to evaluate the potential role of fisetin, a caloric restriction mimetic (CRM), for neuroprotection in D-galactose (D-gal) induced accelerated and natural aging models of rat. Fisetin was supplemented (15mg/kg b.w., orally) to young, D-gal induced aged (D-gal 500mg/kg b.w subcutaneously) and naturally aged rats for 6weeks. Standard protocols were employed to measure pro-oxidants, antioxidants and mitochondrial membrane potential in brain tissues. Gene expression analysis with reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to assess the expression of autophagy, neuronal, aging as well as inflammatory marker genes. We have also evaluated apoptotic cell death and synaptosomal membrane-bound ion transporter activities in brain tissues. Our data demonstrated that fisetin significantly decreased the level of pro-oxidants and increased the level of antioxidants. Furthermore, fisetin also ameliorated mitochondrial membrane depolarization, apoptotic cell death and impairments in the activities of synaptosomal membrane-bound ion transporters in aging rat brain. RT-PCR data revealed that fisetin up-regulated the expression of autophagy genes (Atg-3 and Beclin-1), sirtuin-1 and neuronal markers (NSE and Ngb), and down-regulated the expression of inflammatory (IL-1β and TNF-α) and Sirt-2 genes respectively in aging brain. The present study suggests that fisetin supplementation may provide neuroprotection against aging-induced oxidative stress, apoptotic cell death, neuro-inflammation, and neurodegeneration in rat brain. Copyright © 2017 Elsevier Inc. All rights reserved.

Eicosapentaenoic acid-enriched phosphatidylcholine isolated from Cucumaria frondosa exhibits anti-hyperglycemic effects via activating phosphoinositide 3-kinase/protein kinase B signal pathway.

PubMed

Hu, Shiwei; Xu, Leilei; Shi, Di; Wang, Jingfeng; Wang, Yuming; Lou, Qiaoming; Xue, Changhu

2014-04-01

Eicosapentaenoic acid-enriched phosphatidylcholine was isolated from the sea cucumber Cucumaria frondosa (Cucumaria-PC) and its effects on streptozotocin (STZ)-induced hyperglycemic rats were investigated. Male Sprague-Dawley rats were randomly divided into normal control, model control (STZ), low- and high-dose Cucumaria-PC groups (STZ + Cucumaria-PC at 25 and 75 mg/Kg·b·wt, intragastrically, respectively). Blood glucose, insulin, glycogen in liver and gastrocnemius were determined over 60 days. Insulin signaling in the rats' gastrocnemius was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. The results showed that Cucumaria-PC significantly decreased blood glucose level, increased insulin secretion and glycogen synthesis in diabetic rats. RT-PCR analysis revealed that Cucumaria-PC significantly promoted the expressions of glycometabolism-related genes of insulin receptor (IR), insulin receptor substrate-1 (IRS-1), phosphoinositide 3-kinase (PI3K), protein kinase B (PKB), and glucose transporter 4 (GLUT4) in gastrocnemius. Western blotting assay demonstrated that Cucumaria-PC remarkably enhanced the proteins abundance of IR-β, PI3K, PKB, GLUT4, as well as phosphorylation of Tyr-IR-β, p85-PI3K, Ser473-PKB (P < 0.05 and P < 0.01). These findings suggested that Cucumaria-PC exhibited significant anti-hyperglycemic activities through up-regulating PI3K/PKB signal pathway mediated by insulin. Nutritional supplementation with Cucumaria-PC, if validated for human studies, may offer an adjunctive therapy for diabetes mellitus. Copyright © 2013 The Society for Biotechnology, Japan. All rights reserved.

Characterization of Common Chromosomal Translocations and Their Frequencies in Acute Myeloid Leukemia Patients of Northwest Iran

PubMed Central

Amanollahi Kamaneh, Elnaz; Shams Asenjan, Karim; Movassaghpour Akbari, Aliakbar; Akbarzadeh Laleh, Parvin; Chavoshi, Hadi; Eivazi Ziaei, Jamal; Nikanfar, Alireza; Asvadi Kermani, Iraj; Esfahani, Ali

2016-01-01

Objective Detection of chromosomal translocations has an important role in diagnosis and treatment of hematological disorders. We aimed to evaluate the 46 new cases of de novo acute myeloid leukemia (AML) patients for common translocations and to assess the effect of geographic and ethnic differences on their frequencies. Materials and Methods In this descriptive study, reverse transcriptase-polymerase chain reaction (RT-PCR) was used on 46 fresh bone marrow or peripheral blood samples to detect translocations t (8; 21), t (15; 17), t (9; 11) and inv (16). Patients were classified using the French-American-British (FAB) criteria in to eight sub-groups (M0-M7). Immunophenotyping and biochemical test results of patients were compared with RT-PCR results. Results Our patients were relatively young with a mean age of 44 years. AML was relatively predominant in female patients (54.3%) and most of patients belonged to AML-M2. Translocation t (8; 21) had the highest frequency (13%) and t (15; 17) with 2.7% incidence was the second most frequent. CD19 as an immunophenotypic marker was at a relatively high frequency (50%) in cases with t (8; 21), and patients with this translocation had a specific immunophenotypic pattern of complete expression of CD45, CD38, CD34, CD33 and HLA-DR. Conclusion Similarities and differences of results in Iran with different parts of the world can be explained with ethnic and geographic factors in characterizations of AML. Recognition of these factors especially in other comprehensive studies may aid better diagnosis and management of this disease. PMID:27054117

High expression of AFAP1-AS1 is associated with poor survival and short-term recurrence in pancreatic ductal adenocarcinoma.

PubMed

Ye, Yibiao; Chen, Jie; Zhou, Yu; Fu, Zhiqiang; Zhou, Quanbo; Wang, YingXue; Gao, Wenchao; Zheng, ShangYou; Zhao, Xiaohui; Chen, Tao; Chen, Rufu

2015-04-30

Pancreatic ductal adenocarcinoma (PDAC) is still a lethal malignancy. Long noncoding RNAs (lncRNAs) have been shown to play a critical role in cancer development and progression. Here we identified overexpression of the lncRNA AFAP1-AS1 in PDAC patients and evaluated its prognostic and functional relevance. The global lncRNA expression profile in PDAC was measured by lncRNA microarray. Expression of AFAP1-AS1 was evaluated by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) in 90 PDAC tissue samples and adjacent normal tissues. The impact of AFAP1-AS1 expression on cell proliferation, migration, and invasion were evaluated in vitro using knockdown and ectopic expression strategies. Microarray analysis revealed that up-regulation of AFAP1-AS1 expression in PDAC tissues compared with normal adjacent tissues, which was confirmed by RT-qPCR in 69/90 cases (76.7%). Its overexpression was associated with lymph node metastasis, perineural invasion, and poor survival. When using AFAP1-AS1 as a prognostic marker, the areas under ROC curves were 0.8669 and 0.9370 for predicting tumor progression within 6 months and 1 year, respectively. In vitro functional experiments involving knockdown of AFAP1-AS1 resulted in attenuated PDAC cell proliferation, migration, and invasion. Ectopic expression of AFAP1-AS1 promoted cell proliferation, migration, and invasion. AFAP1-AS1 is a potential novel prognostic marker to predict the clinical outcome of PDAC patients after surgery and may be a rational target for therapy.

A practical tissue sampling method using ordinary paper for molecular detection of infectious bursal disease virus RNA by RT-PCR.

PubMed

Maw, Min Thein; Yamaguchi, Tsuyoshi; Kasanga, Christopher J; Terasaki, Kaori; Fukushi, Hideto

2006-12-01

A practical sampling method for bursal tissue using ordinary paper for molecular diagnosis of infectious bursal disease (IBD) was established. IBD virus-infected bursa was directly smeared on chromatography paper, filter paper, or stationery copy paper and was then fixed with absolute ethanol, Tris-HCl-saturated phenol, or phenol:chloroform:isoamyl alcohol (25:24:1). Flinders Technology Associates (FTA) card, which is designed for the collection of biological samples for molecular detection, was also used. After storage at 37 C for up to 30 days, total RNA directly extracted from the tissue fixed on the papers and FTA card were subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) for detection of IBD virus (IBDV) RNA. In addition, the ability of each chemical used in the fixation and the FTA card to inactivate IBDV was evaluated. Regardless of the paper quality, storage period, and fixation method, IBDV RNA was consistently detected in all of the samples. IBDV in the bursal tissue was inactivated with phenol but not with ethanol or the unknown chemicals in FTA card. These results show that ordinary papers sustain the viral RNA, as does FTA card, but phenol fixation is superior to FTA card in inactivating IBDV. The new sampling method using ordinary paper with phenol fixation is safe, inexpensive, simple, and easy, and is thus suitable for conducting a global survey of IBD even where laboratory resources are limited. This practical method should contribute to the control of IBD worldwide.

Comparison of endogenous feline leukemia virus RNA content in feline vaccine and nonvaccine site-associated sarcomas.

PubMed

Kidney, B A; Ellis, J A; Haines, D M; Jackson, M L

2001-12-01

To determine whether feline vaccine site-associated sarcomas (VSS) contain a higher amount of endogenous FeLV (enFeLV) RNA, compared with feline nonvaccine site-associated sarcomas (non-VSS). Formalin-fixed paraffin-embedded (FFPE) tissues from 50 VSS and 50 cutaneous non-VSS. RNA was extracted from FFPE sections of each tumor, and regions of the long terminal repeat (LTR) and envelope (env) gene of enFeLV were amplified by use of reverse transcriptase-polymerase chain reaction (RT-PCR). The density of each RT-PCR product band for enFeLV was compared with that of a constitutively expressed gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). An integrated density value (IDV) was determined by use of densitometry, and the IDV ratio for enFeLV to GAPDH was calculated for each enFeLV primer set. The median (interquartile range) of the IDV ratio for the enFeLV LTR primer set was 0.52 (0.26 to 1.17) for the VSS group and 0.84 (0.21 to 1.53) for the non-VSS group. The median (interquartile range) of the IDV ratio for the enFeLV env primer set was 0.60 (0.37 to 0.91) for the VSS group and 0.59 (0.36 to 1.09) for the non-VSS group. Because the amount of enFeLV RNA within the LTR and env gene was not significantly different between the VSS and non-VSS groups, enFeLV replication or expression is unlikely to be involved in VSS development.

Somatostatinergic modulation of firing pattern and calcium-activated potassium currents in medium spiny neostriatal neurons.

PubMed

Galarraga, E; Vilchis, C; Tkatch, T; Salgado, H; Tecuapetla, F; Perez-Rosello, T; Perez-Garci, E; Hernandez-Echeagaray, E; Surmeier, D J; Bargas, J

2007-05-11

Somatostatin is synthesized and released by aspiny GABAergic interneurons of the neostriatum, some of them identified as low threshold spike generating neurons (LTS-interneurons). These neurons make synaptic contacts with spiny neostriatal projection neurons. However, very few somatostatin actions on projection neurons have been described. The present work reports that somatostatin modulates the Ca(2+) activated K(+) currents (K(Ca) currents) expressed by projection cells. These actions contribute in designing the firing pattern of the spiny projection neuron; which is the output of the neostriatum. Small conductance (SK) and large conductance (BK) K(Ca) currents represent between 30% and 50% of the sustained outward current in spiny cells. Somatostatin reduces SK-type K(+) currents and at the same time enhances BK-type K(+) currents. This dual effect enhances the fast component of the after hyperpolarizing potential while reducing the slow component. Somatostatin then modifies the firing pattern of spiny neurons which changed from a tonic regular pattern to an interrupted "stuttering"-like pattern. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) tissue expression analysis of dorsal striatal somatostatinergic receptors (SSTR) mRNA revealed that all five SSTR mRNAs are present. However, single cell RT-PCR profiling suggests that the most probable receptor in charge of this modulation is the SSTR2 receptor. Interestingly, aspiny interneurons may exhibit a "stuttering"-like firing pattern. Therefore, somatostatin actions appear to be the entrainment of projection neurons to the rhythms generated by some interneurons. Somatostatin is then capable of modifying the processing and output of the neostriatum.

Identification of the heme acquisition system in Vibrio vulnificus M2799.

PubMed

Kawano, Hiroaki; Miyamoto, Katsushiro; Yasunobe, Megumi; Murata, Masahiro; Yamahata, Eri; Yamaguchi, Ryo; Miyaki, Yuta; Tsuchiya, Takahiro; Tanabe, Tomotaka; Funahashi, Tatsuya; Tsujibo, Hiroshi

2018-04-01

Vibrio vulnificus, the causative agent of serious, often fatal, infections in humans, requires iron for its pathogenesis. As such, it obtains iron via both vulnibactin and heme-mediated iron-uptake systems. In this study, we identified the heme acquisition system in V. vulnificus M2799. The nucleotide sequences of the genes encoding heme receptors HupA and HvtA and the ATP-binding cassette (ABC) transport system proteins HupB, HupC, and HupD were determined, and then used in the construction of deletion mutants developed from a Δics strain, which could not synthesize vulnibactin. Growth experiments using these mutants indicated that HupA and HvtA are major and minor heme receptors, respectively. The expressions of two proteins were analyzed by the quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Furthermore, complementation analyses confirmed that the HupBCD proteins are the only ABC transport system shared by both the HupA and HvtA receptors. This is the first genetic evidence that the HupBCD proteins are essential for heme acquisition by V. vulnificus. Further investigation showed that hupA, hvtA, and hupBCD are regulated by Fur. The qRT-PCR analysis of the heme receptor genes revealed that HupR, a LysR-family positive transcriptional activator, upregulates the expression of hupA, but not hvtA. In addition, ptrB was co-transcribed with hvtA, and PtrB had no influence on growth in low-iron CM9 medium supplemented with hemin, hemoglobin, or cytochrome C. Copyright © 2018 Elsevier Ltd. All rights reserved.

Pathologic findings in red-tailed hawks (Buteo jamaicensis) and Cooper's hawks (Accipiter cooper) naturally infected with West Nile virus.

PubMed

Wünschmann, Arno; Shivers, Jan; Bender, Jeff; Carroll, Larry; Fuller, Susan; Saggese, Miguel; van Wettere, Arnaud; Redig, Pat

2004-09-01

Carcasses of 13 red-tailed hawks (RTHAs) and 11 Cooper's hawks (COHAs) were tested for West Nile virus (WNV) using WNV-specific reverse transcriptase-polymerase chain reaction (RT-PCR) on fresh brain tissue and WNV-specific immunohistochemistry (IHC) on various organs. Ten COHAs (91%) and 11 RTHAs (85%) were positive for WNV RNA by RT-PCR. All 11 COHAs (100%) and 10 RTHAs (77%) were positive for WNV antigen by IHC. A triad of inflammatory lesions, including chronic lymphoplasmacytic and histiocytic encephalitis, endophthalmitis, and myocarditis, was common in both species. In COHAs, the heart (54%), cerebrum (50%), and eye (45%) were the organs that most commonly contained WNV antigen. The amount of WNV antigen was usually small. In RTHAs, the kidney (38%), cerebrum (38%), cerebellum (38%), and eye (36%) were the organs most commonly containing WNV antigen. Unlike COHAs, larger amounts of WNV antigen were present in the cerebrum of RTHAs. WNV antigen was detected in similar cell populations in both species, including neurons of brain, spinal cord, and retina, pigmented epithelial cells of the retina, epithelial cells of renal medullary tubules, cardiomyocytes, endothelial cells and smooth muscle cells of arteries, dendritic cells of splenic lymph follicles, exocrine pancreatic cells, adrenal cells, and keratinocytes of the skin. The study presents strong evidence that WNV can cause a chronic fatal disease in RTHAs and COHAs. The lesion distribution of WNV infection in both species is variable, but inflammatory lesions are common, and a triad of lesions including encephalitis, myocarditis, and endophthalmitis is indicative of WNV infection in both species.

Pannus invasion and cartilage degradation in rheumatoid arthritis: involvement of MMP-3 and interleukin-1beta.

PubMed

Ainola, M M; Mandelin, J A; Liljeström, M P; Li, T F; Hukkanen, M V J; Konttinen, Y T

2005-01-01

Synovial inflammation in rheumatoid arthritis (RA) leads to pannus tissue invasion and destruction of cartilage/bone matrix by proteinases. Our intention was to analyze some of the key matrix metalloproteinases (MMPs) in pannus tissue overlying evolving cartilage erosions in RA. Frozen tissue samples of pannus and synovium from advanced RA and synovium from osteoarthritic patients were used for immunohistochemical, western blotting and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis of MMP-1, -3, -13 and -14. Synovial fibroblast cultures, stimulated with tumour necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1beta), were analyzed with enzyme-linked immunosorbent assays (ELISA) and quantitative RT-PCR. MMP-3 was highly expressed in pannus tissue compared with significantly lower expression levels of MMP-1, -13 and -14. In fibroblast cultures IL-1beta was a potent stimulus for MMP-3, whereas TNF-alpha was more potent for MMP-1. This is the first study to demonstrate quantitatively in real time that MMP-3 mRNA expression is clearly higher in advanced RA pannus tissue compared to parallel RA or osteoarthritic synovium. MMP-3 mRNA levels were also clearly overexpressed in RA pannus compared to MMP-1, -13 and -14. Advanced RA has previously been found to overexpress IL-1beta. The high expression of MMP-3 in pannus and IL-1beta, mediated stimulation of MMP-3 suggest that MMP-3 plays a significant role in the progression of erosions through the proteoglycan-rich cartilage matrix.

The Prognostic Value of Epithelial Membrane Protein 1 (EMP-1) in Patients with Laryngeal Carcinoma

PubMed Central

Liu, Chang; Wei, Xiaojun; Li, Feng; Wang, Li; Ruan, Xinjian; Jia, Jia; Zhang, Xia

2017-01-01

Background In the present study, we aimed to investigate the prognostic value of epithelial membrane protein 1 (EMP-1) gene in patients diagnosed with laryngeal carcinoma (LC). Material/Methods Patients who were pathologically diagnosed with LC were enrolled in the present study. The expression levels of EMP-1 in tumor tissues and corresponding normal tissues collected from the LC patients were detected by semi-reverse transcriptase polymerase chain reaction (semi-RT-PCR). Chi-square analysis was used to evaluate the relationship between EMP-1 expression level and clinical characteristics. Survival analysis for the study population was analyzed by Kaplan-Meier method with log rank test. Additionally, Cox regression model was applied to evaluate the prognostic value of EMP-1 in LC patients. Results 106 LC patients, including 55 men and 51 women, were enrolled in the present study. Semi-RT-PCR demonstrated that the expression level of EMP-1 was decreased in tumor tissues, compared with adjacent normal tissues (p<0.001). Moreover, the level was significantly associated with lymph node metastasis, histological grade, and clinical stage (p<0.05 for all). In addition, low levels of EMP-1 was significantly correlated with poor survival rate (log rank test, p=0.020). Cox regression analysis indicated that EMP-1 was an independent marker for LC prognosis (HR=2.755, 95% CI=1.123–6.760, p=0.027). Conclusions The abnormal expression of EMP-1 may be associated with progression of LC and the gene may act as a prognostic marker for LC. PMID:28779068

The Prognostic Value of Epithelial Membrane Protein 1 (EMP-1) in Patients with Laryngeal Carcinoma.

PubMed

Liu, Chang; Wei, Xiaojun; Li, Feng; Wang, Li; Ruan, Xinjian; Jia, Jia; Zhang, Xia

2017-08-05

BACKGROUND In the present study, we aimed to investigate the prognostic value of epithelial membrane protein 1 (EMP-1) gene in patients diagnosed with laryngeal carcinoma (LC). MATERIAL AND METHODS Patients who were pathologically diagnosed with LC were enrolled in the present study. The expression levels of EMP-1 in tumor tissues and corresponding normal tissues collected from the LC patients were detected by semi-reverse transcriptase polymerase chain reaction (semi-RT-PCR). Chi-square analysis was used to evaluate the relationship between EMP-1 expression level and clinical characteristics. Survival analysis for the study population was analyzed by Kaplan-Meier method with log rank test. Additionally, Cox regression model was applied to evaluate the prognostic value of EMP-1 in LC patients. RESULTS 106 LC patients, including 55 men and 51 women, were enrolled in the present study. Semi-RT-PCR demonstrated that the expression level of EMP-1 was decreased in tumor tissues, compared with adjacent normal tissues (p<0.001). Moreover, the level was significantly associated with lymph node metastasis, histological grade, and clinical stage (p<0.05 for all). In addition, low levels of EMP-1 was significantly correlated with poor survival rate (log rank test, p=0.020). Cox regression analysis indicated that EMP-1 was an independent marker for LC prognosis (HR=2.755, 95% CI=1.123-6.760, p=0.027). CONCLUSIONS The abnormal expression of EMP-1 may be associated with progression of LC and the gene may act as a prognostic marker for LC.

Emergence of West Nile virus in West Bengal, India: a new report.

PubMed

Khatun, Tanuja; Chatterjee, Shyamalendu

2017-04-01

The ICMR virus unit in Kolkata functions as an Appex Referral Laboratory for the detection of dengue (DENV) and chikungunya (CHIKV) infections in the eastern part of India. In spite of efforts for confirmatory diagnosis, some samples remain undiagnosed every year. West Nile virus (WNV) infection may mimic either dengue (flavivirus) or chikungunya (alphavirus) like illness. WNV is endemic in the tropical region where its principal/potential vectors are Aedes and Culex. We explored the existence of WNV within undiagnosed samples to identify the emergence of a new public health problem. Of 1278 sera samples, 574 were negative for DENV and CHIKV either by ELISA or by reverse transcriptase (RT)-PCR. Of these 574 negative samples, 83 (14.5%) and 141 (24.56%) were positive for WNV by ELISA and RT-PCR, respectively; no samples were positive for WNV by both methods. After assembling raw sequencing data, partial envelope genome sequence of West Bengal isolates, WNV was compared through BLAST with other WNV Indian strains and 98% homology detected. Phylogenetic analysis of one West Bengal isolates (Accession No. KY421790) and 28 Indian isolates available in GenBank, indicated close clustering. The serological and molecular approaches have clearly established the emergence of WNV in West Bengal. Hence, for proper case management, detection of WNV in common febrile illness is strongly recommended. © The Author 2017. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

First round of external quality assessment of dengue diagnostics in the WHO Western Pacific Region, 2013.

PubMed

Pok, Kwoon Yong; Squires, Raynal C; Tan, Li Kiang; Takasaki, Tomohiko; Abubakar, Sazaly; Hasebe, Futoshi; Partridge, Jeffrey; Lee, Chin Kei; Lo, Janice; Aaskov, John; Ng, Lee Ching; Konings, Frank

2015-01-01

Accurate laboratory testing is a critical component of dengue surveillance and control. The objective of this programme was to assess dengue diagnostic proficiency among national-level public health laboratories in the World Health Organization (WHO) Western Pacific Region. Nineteen national-level public health laboratories performed routine dengue diagnostic assays on a proficiency testing panel consisting of two modules: one containing commercial serum samples spiked with cultured dengue viruses for the detection of nucleic acid and non-structural protein 1 (NS1) (Module A) and one containing human serum samples for the detection of anti-dengue virus antibodies (Module B). A review of logistics arrangements was also conducted. All 16 laboratories testing Module A performed reverse transcriptase polymerase chain reaction (RT-PCR) for both RNA and serotype detection. Of these, 15 had correct results for RNA detection and all 16 correctly serotyped the viruses. All nine laboratories performing NS1 antigen detection obtained the correct results. Sixteen of the 18 laboratories using IgM assays in Module B obtained the correct results as did the 13 laboratories that performed IgG assays. Detection of ongoing/recent dengue virus infection by both molecular (RT-PCR) and serological methods (IgM) was available in 15/19 participating laboratories. This first round of external quality assessment of dengue diagnostics was successfully conducted in national-level public health laboratories in the WHO Western Pacific Region, revealing good proficiency in both molecular and serological testing. Further comprehensive diagnostic testing for dengue virus and other priority pathogens in the Region will be assessed during future rounds.

Low prevalence of avian influenza virus in shorebirds on the Pacific coast of North America

USGS Publications Warehouse

Iverson, Samuel A.; Takekawa, John Y.; Schwarzbach, Steven; Cardona, Carol J.; Warnock, Nils; Bishop, Mary Anne; Schirato, Greg A.; Paroulek, Sara; Ackerman, Joshua T.; Ip, Hon S.; Boyce, Walter M.

2008-01-01

The emergence of highly pathogenic avian influenza (HPAI) H5N1 has elevated concerns about wild birds as virus hosts; however, little is known about the ecological and epidemiological factors of transmission by shorebirds. Here we summarize results for 2,773 shorebirds that were live-trapped on the Pacific coast of the United States during 2006-2007 and tested for avian influenza virus using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and virus isolation. As was the case throughout North America, HPAI H5N1 was not detected in shorebirds during this interval. Contrary to other wild bird groups, most notably waterfowl, the prevalence of even low pathogenicity virus among shorebirds in our study areas in California, Washington, and Alaska was extremely low (0.5%). Virus was detected by RT-PCR from four different species, including, Dunlin (Calidris alpina; N = 3), Western Sandpiper (C. mauri; N = 8), Long-billed Dowitcher (Limnodromus scolopaceus; N = 1), and American Avocet (Recurvirostra americana; N = 1), with the detections in the latter three constituting the first published records for these birds. Based on studies in the eastern United States, we expected, but did not detect (H1 = 1.6, P = 0.21) elevated avian influenza prevalence among shorebirds during spring migration. Diagnostic tests, which were designed to evaluate testing and sampling methods, indicated poor functioning of traditional virus isolation methods and no improvement in detection likelihood by collecting oropharyngeal swabs in addition to cloacal swab samples for low pathogenicity viruses (Z1 = 0.7, P = 0.48).

Elimination of PPV and PNRSV through thermotherapy and meristem-tip culture in nectarine.

PubMed

Manganaris, G A; Economou, A S; Boubourakas, I N; Katis, N I

2003-10-01

The plum pox virus (PPV) and prunus necrotic ringspot virus (PNRSV) cause serious disease problems in stone-fruit trees. In this work, the possibility of obtaining plant material free from these viruses through thermotherapy and meristem-tip culture from infected nectarine shoots (Prunus persica var. nectarina Max, cv. 'Arm King') was studied. In addition, the detection of these viruses in in vitro cultures and young acclimatized plantlets with double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) and multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) was studied. Meristem-tip explants (0.8-1.3 mm) derived from sprouted buds of winter wood and spring shoots from field grown plants had a 2-5% regeneration response. However, application of thermotherapy to potted nectarine trees (3 weeks at a maximum temperature of 35 degrees C) facilitated excision of longer meristem tips (1.3-2.0 mm) that resulted in a significantly higher regeneration response (38%) in woody plant medium (WPM) without plant growth regulators. Such explants formed multiple shoots with the addition of 8 microM benzylaminopurine and 0.8 microM indoleacetic acid. When they were tested for the presence of PPV and PNRSV, 86% and 81% were found to be virus-free as detected by DAS-ELISA and multiplex RT-PCR, respectively. Individual shoots excised from virus-free cultures readily rooted in vitro (half-strength WPM plus 2 microM indolebutyric acid) and grew to plantlets. The combination of an efficient protocol for virus elimination and the establishment of highly sensitive diagnostics resulted in the production of nectarine plants free from PPV and PNRSV.

Detection of the Single Nucleotide Polymorphism at Position rs2735940 in the Human Telomerase Reverse Transcriptase Gene by the Introduction of a New Restriction Enzyme Site for the PCR-RFLP Assay.

PubMed

Wang, Sihua; Ding, Mingcui; Duan, Xiaoran; Wang, Tuanwei; Feng, Xiaolei; Wang, Pengpeng; Yao, Wu; Wu, Yongjun; Yan, Zhen; Feng, Feifei; Yu, Songcheng; Wang, Wei

2017-09-01

It has been shown that the single nucleotide polymorphism (SNP) of the rs2735940 site in the human telomerase reverse transcriptase ( hTERT ) gene is associated with increased cancer risk. The traditional method to detect SNP genotypes is polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). However, there is a limitation to utilizing PCR-RFLP due to a lack of proper restriction enzyme sites at many polymorphic loci. This study used an improved PCR-RFLP method with a mismatched base for detection of the SNP rs2735940. A new restriction enzyme cutting site was created by created restriction site PCR (CRS-PCR), and in addition, the restriction enzyme Msp I for CRS-PCR was cheaper than other enzymes. We used this novel assay to determine the allele frequencies in 552 healthy Chinese Han individuals, and found the allele frequencies to be 63% for allele C and 37% for allele T In summary, the modified PCR-RFLP can be used to detect the SNP of rs2735940 with low cost and high efficiency. © 2017 by the Association of Clinical Scientists, Inc.

Coal-burning endemic fluorosis is associated with reduced activity in antioxidative enzymes and Cu/Zn-SOD gene expression.

PubMed

Wang, Qi; Cui, Kang-ping; Xu, Yuan-yuan; Gao, Yan-ling; Zhao, Jing; Li, Da-sheng; Li, Xiao-lei; Huang, Hou-jin

2014-02-01

To study the effect of fluorine on the oxidative stress in coal-burning fluorosis, we investigated the environmental characteristics of coal-burning endemic fluorosis combined with fluorine content surveillance in air, water, food, briquette, and clay binder samples from Bijie region, Guizhou Province, southwest of China. The activities of antioxidant enzymes including copper/zinc superoxide dismutase (Cu/Zn-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and level of lipid peroxidation such as malondialdehyde (MDA) were measured in serum samples obtained from subjects residing in the Bijie region. Expression of the Cu/Zn-SOD gene was assessed by quantitative reverse transcriptase PCR (qRT-PCR). Our results showed that people suffering from endemic fluorosis (the high and low exposure groups) had much higher MDA level. Their antioxidant enzyme activities and Cu/Zn-SOD gene expression levels were lower when compared to healthy people (the control group). Fluorosis can decrease the activities of antioxidant enzymes, which was associated with exposure level of fluorine. Down-regulation of Cu/Zn-SOD expression may play an important role in the aggravation of oxidative stress in endemic fluorosis.