Microarray analysis and quantitative real-time RT-PCR are the major high-throughput techniques that are used to study transcript profiles. One of the major limitations in these technologies is the isolation maximum yield of highly-pure RNA from plant tissues rich in complex polysaccharides, polyphen...
Ghorashi, Seyed A; O'Rourke, Denise; Ignjatovic, Jagoda; Noormohammadi, Amir H
Differentiation of infectious bursal disease virus (IBDV) strains is crucial for effective vaccination programs and epidemiological investigations. In this study, a combination of real-time RT-PCR and high resolution melt (HRM) curve analysis was developed for simultaneous detection and differentiation of IBDV strains/isolates. The hypervariable region of VP2 gene was amplified from several IBDV strains and subjected to HRM curve analysis. The method could readily differentiate between classical vaccines/isolates and variants. Analysis of the nucleotide sequence of the amplicons from each strain revealed that each melt curve profile was related to a unique DNA sequence. The real-time RT-PCR HRM curve analysis was also able to differentiate IBDV strains/isolates directly in bursal tissues from field submissions and from vaccinated commercial flocks. The differences between melting peaks generated from IBDV strains were significantly different (P<0.0001) demonstrating the high discriminatory power of this technique. The results presented in this study indicated that real-time RT-PCR followed by HRM curve analysis provides a rapid and robust technique for genotyping IBDV isolates/strains and can contribute to effective control of IBDV outbreaks.
Yuwen, Yan-Qing; Dong, Zi-Mei; Wang, Qing-Hua; Sun, Xiao-Juan; Shi, Chang-Ying; Chen, Guang-Wen
It is important that endogenous reference genes for real-time RT-PCR be empirically evaluated for stability in different cell types, developmental stages, and/or sample treatment. To select the most stable endogenous reference genes during planarian regeneration, three housekeeping genes, 18S rRNA, ACTB and DjEF2, were identified and established expression levels by real-time RT-PCR. The data were analyzed by GeNorm and NormFinder software. Expression levels of the Djsix-1 gene were studied in parallel with ACTB and DjEF2 both or each and 18S rRNA as reference during regeneration. The results showed that ACTB was the most stable expressed reference gene in the planarian regeneration.
Wacker, Michael J; Godard, Michael P
Real-time reverse transcription polymerase chain reaction (RT-PCR) is a commonly used technique to analyze gene expression. There has been little research conducted to test if SuperScript III quantitative one-step (reverse transcription carried out in the same tube as PCR) and two-step (reverse transcription carried out in a separate reaction) RT-PCR systems provide similar real-time results. In this study, real-time reactions were set up using the housekeeping genes glyceraldehyde phosphate dehydrogenase (GAPDH), beta2-microglobulin (B2M), and RNA polymerase 2 subunit A (PolR2A). Reaction efficiencies were determined by generating standard curves using total RNA isolated from human skeletal muscle and brain. Reaction efficiencies ranged from 97.7+/-0.9% to 99.4+/-1.8% for one-step and 98.0+/-0.2% to 102.6+/-1.3% for two-step RT-PCR (R2 values for all reactions>or=0.995). The sensitivities of one-step and two-step methods, as measured by cycle threshold values, were similar for GAPDH and B2M. However, for the lesser expressed PolR2A mRNA there was a 5 cycle lower threshold for one-step. In summary, both SuperScript III one-step and two-step methods yield reaction efficiencies close to 100% and produce similar, accurate, linear standard curves. However, using the one-step method with gene-specific priming may be more sensitive for quantification of certain genes such as PolR2A.
In this study, real-time RT-PCR assays were combined with high resolution melting (HRM) analysis for the simultaneous detection of Cherry necrotic rusty mottle virus (CNRMV) and Cherry green ring mottle virus (CGRMV) infection in sweet cherry trees. Detection of CNRMV and CGRMV was performed using a...
Martínez, E; Riera, P; Sitjà, M; Fang, Y; Oliveira, S; Maldonado, J
The feasibility of using a SYBR Green-based real-time RT-PCR assay (SYBR Green ReTi RT-PCR) followed by melting curve analysis (MCA) for detecting and genotyping porcine reproductive and respiratory syndrome virus (PRRSV) was assessed. The SYBR Green ReTi RT-PCR and a previously reported two-step, non-nested RT-PCR assays were simultaneously tested on selected European (EU) and North American (US) PRRSV strains and isolates collected from diverse clinical, temporal, and geographical origins. The validation experiments showed that the optimised SYBR Green ReTi RT-PCR can sensitively and specifically detect PRRSV, consistently detecting as little as 0.03TCID(50)/sample of each virus genotype, with no type-bias and no amplification signal for other swine pathogens. After MCA, two well-differentiated melting temperature (T(m)) profiles for each virus genotype were obtained, as sequencing confirmed it. High repeatability was obtained for the T(m) values, with intra-run coefficients of variation (CoVs) of 0.25 and 0.32 and inter-run CoVs of 0.42 and 0.52 for EU and US genotypes, respectively. The sensitivity of the SYBR Green ReTi RT-PCR (100%) was higher than that of the RT-PCR (95.7%) when testing field isolates. This greater sensitivity of the SYBR Green ReTi RT-PCR was further confirmed by the detection of a higher proportion of PRRSV-positive diagnostic specimens (29.7%) than by the RT-PCR (28.5%). The SYBR Green ReTi RT-PCR test detected infection as early as 2 dpi in the sera of experimentally infected pigs regardless of virus genotype, and discriminated negative (non-inoculated), EU- and US-infected pigs. In conclusion, the reported SYBR Green ReTi RT-PCR assay coupled with MCA can detect and type PRRSV and may be useful as an alternative diagnostic assay in diverse PRRSV epidemiological circumstances.
Yin, Lihua; Ke, Xiwang; Han, Dong
Adzuki bean (Vigna angularis) is one of the most important legume crops in Asian countries like China, Japan and Korea due to its nutritious protein and starch contents. In spite of its economic importance, gene expression analysis system for gene function verification of adzuki bean is still absent. Therefore, reference genes for gene expression analysis based on the quantitative real time PCR (qRT-PCR) were screened in current study. A total of nine general housekeeping genes, including ACT, Fbox, ZMPP, GAPDH, EF, PP2A, UBC, UBN and PTB were evaluated for their expression stability by qRT-PCR in four adzuki bean cultivars, three different tissues, four abiotic stress and one biotic stress. The best group of candidates as reference genes were as follows: PTB and ACT for different cultivars; EF and UBN for different tissues; ACT and ZMPP for biotic stress and waterlogging stress; Fbox and UBC for salinity-alkalinity stress; Fbox and PTB for drought stress. Our results will provide a more accurate and reliable normalization of qRT-PCR data in adzuki bean. PMID:27992593
Choudhary, Manohar Lal; Anand, Siddharth P; Sonawane, Nupoor S; Chadha, Mandeep S
Human metapneumovirus (HMPV) is an important respiratory virus implicated in respiratory infections. The purpose of this study was to develop a one-step real-time RT-PCR assay that can detect all four lineages of HMPV and to identify the HMPV lineages circulating in Pune, India. Conserved regions of the nucleoprotein gene were used to design real-time primers and a probe. A total of 224 clinical samples that were positive for different respiratory viruses (including 51 samples that were positive for HMPV) were tested using the real time RT-PCR assay, and the specificity of the assay was observed to be 100 %. Using in vitro-synthesized RNA, the sensitivity of the assay was ascertained to be 100 copies of the target gene per reaction. Phylogenetic analysis of the nucleoprotein (N) and attachment glycoprotein (G) genes confirmed that this assay detected all lineages of HMPV. A2, B1 and B2 strains were observed during the study period. Our assay is highly sensitive and specific for all known lineages of HMPV, making it a valuable tool for rapid detection of the virus. A2 and B2 were the predominant subtypes circulating in Pune, Western India.
Svingen, T; Spiller, C M; Kashimada, K; Harley, V R; Koopman, P
In biological research, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assays are commonly employed to study mRNA abundance in cells and tissues. This type of assay usually relies on assessing transcript abundance relative to constitutively expressed endogenous reference genes. Therefore, it is important that the reference genes themselves are stably expressed in the cells or tissues analyzed, independent of factors such as age, sex, disease or experimental manipulations. Since no gene is expressed at the same level in all cells at all times, suitable reference genes must be identified for the specific cellular system or tissue being investigated. Here, we sought to identify stably expressed endogenous reference genes during embryonic gonad development in the mouse. We measured the transcript abundance of 10 frequently employed normalizing genes, of which 4 were stably expressed in fetal gonads from 11.5 to 14.5 dpc irrespective of sex. Based on our analysis, we suggest that Rn18s, Rps29, Tbp and Sdha are suitable reference genes for qRT-PCR expression studies during early gonad differentiation in the mouse.
Yokoi, Hajime; Tanaka, Toshimitsu; Mizumura, Ayano; Kitahashi, Tomoko
The TaqMan-based quantitative real-time RT-PCR assay we developed uses specific probes to identify respiratory syncytial virus (RSV) and to distinguish RSV subgroups A (RSV-A) and B (RSV-B). We selected conserved regions of the F gene as assay targets and designed new primers and TaqMan MGB probes to detect RSV-A and B. RSV-A and B control plasmids confirmed real-time reverse transcription polymerase chain reaction (RT-PCR) reactivity whose efficiency was 2.5 x 10(1) to 2.5 x 10(7) copies/tube. The assay detection limit was 10 to 10(2) times higher than that of the conventional RT-PCR assay and was equal to the nested PCR assay. No cross-reactions occurred against other respiratory viruses, including influenza virus, metapneumovirus, measles virus, coxsackievirus, enterovirus, echovirus, mumps virus, parainfluenza virus, and rhinovirus. Of 154 clinical specimens derived from subjects with acute respiratory infection and tested by using both real-time RT-PCR and nested PCR, 40 were RSV-positive in both assays. Of these, 25 were identified as RSV-A and 15 as RSV-B by both assays. There was 100% concordance in RSV subgroup identification between real-time RT-PCR and nested PCR assays. These results indicate that our real-time RT-PCR assay can be used for rapid detection, quantitative analysis and subgrouping of RSV-A and RSV-B.
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...
Deubelbeiss, A.; Zahno, M.-L.; Zanoni, M.; Bruegger, D.; Zanoni, R.
The causative agents of rabies are single-stranded, negative-sense RNA viruses in the genus Lyssavirus of Rhabdoviridae, consisting of twelve classified and three as yet unclassified species including classical rabies virus (RABV). Highly neurotropic RABV causes rapidly progressive encephalomyelitis with nearly invariable fatal outcome. Rapid and reliable diagnosis of rabies is highly relevant for public and veterinary health. Due to growing variety of the genus Lyssavirus observed, the development of suitable molecular assays for diagnosis and differentiation is challenging. This work focused on the establishment of a suitable real-time RT-PCR technique for rabies diagnosis as a complement to fluorescent antibody test and rabies tissue culture infection test as gold standard for diagnosis and confirmation. The real-time RT-PCR was adapted with the goal to detect the whole spectrum of lyssavirus species, for nine of which synthesized DNA fragments were used. For the detection of species, seven probes were developed. Serial dilutions of the rabies virus strain CVS-11 showed a 100-fold higher sensitivity of real-time PCR compared to heminested RT-PCR. Using a panel of thirty-one lyssaviruses representing four species, the suitability of the protocol could be shown. Phylogenetic analysis of the sequences obtained by heminested PCR allowed correct classification of all viruses used. PMID:26464934
Balasuriya, Udeni B R
Equine influenza (EI) is a highly contagious disease of horses caused by the equine influenza virus (EIV) H3N8 subtype. EI is the most important respiratory virus infection of horses and can disrupt major equestrian events and cause significant economic losses to the equine industry worldwide. Influenza H3N8 virus spreads rapidly in susceptible horses and can result in very high morbidity within 24-48 h after exposure to the virus. Therefore, rapid and accurate diagnosis of EI is critical for implementation of prevention and control measures to avoid the spread of EIV and to reduce the economic impact of the disease. The probe-based real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assays targeting various EIV genes are reported to be highly sensitive and specific compared to the Directigen Flu A(®) test and virus isolation in embryonated hens' eggs. Recently, a TaqMan(®) probe-based insulated isothermal RT-PCR (iiRT-PCR) assay for the detection of EIV H3N8 subtype has been described. These molecular based diagnostic assays provide a fast and reliable means of EIV detection and disease surveillance.
1 Real - time PCR (RT-PCR) Assays for Burkholderia mallei and B. pseudomallei Vipin K. Rastogi1, Tu-chen Cheng1, Lisa Collins1 and Jennifer Bagley2 1...A 3. DATES COVERED - 4. TITLE AND SUBTITLE Real - time PCR (RT-PCR) Assays for Burkholderia mallei and B.pseudomallei 5a. CONTRACT NUMBER 5b...risk. There is currently no real - time PCR assay for detection of both of these pathogens. Primers and probes corresponding to specific genomic regions
Xu, Ming-Yue; Liu, Si-Qing; Deng, Cheng-Lin; Zhang, Qiu-Yan; Zhang, Bo
The ongoing Zika virus (ZIKV) outbreak has rapidly spread to new areas of Americas, which were the first transmissions outside its traditional endemic areas in Africa and Asia. Due to the link with newborn defects and neurological disorder, numerous infected cases throughout the world and various mosquito vectors, the virus has been considered to be an international public health emergency. In the present study, we developed a SYBR Green based one-step real-time RT-PCR assay for rapid detection of ZIKV. Our results revealed that the real-time assay is highly specific and sensitive in detection of ZIKV in cell samples. Importantly, the replication of ZIKV at different time points in infected cells could be rapidly monitored by the real-time RT-PCR assay. Specifically, the real-time RT-PCR showed acceptable performance in measurement of infectious ZIKV RNA. This assay could detect ZIKV at a titer as low as 1PFU/mL. The real-time RT-PCR assay could be a useful tool for further virology surveillance and diagnosis of ZIKV.
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
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.
For universal detection of Citrus tristeza virus (CTV) strains by real time RT-PCR, a protocol was developed based on a set of primers and a Cy5-labeled TaqMan probe. This test included primers and a TET-labeled TaqMan probe selected on the mitochondrial nad5 gene for the simultaneous detection of ...
Luan, Huaibiao; Wang, Yixin; Li, Yang; Cui, Zhizhong; Chang, Shuang; Zhao, Peng
Based on the published Avian reticuloendotheliosis virus (REV) whole genome sequence, primers and TaqMan probes were designed and synthesized, and the TaqMan probe fluorescence real-time quantitative RT-PCR (qRT-PCR) method for detecting the REV pol gene was established by optimizing the reaction conditions. Sensitivity analysis showed that the qRT-PCR method had a sensitivity that was 1,000-fold higher than conventional PCR. Additionally, no amplification signals were obtained when we attempted to detect DNA or cDNA of ALV-A/B/J, MDV, CIAV, IBDV, ARV, NDV, AIV, or other viruses, suggesting a high specificity for our method. Various titers of REV were artificially "spiked" into the FPV and MDV vaccines to simulate REV contamination in attenuated vaccines to validate this qRT-PCR method. Our findings indicated that this qRT-PCR method could detect REV contamination at a dose of 1 TCID50/1,000 feathers, which was 10,000-fold more sensitive than the regular RT-PCR detection (10(4) TCID50/1000 feathers).
Choudhary, Manohar L; Anand, Siddharth P; Tikhe, Shamal A; Walimbe, Atul M; Potdar, Varsha A; Chadha, Mandeep S; Mishra, Akhilesh C
Detection of respiratory viruses using polymerase chain reaction (PCR) is sensitive, specific and cost effective, having huge potential for patient management. In this study, the performance of an in-house developed conventional multiplex RT-PCR (mRT-PCR), real time RT-PCR (rtRT-PCR) and Luminex xTAG(®) RVP fast assay (Luminex Diagnostics, Toronto, Canada) for the detection of respiratory viruses was compared. A total 310 respiratory clinical specimens predominantly from pediatric patients, referred for diagnosis of influenza A/H1N1pdm09 from August 2009 to March 2011 were tested to determine performance characteristic of the three methods. A total 193 (62.2%) samples were detected positive for one or more viruses by mRT-PCR, 175 (56.4%) samples by real time monoplex RT-PCR, and 138 (44.5%) samples by xTAG(®) RVP fast assay. The overall sensitivity of mRT-PCR was 96.9% (95% CI: 93.5, 98.8), rtRT-PCR 87.9% (95% CI: 82.5, 92.1) and xTAG(®) RVP fast was 68.3% (95% CI: 61.4, 74.6). Rhinovirus was detected most commonly followed by respiratory syncytial virus group B and influenza A/H1N1pdm09. The monoplex real time RT-PCR and in-house developed mRT-PCR are more sensitive, specific and cost effective than the xTAG(®) RVP fast assay.
Capote, Nieves; Bertolini, Edson; Olmos, Antonio; Vidal, Eduardo; Martínez, Maria Carmen; Cambra, Mariano
Direct systems to process plant materials allowed high-throughput testing of Plum pox virus (PPV) by real-time reverse transcription (RT)-PCR without nucleic acids purification. Crude plant extracts were diluted in buffer or spotted on membranes to be used as templates. Alternatively, immobilized PPV targets were amplified from fresh sections of plant tissues printed or squashed onto the same supports, without extract preparation. Spot real-time RT-PCR was validated as a PPV diagnostic method in samples collected during the dormancy period and showed high sensitivity (93.6%), specificity (98.0%), and post-test probability (97.9%) towards sharka disease. In an analysis of 2919 Prunus samples by spot real-time RT-PCR and DASI-ELISA 90.8% of the results coincided, demonstrating high agreement (k = 0.77 +/- 0.01) between the two techniques. These results validate the use of immobilized PPV targets and spot real-time RT-PCR as screening method for largescale analyses.
Soler, Marçal; Serra, Olga; Molinas, Marisa; García-Berthou, Emili; Caritat, Antònia; Figueras, Mercè
The molecular processes underlying cork biosynthesis and differentiation are mostly unknown. Recently, a list of candidate genes for cork biosynthesis and regulation was made available opening new possibilities for molecular studies in cork oak (Quercus suber L.). Based on this list, we analyzed the seasonal variation in mRNA abundance in cork tissue of selected genes by real time reverse-transcriptase polymerase chain reaction (RT-PCR). Relative transcript abundance was evaluated by principal component analysis and genes were clustered in several functional subgroups. Structural genes of suberin pathways such as CYP86A1, GPAT and HCBT, and regulatory genes of the NAM and WRKY families showed highest transcript accumulation in June, a crucial month for cork development. Other cork structural genes, such as FAT and F5H, were significantly correlated with temperature and relative humidity. The stress genes HSP17.4 and ANN were strongly positively correlated to temperature, in accord with their protective role.
Mourya, Devendra T; Yadav, Pragya D; Mehla, Rajeev; Barde, Pradip V; Yergolkar, Prasanna N; Kumar, Sandeep R P; Thakare, Jyotsna P; Mishra, Akhilesh C
Kyasanur forest disease (KFD) is a zoonotic viral disease caused by infection by a Flavivirus, a member of the family Flaviviridae. KFD is a public health concern in the Karnataka State in southern India. Available conventional diagnostic tests such as virus isolation and serological tests, such as haemagglutination inhibition and complement fixation tests are time consuming. This study reports the development of a nested RT-PCR [nRT-PCR] and a TaqMan-based real-time RT-PCR and IgM antibodies capture ELISA [MAC-ELISA] for rapid and accurate diagnosis of suspected KFD cases. The nRT-PCR and the TaqMan-based real-time RT-PCR assays were developed using gene sequences of the NS-5/non-coding region. Both the assays detected KFD viral RNA in acute phase human serum samples and can provide early diagnosis of infection. Real-time RT-PCR was found to be more sensitive than nRT-PCR, which could detect 38 copies of KFDV RNA. MAC-ELISA was developed for the detection of recent infections. Although real-time RT-PCR and nRT-PCR require expensive reagents, expensive equipment and trained personnel, the developed MAC-ELISA can be used easily in the affected areas. These tests add to the existing diagnosis arsenal against haemorrhagic viruses that are prevalent in India. These assays will also help to extend our knowledge of the pathology of KFD virus and its associated clinical features, by measuring the viral titre during infection and at the time of seroconversion. Information, which is not available currently because of the lack of appropriate diagnostic methods. In addition, early laboratory diagnosis of KFDV infection will help in the application of appropriate control measures and management of KFD cases.
Sahiner, Fatih; Avcı, Ismail Yaşar; Bedir, Orhan; Koru, Ozgür; Sener, Kenan; Yapar, Mehmet; Kubar, Ayhan
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.
Lu, Rou-jian; Zhang, Ling-lin; Tan, Wen-jie; Zhou, Wei-min; Wang, Zhong; Peng, Kun; Ruan, Li
We designed specific primers and fluorescence-labeled probes to develop real-time and conventional RT-PCR assays for detection of human coronavirus NL63 or HKU1. Subsequently, experiments were undertaken to assess diagnostic criteria such as specificity, sensitivity and reproducibility. The detection limit of the real-time RT-PCR assays was 10 RNA copies per reaction mixture. No cross-reactivity was observed between RNA samples derived from designed HCoV and other HCoV or human metapneumovirus. A total of 158 nasopharyngeal swab specimens collected from adult patients with acute respiratory tract infection in Beijing were screened for the presence of human coronavirus NL63 and HKU1 by using real-time RT-PCR and conventional RT-PCR method. The fluorescence quantitative RT-PCR method detected six specimens positive for human coronavirus NL63, five specimens positive for human coronavirus HKU1; and conventional RT-PCR method detected three HCoV-NL63 positive and three HCoV-HKU1 positive, respectively. The convention RT-PCR products of positive samples were obtained and sequence analysis confirmed the reliability of the above methods. In summary, the real-time RT-PCR assay for HCoV- NL63 or HKU1 was more sensitive than conventional RT-PCR and with less time (less than 4 hours) for completion. It may be suitable for molecular epidemiological surveillance and clinical diagnosis for human coronavirus NL63 and HKU1.
Yang, Chang Geng; Wang, Xian Li; Tian, Juan; Liu, Wei; Wu, Fan; Jiang, Ming; Wen, Hua
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.
De Regge, N; Deblauwe, I; De Deken, R; Vantieghem, P; Madder, M; Geysen, D; Smeets, F; Losson, B; van den Berg, T; Cay, A B
To identify possible vectors of Schmallenberg virus (SBV), we tested pools containing heads of biting midges (Culicoides) that were caught during the summer and early autumn of 2011 at several places in Belgium by real-time RT-PCR. Pools of heads originating from following species: C. obsoletus complex, C. dewulfi and C. chiopterus were found positive, strongly indicating that these species are relevant vectors for SBV.
Camenisch, Glauco; Bandli, Risch; Hoop, Richard
Wild birds are considered to be the natural reservoir of the Newcastle disease virus (NDV; avian paramyxovirus-1) causing New-castle disease, and are often suspected to be involved in outbreaks in domesticated birds. To assess the epidemiologic status of wild birds living, or overwintering, in Switzerland, 3,049 cloacal swabs covering the period 2003-2006 were screened for NDV, using real time RT-PCR. All samples were negative. This result seems in contrast with previously performed serologic screenings of wild birds.
Mo, Qiu-Hua; Wang, Hai-Bo; Tan, Hua; Wu, Bi-Mei; Feng, Zi-Li; Wang, Qi; Lin, Ji-Can; Yang, Ze
Rotavirus is one of the major viral pathogens leading to diarrhea. Diagnosis has been conducted by either traditional cultural, serological methods or molecular biology techniques, which include RT-PCR and nucleic acid sequence-based amplification (NASBA). However, their differences regarding accuracy and sensitivity remain unknown. In this study, an in-house conventional RT-PCR assay and more importantly, an in-house real-time NASBA (RT-NASBA) were established, and compared with a commercial TaqMan RT-PCR assay. The results showed that all of these methods were able to detect and distinguish rotavirus from other diarrhea viruses with a 100% concordance rate during the course of an evaluation on 20 clinical stool samples. However, RT-NASBA was much quicker than the other two methods. More importantly, the limit of detection of RT-NASBA could reach seven copies per reaction and was one to two logs lower than that of conventional RT-PCR and TaqMan RT-PCR. These results indicate that this in-house assay was more sensitive, and thus could be used as an efficient diagnosis tool for rotavirus. To the best of our knowledge, this is the first direct comparison among three different assays for the detection of rotavirus. These findings would provide implication for the rational selection of diagnosis tool for rotavirus.
Kianianmomeni, Arash; Hallmann, Armin
Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) is a sensitive technique for analysis of gene expression under a wide diversity of biological conditions. However, the identification of suitable reference genes is a critical factor for analysis of gene expression data. To determine potential reference genes for normalization of qRT-PCR data in the green alga Volvox carteri, the transcript levels of ten candidate reference genes were measured by qRT-PCR in three experimental sample pools containing different developmental stages, cell types and stress treatments. The expression stability of the candidate reference genes was then calculated using the algorithms geNorm, NormFinder and BestKeeper. The genes for 18S ribosomal RNA (18S) and eukaryotic translation elongation factor 1α2 (eef1) turned out to have the most stable expression levels among the samples both from different developmental stages and different stress treatments. The genes for the ribosomal protein L23 (rpl23) and the TATA-box binding protein (tbpA) showed equivalent transcript levels in the comparison of different cell types, and therefore, can be used as reference genes for cell-type specific gene expression analysis. Our results indicate that more than one reference gene is required for accurate normalization of qRT-PCRs in V. carteri. The reference genes in our study show a much better performance than the housekeeping genes used as a reference in previous studies.
Muhd Radzi, Siti Fatimah; Rückert, Claudia; Sam, Sing-Sin; Teoh, Boon-Teong; Jee, Pui-Fong; Phoon, Wai-Hong; Abubakar, Sazaly; Zandi, Keivan
Langat virus (LGTV), one of the members of the tick-borne encephalitis virus (TBEV) complex, was firstly isolated from Ixodes granulatus ticks in Malaysia. However, the prevalence of LGTV in ticks in the region remains unknown. Surveillance for LGTV is therefore important and thus a tool for specific detection of LGTV is needed. In the present study, we developed a real-time quantitative reverse-transcription-polymerase chain reaction (qRT-PCR) for rapid detection of LGTV. Our findings showed that the developed qRT-PCR could detect LGTV at a titre as low as 0.1 FFU/ml. The detection limit of the qRT-PCR assay at 95% probability was 0.28 FFU/ml as determined by probit analysis (p ≤ 0.05). Besides, the designed primers and probe did not amplify ORF of the E genes for some closely related and more pathogenic viruses including TBEV, Louping ill virus, Omsk hemorrhagic fever virus (OHFV), Alkhurma virus (ALKV), Kyasanur Forest Disease virus (KFDV) and Powassan virus (POWV) which showed the acceptable specificity of the developed assay. The sensitivity of the developed method also has been confirmed by determining the LGTV in infected tick cell line as well as LGTV- spiked tick tissues. PMID:26360297
Simonet, Gert; Breugelmans, Bert; Proost, Paul; Claeys, Ilse; Van Damme, Jozef; De Loof, Arnold; Vanden Broeck, Jozef
In the last decade, a new serine protease inhibitor family has been described in arthropods. Eight members of the family were purified from locusts and share a conserved cysteine array (Cys-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-6)-Cys-Thr-Xaa3-Cys) with nine inhibitory domains of the light chain of the crayfish protease inhibitor, pacifastin (PLDs; pacifastin light chain domains). Using cDNA cloning, several pacifastin-related precursors have been identified, encoding additional PLD-related peptides in different insect species. In the present study, two isoforms of a novel pacifastin-related precursor (SGPP-4) have been identified in the desert locust, predicting the previously identified SGPI-5 (Schistocerca gregaria PLD-related inhibitor-5) peptide and two novel PLD-related peptide sequences. One novel peptide (SGPI-5A) was synthesized chemically, and its inhibitory activity was assessed in vitro. Although proteases from a locust midgut extract were very sensitive to SGPI-5A, the same peptide proved to be a relatively poor inhibitor of bovine trypsin. By an in silico datamining approach, a novel pacifastin-related precursor with seven PLD-related domains was identified in the mosquito, Aedes aegypti. As in other insect pacifastin-related precursors, the Aedes precursor showed a particular domain architecture that is not encountered in other serine protease inhibitor families. Finally, a comparative real-time RT-PCR analysis of SGPP-4 transcripts in different tissues of isolated- (solitarious) and crowded-reared (gregarious) locusts was performed. This showed that SGPP-4 mRNA levels are higher in the brain, testes and fat body of gregarious males than of solitarious males. These results have been compared with data from a similar study on SGPP-1-3 transcripts and discussed with respect to a differential regulation of serine-protease-dependent pathways as a possible mechanism underlying locust phase polymorphism.
Waggoner, Jesse J.; Sahadeo, Nikita S. D.; Brown, Arianne; Mohamed-Hadley, Alisha; Hadley, Dexter; Carrington, Leslie; Carrington, Christine V. F.; Pinsky, Benjamin A.
Dengue virus (DENV) transmission occurs throughout the Caribbean, though laboratory confirmation and epidemiologic surveillance is limited by the availability of serotype-specific molecular diagnostics. In this study, we show that a serotype-specific DENV multiplex, real-time RT-PCR detected DENV RNA in significantly more samples (82/182) than a reference hemi-nested RT-PCR (57/182; p=0.01). PMID:25533614
Rupprom, Kitwadee; Chavalitshewinkoon-Petmitr, Porntip; Diraphat, Pornphan; Kittigul, Leera
Noroviruses are the leading cause of acute gastroenteritis in humans. Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) is a promising molecular method for the detection of noroviruses. In this study, the performance of three TaqMan real-time RT-PCR assays was assessed, which were one commercially available real-time RT-PCR kit (assay A: Norovirus Real Time RT-PCR kit) and two in-house real-time RT-PCR assays (assay B: LightCycler RNA Master Hybprobe and assay C: RealTime ready RNA Virus Master). Assays A and B showed higher sensitivity than assay C for norovirus GI, while they all had the same sensitivity (10(3) DNA copies/mL) for GII DNA standard controls. Assay B had the highest efficiency for both genogroups. No cross-reactivity was observed among GI and GII noroviruses, rotavirus, hepatitis A virus, and poliovirus. The detection rates of these assays in GI and GII norovirus-positive fecal samples were not significantly different. However, the mean quantification cycle (Cq) value of assay B for GII was lower than assays A and C with statistical significance (P-value, 0.000). All three real-time RT-PCR assays could detect a variety of noroviruses including GI.2, GII.2, GII.3, GII.4, GII.6, GII.12, GII.17, and GII.21. This study suggests assay B as a suitable assay for the detection and quantification of noroviruses GI and GII due to good analytical sensitivity and higher performance to amplify norovirus on DNA standard controls and clinical samples.
Elmas, Colette R; Koenig, Judith B; Bienzle, Dorothee; Cribb, Nicola C; Cernicchiaro, Natalia; Coté, Nathalie M; Weese, J Scott
Septic synovitis is a potentially debilitating and life-threatening disorder in horses. We hypothesized that a universal bacterial real-time PCR (RT-PCR) assay would have improved sensitivity and decreased turn-around time for detection of bacteria in synovial fluid (SF) samples. Forty-eight SF samples were collected from 36 horses that presented to two referral institutions with suspected septic synovitis. Universal RT-PCR, bacterial culture and SF analysis were performed on all samples, and an interpretation on the sample being septic or not was derived by three board certified specialists from the history, clinical assessment and SF characteristics. RT-PCR results were compared to a composite standard comprised of positive culture and interpretation by all three specialists of samples as "septic". For 41 of 48 samples (85%), culture and RT-PCR results were concordant. Compared to the composite standard, 83% of samples were correctly classified by RT-PCR (turn-around time of approximately 4 hours). Relative sensitivity and specificity of RT-PCR were 87% and 72% respectively, and 56% and 86% for culture. Hence, universal RT-PCR was a rapid and highly sensitive test, which may accelerate diagnosis and improve outcome for horses with septic synovitis.
Carolan, Louise A; Butler, Jeff; Rockman, Steve; Guarnaccia, Teagan; Hurt, Aeron C; Reading, Patrick; Kelso, Anne; Barr, Ian; Laurie, Karen L
The ferret is an excellent model for many human infectious diseases including influenza, SARS-CoV, henipavirus and pneumococcal infections. The ferret is also used to study cystic fibrosis and various cancers, as well as reproductive biology and physiology. However, the range of reagents available to measure the ferret immune response is very limited. To address this deficiency, high-throughput real time RT-PCR TaqMan assays were developed to measure the expression of fifteen immune mediators associated with the innate and adaptive immune responses (IFNα, IFNβ, IFNγ, IL1α, IL1β, IL2, IL4, IL6, IL8, IL10, IL12p40, IL17, Granzyme A, MCP1, TNFα), as well as four endogenous housekeeping genes (ATF4, HPRT, GAPDH, L32). These assays have been optimized to maximize reaction efficiency, reduce the amount of sample required (down to 1ng RNA per real time RT-PCR reaction) and to select the most appropriate housekeeping genes. Using these assays, the expression of each of the tested genes could be detected in ferret lymph node cells stimulated with mitogens or infected with influenza virus in vitro. These new tools will allow a more comprehensive analysis of the ferret immune responses following infection or in other disease states.
Elizalde, Maia; Agüero, Montserrat; Buitrago, Dolores; Yuste, María; Arias, María Luisa; Muñoz, María Jesús; Lelli, Davide; Pérez-Ramírez, Elisa; Moreno-Martin, Ana María; Fernández-Pinero, Jovita
Sixteen haemagglutinin (HA) subtypes of avian influenza viruses (AIV) have been described to date. Rapid subtype identification of any AIV is of major interest because of the possible serious consequences for the poultry industry and even public health. Molecular techniques currently allow immediate accurate subtype characterisation prior to virus isolation. In this study, a set of fourteen specific real-time RT-PCR methods were developed and evaluated for AIV HA subtyping (H1-H4, H6-H8, H10-H16), H5 and H9 being excluded on the basis of the current validity of the European Union (EU) recommended specific assays. Specific primers and probes sets for each HA-subtype were designed to hybridise the largest isolates range within each single subtype, considering the Eurasian lineage as a major target. The robustness and general application of the 14 HA-subtype methods were verified by the analysis of 110 AIV isolates belonging to all 16 HA-subtypes, performed in different laboratories. The developed real-time RT-PCR assays proved to be highly specific and revealed suitable sensitivity, allowing direct HA-subtyping of clinical material. In summary, this study provides for the first time a panel of molecular tests using specific hydrolysis probes for rapid and complete AIV HA-subtype identification.
Noutsias, Michel; Rohde, Maria; Block, Andrea; Klippert, Katrin; Lettau, Olga; Blunert, Katja; Hummel, Michael; Kühl, Uwe; Lehmkuhl, Hans; Hetzer, Roland; Rauch, Ursula; Poller, Wolfgang; Pauschinger, Matthias; Schultheiss, Heinz P; Volk, Hans D; Kotsch, Katja
Background Due to the limited RNA amounts from endomyocardial biopsies (EMBs) and low expression levels of certain genes, gene expression analyses by conventional real-time RT-PCR are restrained in EMBs. We applied two preamplification techniques, the TaqMan® PreAmp Master Mix (T-PreAmp) and a multiplex preamplification following a sequence specific reverse transcription (SSRT-PreAmp). Results T-PreAmp encompassing 92 gene assays with 14 cycles resulted in a mean improvement of 7.24 ± 0.33 Ct values. The coefficients for inter- (1.89 ± 0.48%) and intra-assay variation (0.85 ± 0.45%) were low for all gene assays tested (<4%). The PreAmp uniformity values related to the reference gene CDKN1B for 91 of the investigated gene assays (except for CD56) were -0.38 ± 0.33, without significant differences between self-designed and ABI inventoried Taqman® gene assays. Only two of the tested Taqman® ABI inventoried gene assays (HPRT-ABI and CD56) did not maintain PreAmp uniformity levels between -1.5 and +1.5. In comparison, the SSRT-PreAmp tested on 8 self-designed gene assays yielded higher Ct improvement (9.76 ± 2.45), however was not as robust regarding the maintenance of PreAmp uniformity related to HPRT-CCM (-3.29 ± 2.40; p < 0.0001), and demonstrated comparable intra-assay CVs (1.47 ± 0.74), albeit higher inter-assay CVs (5.38 ± 2.06; p = 0.01). Comparing EMBs from each 10 patients with dilated cardiomyopathy (DCM) and inflammatory cardiomyopathy (DCMi), T-PreAmp real-time RT-PCR analyses revealed differential regulation regarding 27 (30%) of the investigated 90 genes related to both HPRT-CCM and CDKN1B. Ct values of HPRT and CDKN1B did not differ in equal RNA amounts from explanted DCM and donor hearts. Conclusion In comparison to the SSRT-PreAmp, T-PreAmp enables a relatively simple workflow, and results in a robust PreAmp of multiple target genes (at least 92 gene assays as tested here) by a mean Ct improvement around 7 cycles, and in a lower inter
Elia, Gabriella; Decaro, Nicola; Martella, Vito; Cirone, Francesco; Lucente, Maria Stella; Lorusso, Eleonora; Di Trani, Livia; Buonavoglia, Canio
Canine distemper virus is the etiological agent of a severe disease in dogs and many other carnivores. Clinical diagnosis of canine distemper is difficult due to the broad spectrum of signs that may be confounded with other respiratory and enteric diseases of dogs. Accordingly, a laboratory confirmation is required for suspected cases. In this study a real-time RT-PCR assay was developed for detection and quantitation of canine distemper virus. The assay exhibited high specificity as all the negative controls (no-template-controls and samples from healthy sero-negative dogs) and other canine pathogens were not misdetected. Up to 1 x 10(2) copies of RNA were detected by the TaqMan assay, thus revealing a high sensitivity. Quantitative TaqMan was validated on clinical samples, including various tissues and organs collected from dogs naturally infected by canine distemper virus. Urines, tonsil, conjunctival swabs and whole blood were found to contain high virus loads and therefore proved to be suitable targets for detection of canine distemper virus RNA.
Pico de Coaña, Yago; Barrero, Carlos; Cajiao, Isabela; Mosquera, Catalina; Patarroyo, Manuel Elkin; Patarroyo, Manuel Alfonso
Aotus spp. monkeys are considered the ideal model for studying the progress of malarial infection and the immune response it elicits. We describe the use of a recently developed technique, real-time quantitative RT-PCR, to quantify several Aotus monkey cytokine mRNAs involved in Th1/Th2 responses (IL-4, IL-10, TNF-beta and IFN-gamma). Specific primers were designed for each cytokine and standard curves were constructed using serial dilutions of pDNA containing each target sequence. Results were normalized to GAPDH housekeeping gene expression levels. Standard curves showed high correlation coefficients and were linear over a wide range of copy numbers. Quantification of Aotus samples showed little intra- and inter-experiment variation, thus, the technique has proven to be highly reproducible and sensitive allowing us to detect as little as 25 copies/microl of target DNA. This technique will allow studying Th1 and Th2 cytokine patterns elicited in response to infection for prospectively evaluating the efficacy of malarial vaccines.
Bachanek-Bankowska, Katarzyna; Maan, Sushila; Castillo-Olivares, Javier; Manning, Nicola M.; Maan, Narender Singh; Potgieter, Abraham C.; Di Nardo, Antonello; Sutton, Geoff; Batten, Carrie; Mertens, Peter P. C.
Although African horse sickness (AHS) can cause up to 95% mortality in horses, naïve animals can be protected by vaccination against the homologous AHSV serotype. Genome segment 2 (Seg-2) encodes outer capsid protein VP2, the most variable of the AHSV proteins. VP2 is also a primary target for AHSV specific neutralising antibodies, and consequently determines the identity of the nine AHSV serotypes. In contrast VP1 (the viral polymerase) and VP3 (the sub-core shell protein), encoded by Seg-1 and Seg-3 respectively, are highly conserved, representing virus species/orbivirus-serogroup-specific antigens. We report development and evaluation of real-time RT-PCR assays targeting AHSV Seg-1 or Seg-3, that can detect any AHSV type (virus species/serogroup-specific assays), as well as type-specific assays targeting Seg-2 of the nine AHSV serotypes. These assays were evaluated using isolates of different AHSV serotypes and other closely related orbiviruses, from the ‘Orbivirus Reference Collection’ (ORC) at The Pirbright Institute. The assays were shown to be AHSV virus-species-specific, or type-specific (as designed) and can be used for rapid, sensitive and reliable detection and identification (typing) of AHSV RNA in infected blood, tissue samples, homogenised Culicoides, or tissue culture supernatant. None of the assays amplified cDNAs from closely related heterologous orbiviruses, or from uninfected host animals or cell cultures. PMID:24721971
Pabbaraju, Kanti; Wong, Sallene; Wong, Anita A; Tellier, Raymond
Detection of all enteroviruses while excluding cross-detection of rhinoviruses is challenging because of sequence similarities in the commonly used conserved targets for molecular assays. In addition, simultaneous detection and differentiation of enteroviruses and parechoviruses would be beneficial because of a similar clinical picture presented by these viruses. A sensitive and specific real-time RT-PCR protocol that can address these clinical needs would be valuable to molecular diagnostic laboratories. Here we report a multiplex nucleic acid based assay using hydrolysis probes targeting the 5' non-translated region for the detection and differentiation of enteroviruses and parechoviruses without cross-detection of rhinoviruses. This assay has been shown to detect enteroviruses belonging to the different species in a variety of specimen types without detecting the different species of rhinoviruses. Laboratory validation shows the assay to be sensitive, specific, reproducible, easy to set up and uses generic cycling conditions. This assay can be implemented for diagnostic testing of patient samples in a high throughput fashion.
Liu, Qingtian; Yang, Zengqi; Hao, Huafang; Cheng, Shenli; Fan, Wentao; Du, Enqi; Xiao, Sa; Wang, Xinglong; Zhang, Shuxia
Avian encephalomyelitis virus (AEV) causes epidemic diseases in poultry worldwide. A SYBR Green real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay was developed for the rapid detection and quantitation of AEV in this study. A pair of specific primers was designed in the highly conserved VP1 gene of this virus. When comparing this assay with conventional RT-PCR, the rRT-PCR assay was 100 times more sensitive and could detect levels as low as 10 standard DNA copies of the AEV SX strain. The specificity of this technique was evaluated in five other avian pathogens. The AEV RNA was detected as early as three days post-infection in chicken embryos. All 18 clinical chicken brains collected from an AEV outbreak in Northwestern China were detected to be positive (100%) using the rRT-PCR assay. However, only 5 of the 18 samples were positive (28%) using the conventional RT-PCR. The results were confirmed by virus isolation in chicken embryos. This high sensitivity, specificity, and simplicity of the SYBR Green rRT-PCR approach can be a more effective method than the conventional one for AEV diagnosis and surveillance.
Zhou, Xinrong; Zhang, Tiansheng; Song, Deping; Huang, Tao; Peng, Qi; Chen, Yanjun; Li, Anqi; Zhang, Fanfan; Wu, Qiong; Ye, Yu; Tang, Yuxin
Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV) is a highly contagious intestinal disease, resulting in substantial economic losses to the swine industry worldwide. In this study, three assays, namely a conventional reverse transcription-polymerase chain reaction (RT-PCR), a SYBR Green I real-time RT-PCR and a TaqMan real-time RT-PCR targeting the highly conserved M gene of PEDV, were developed and evaluated. Then, the analytical specificity, sensitivity and reproducibility of these assays were determined and compared. The TaqMan real-time RT-PCR was 100-fold and 10,000-fold more sensitive than that of the SYBR Green I real-time RT-PCR and the conventional RT-PCR, respectively. The analytical sensitivity of TaqMan real-time RT-PCR was 10 copies/μl of target gene and no cross amplification with other viruses tested was observed. With the features of high specificity, sensitivity, and reproducibility, the TaqMan real-time RT-PCR established in this study could be a useful tool for clinical diagnosis, epidemiological surveys and outbreak investigations of PED.
Knowledge of how birds react to infection from avian influenza virus is critical to understanding disease pathogenesis and host response. The use of real-time (R), reverse-transcriptase (RT), PCR to measure innate immunity, including cytokine and interferon gene expression, has become a standard tec...
López-Fabuel, Irene; Wetzel, Thierry; Bertolini, Edson; Bassler, Alexandra; Vidal, Eduardo; Torres, Luis B; Yuste, Alberto; Olmos, Antonio
A real-time multiplex RT-PCR has been developed for the simultaneous detection and identification of the major RNA viruses that infect grapevines (Grapevine fanleaf virus, Arabis mosaic virus, Grapevine leafroll-associated virus 1, Grapevine leafroll-associated virus 3 and Grapevine fleck virus). Serial dilutions of infected plant extracts were tested using the new method, and the results were compared with those obtained using a commercially available ELISA and real-time singleplex RT-PCR. The two real-time RT-PCR versions detected up to the same level of dilution and were at least 10,000 times more sensitive than the ELISA. In addition, 158 grapevine plants collected in a survey of the Protected Designation of Origin in Alicante, Spain were compared using the three methods. The results of the molecular methods were very similar, with only four discordant results, and both were able to detect many more infected plants than the ELISA. The high prevalence of Grapevine fleck virus, Grapevine leafroll-associated virus 3 and Grapevine fanleaf virus suggests that the main pathways of viral introduction are infected plant material that has escaped controls and/or uncontrolled traffic of propagating plant material. Real-time multiplex RT-PCR could be used to facilitate a better control of grapevine viruses.
Kumthip, Kattareeya; Khamrin, Pattara; Saikruang, Wilaiporn; Supadej, Kanittapon; Ushijima, Hiroshi; Maneekarn, Niwat
Immunochromatographic (IC) test for norovirus detection is a rapid and simple detection method. This study evaluated the sensitivity and specificity of a recent version of R-Biopharm RIDA®QUICK Norovirus IC assay for norovirus detection in fecal specimens from children hospitalized with acute gastroenteritis. Fecal specimens were tested by IC kit in comparison with gold standard reverse transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR. The IC kit showed high sensitivity and specificity comparable with PCR-based methods. None of false positive and false negative was found and the assay did not cross-react with other gastroenteritis viruses. The IC assay could detect genogroup I.5 (GI.5) and a wide range of genotypes in the GII noroviruses including GII.3, GII.4, GII.6, GII.7, GII.14, GII.15, GII.21, and also newly emerging GII.17 norovirus. In conclusion, this norovirus IC kit could be an alternative choice for rapid screening or a quick diagnostic tool for norovirus detection in fecal specimens of acute gastroenteritis patients.
Maquart, Marianne; Temmam, Sarah; Héraud, Jean-Michel; Leparc-Goffart, Isabelle; Cêtre-Sossah, Catherine; Dellagi, Koussay; Cardinale, Eric; Pascalis, Hervé
In recent years, Madagascar and the Comoros archipelago have been affected by epidemics of Rift Valley fever (RVF), however detection of Rift Valley fever virus (RVFV) in zebu, sheep and goats during the post epidemic periods was frequently unsuccessful. Thus, a highly sensitive real-time RT-PCR assay was developed for the detection of RVFV at low viral loads. A new RVF SYBR Green RT-PCR targeting the M segment was tested on serum from different RVF seronegative ruminant species collected from May 2010 to August 2011 in Madagascar and the Comoros archipelago and compared with a RVF specific quantitative real time RT-PCR technique, which is considered as the reference technique. The specificity was tested on a wide range of arboviruses or other viruses giving RVF similar clinical signs. A total of 38 out of 2756 serum samples tested positive with the new RT-PCR, whereas the reference technique only detected 5 out of the 2756. The described RT-PCR is an efficient diagnostic tool for the investigation of enzootic circulation of the RVF virus. It allows the detection of low viral RNA loads adapted for the investigations of reservoirs or specific epidemiological situations such as inter-epizootic periods.
Sakurada, Koichi; Akutsu, Tomoko; Watanabe, Ken; Yoshino, Mineo
A new approach for the identification of body fluid stains by comparing specific mRNA expression levels has been extensively studied in recent years. Here, we examine whether nasal blood, which is regarded as one of the most difficult types of blood to identify, can be identified by comparing mRNA expression levels of target genes specific to saliva, nasal secretion, and blood. The saliva-specific statherin gene (STATH) was found to be expressed at high levels in not only saliva (dCt value: 1.32±1.39, n=5), but also nasal secretions (dCt value: 0.90±1.14, n=5), while the histatin gene (HTN3) was only expressed at high levels in saliva (dCt value: 1.08±2.35, n=5). We also confirmed that the hemoglobin-beta gene (HBB) showed high expression levels in blood (dCt value: -9.51±0.40, n=5). Four nasal blood stains were found to highly express STATH (dCt value: 5.65±3.98) and HBB (dCt value: -8.79±1.67) but not HTN3, suggesting that the stain samples contained both nasal secretions and blood and can therefore be identified as nasal blood stains. Although menstrual blood showed the same expression pattern as nasal blood, the menstrual blood-specific protein matrix metallopeptidase 7 (MMP7) was not expressed in all nasal blood stain samples. Therefore, its expression levels could be used to discriminate between nasal and menstrual blood. In conclusion, real-time RT-PCR was able to identify nasal blood, although the stability of gene expression in nasal blood stains was low over time, suggesting that this assay may not be effective for older stains. Future work should examine the usefulness of this assay under various environmental conditions.
Machado, Alex Martins; de Souza, William Marciel; de Pádua, Michelly; da Silva Rodrigues Machado, Aline Rafaela; Figueiredo, Luiz Tadeu Moraes
Hantaviruses are members of the family Bunyaviridae and are an emerging cause of disease worldwide with high lethality in the Americas. In Brazil, the diagnosis for hantaviruses is based on immunologic techniques associated with conventional RT-PCR. A novel one-step SYBR Green real-time RT-PCR was developed for the detection and quantitation of Araraquara (ARAV) and Rio Mamore hantavirus (RIOMV). The detection limit of assay was 10 copies/μL of RNA in vitro transcribed of segment S. The specificity of assay was evaluated by melting curve analysis, which showed that the Araraquara virus amplified product generated a melt peak at 80.83 ± 0.89 °C without generating primer-dimers or non-specific products. The assay was more sensitive than conventional RT-PCR and we detected two samples undetected by conventional RT-PCR. The one-step SYBR Green real-time quantitative RT-PCR is specific, sensible and reproducible, which makes it a powerful tool in both diagnostic applications and general research of ARAV and RIOMV and possibly other Brazilian hantaviruses.
Zhang, Yongjiang; Zhao, Wenjun; Li, Mingfu; Chen, Hongjun; Zhu, Shuifang; Fan, Zaifeng
Maize chlorotic mottle virus (MCMV) causes corn lethal necrosis disease, and can be transmitted through infected maize seeds. It remains a challenge to detect this virus in the seeds to prevent its introduction and infection. For this purpose, a real-time TaqMan RT-PCR procedure for efficient detection of MCMV was developed. The sensitivity of the method was 4 fg of total RNA or 25 copies of RNA transcripts, which was approximately ten-fold higher than conventional RT-PCR gel electrophoresis method. The successful detection of MCMV in maize seeds suggested the feasibility of this procedure for routine testing.
Muller, Patrick Y; Janovjak, Harald; Miserez, André R; Dobbie, Zuzana
Quantitative real-time PCR represents a highly sensitive and powerful technique for the quantitation of nucleic acids. It has a tremendous potential for the high-throughput analysis of gene expression in research and routine diagnostics. However, the major hurdle is not the practical performance of the experiments themselves but rather the efficient evaluation and the mathematical and statistical analysis of the enormous amount of data gained by this technology, as these functions are not included in the software provided by the manufacturers of the detection systems. In this work, we focus on the mathematical evaluation and analysis of the data generated by quantitative real-time PCR, the calculation of the final results, the propagation of experimental variation of the measured values to the final results, and the statistical analysis. We developed a Microsoft Excel-based software application coded in Visual Basic for Applications, called Q-Gene, which addresses these points. Q-Gene manages and expedites the planning, performance, and evaluation of quantitative real-time PCR experiments, as well as the mathematical and statistical analysis, storage, and graphical presentation of the data. The Q-Gene software application is a tool to cope with complex quantitative real-time PCR experiments at a high-throughput scale and considerably expedites and rationalizes the experimental setup, data analysis, and data management while ensuring highest reproducibility.
Waggoner, Jesse J.; Abeynayake, Janaki; Sahoo, Malaya K.; Gresh, Lionel; Tellez, Yolanda; Gonzalez, Karla; Ballesteros, Gabriela; Pierro, Anna M.; Gaibani, Paolo; Guo, Frances P.; Sambri, Vittorio; Balmaseda, Angel; Karunaratne, Kumudu; Harris, Eva; Pinsky, Benjamin A.
Background Dengue fever results from infection with one or more of four different serotypes of dengue virus (DENV). Despite the widespread nature of this infection, available molecular diagnostics have significant limitations. The aim of this study was to develop a multiplex, real-time, reverse transcriptase-PCR (rRT-PCR) for the detection, quantitation, and serotyping of dengue viruses in a single reaction. Methodology/Principal Findings An rRT-PCR assay targeting the 5′ untranslated region and capsid gene of the DENV genome was designed using molecular beacons to provide serotype specificity. Using reference DENV strains, the assay was linear from 7.0 to 1.0 log10 cDNA equivalents/µL for each serotype. The lower limit of detection using genomic RNA was 0.3, 13.8, 0.8, and 12.4 cDNA equivalents/µL for serotypes 1–4, respectively, which was 6- to 275-fold more analytically sensitive than a widely used hemi-nested RT-PCR. Using samples from Nicaragua collected within the first five days of illness, the multiplex rRT-PCR was positive in 100% (69/69) of specimens that were positive by the hemi-nested assay, with full serotype agreement. Furthermore, the multiplex rRT-PCR detected DENV RNA in 97.2% (35/36) of specimens from Sri Lanka positive for anti-DENV IgM antibodies compared to just 44.4% (16/36) by the hemi-nested RT-PCR. No amplification was observed in 80 clinical samples sent for routine quantitative hepatitis C virus testing or when genomic RNA from other flaviviruses was tested. Conclusions/Significance This single-reaction, quantitative, multiplex rRT-PCR for DENV serotyping demonstrates superior analytical and clinical performance, as well as simpler workflow compared to the hemi-nested RT-PCR reference. In particular, this multiplex rRT-PCR detects viral RNA and provides serotype information in specimens collected more than five days after fever onset and from patients who had already developed anti-DENV IgM antibodies. The implementation of this
Viarouge, Cyril; Breard, Emmanuel; Zientara, Stephan; Vitour, Damien; Sailleau, Corinne
Epizootic haemorrhagic disease virus (EHDV) may cause severe clinical episodes in some species of deer and sometimes in cattle. Laboratory diagnosis provides a basis for the design and timely implementation of disease control measures. There are seven distinct EHDV serotypes, VP2 coding segment 2 being the target for serotype specificity. This paper reports the development and validation of eight duplex real-time RT-PCR assays to simultaneously amplify the EHDV target (S9 for the pan-EHDV real-time RT-PCR assay and S2 for the serotyping assays) and endogenous control gene Beta-actin. Analytical and diagnostic sensitivity and specificity, inter- and intra-assay variation and efficiency were evaluated for each assay. All were shown to be highly specific and sensitive. PMID:26161784
Payungporn, Sunchai; Chutinimitkul, Salin; Chaisingh, Arunee; Damrongwantanapokin, Sudarat; Buranathai, Chantanee; Amonsin, Alongkorn; Theamboonlers, Apiradee; Poovorawan, Yong
H5N1 influenza A virus causes a rapidly fatal systemic disease in domestic poultry and spreads directly from poultry to mammalian species such as leopards, tigers and humans. The aim of this study was to develop a multiplex real-time RT-PCR for rapid detection of H5N1 influenza A virus. The selected primers and various labeled TaqMan MGB reporter probes corresponding to M, H5 and N1 were used in a single step multiplex real-time RT-PCR to simultaneously detect triple fluorescent signals. In order to validate the method, 75 clinical specimens infected with H5N1 isolated from both poultry and mammals, as well as various specimens of other subtypes and RNA from other viral pathogens of poultry and human were tested. The results showed that the multiplex real-time RT-PCR assays can be applied to detect virus suspensions of H5N1 influenza A virus from a wide host range and demonstrated the sensitivity of the assay amounted to approximately 10(2)-10(3)copies/mul. In conclusion, the highlights of this particular method lie in its rapidity, specificity and sensitivity thus rendering it feasible and effective for large-scale screening at times of H5N1 influenza A virus outbreaks.
Park, Sang-Ik; Park, Da-Hae; Saif, Linda J; Jeong, Young-Ju; Shin, Dong-Jun; Chun, Young-Hyun; Park, Su-Jin; Kim, Hyun-Jeong; Hosmillo, Myra; Kwon, Hyung-Jun; Kang, Mun-Il; Cho, Kyoung-Oh
Unclassified bovine enteric calicivirus (BECV) is a newly recognized bovine enteric calicivirus that differs from bovine norovirus, and which causes diarrhea in the small intestines of calves. To date, methods such as real-time reverse transcription-polymerase chain reaction (RT-PCR) have not been developed for the rapid detection, quantitation and diagnosis of BECV. Presently, a BECV-specific SYBR Green real-time RT-PCR assay was evaluated and optimized. Diarrheic specimens (n=118) collected from 2004 to 2005 were subjected to RT-PCR, nested PCR and SYBR Green real-time RT-PCR. By conventional RT-PCR and nested PCR, 9 (7.6%) and 59 (50%) samples tested positive, respectively, whereas the SYBR Green assay detected BECV in 91 (77.1%) samples. Using BECV RNA standards generated by in vitro transcription, the SYBR Green real-time RT-PCR assay sensitively detected BECV RNA to 1.1 x 10(0)copies/microl (correlation coefficiency=0.98). The detection limits of the RT-PCR and nested PCR were 1.1 x 10(5) and 1.1 x 10(2)copies/microl, respectively. These results indicate that the SYBR Green real-time RT-PCR assay is more sensitive than conventional RT-PCR and nested PCR assays, and has potential as a reliable, reproducible, specific, sensitive and rapid tool for the detection, quantitation and diagnosis of unclassified BECV.
Tan, Irene L; Dimamay, Mark Pierre S; Buerano, Corazon C; Alfon, Jhoe Anthony R; Tanig, Carol Z; Matias, Ronald R; Natividad, Filipinas F
A dengue-3-specific real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was developed using the novel Light Upon eXtension (LUX™) fluorogenic technology. A labeled forward primer and a standard reverse primer that target a conserved region within the non-structural 1 (NS1) gene of dengue 3 strains were designed. The dengue-3-specific assay did not recognize other dengue serotypes and related flaviviruses. Using a tenfold serial dilution of plasmid DNA containing the dengue 3 NS1 gene as standards, the range of dengue virus detection was determined to be from 10(3) to 10(9) copies/ml or from 80 to 8 × 10(7) copies/reaction with an average correlation coefficient of ≥ 0.99. The mean intra-assay coefficient of variation (CV) at 2.01% and the mean inter-assay CV at 2.68% suggest the repeatability of the procedure. Moreover, the fluorogenic assay was evaluated by using clinical specimens and comparing test results with historical data obtained from conventional RT-PCR, which served as the criterion standard. Using patient sera as test samples, the assay demonstrated 95.45% sensitivity and 100% specificity, respectively. These results reveal that the real-time RT-PCR assay may be utilized as a rapid, convenient, and sensitive tool for the detection of dengue 3 in clinical and laboratory specimens.
Cecilia, D; Kakade, M; Alagarasu, K; Patil, J; Salunke, A; Parashar, D; Shah, P S
Dengue and chikungunya viruses co-circulate and cause infections that start with similar symptoms but progress to radically different outcomes. Therefore, an early diagnostic test that can differentiate between the two is needed. A single-step multiplex real-time RT-PCR assay was developed that can simultaneously detect and quantitate RNA of all dengue virus (DENV) serotypes and chikungunya virus (CHIKV). The sensitivity was 100 % for DENV and 95.8 % for CHIKV, whilst the specificity was 100 % for both viruses when compared with conventional RT-PCR. The detection limit ranged from 1 to 50 plaque-forming units. The assay was successfully used for differential diagnosis of dengue and chikungunya in Pune, where the viruses co-circulate.
Background Reverse transcription PCR (RT-PCR) and real time RT-PCR (rRT-PCR) have been indispensable methods for influenza surveillance, especially for determination of avian influenza. The movement of testing beyond reference lab introduced the need of quality control, including the implementation of an evaluation system for validating personal training and sample proficiency testing. Methods We developed a panel with lysates of seasonal influenza virus (H1N1, H3N2 and B), serials of diluted H5N1 virus lysates, and in-vitro transcribed H5 hemaglutinin (HA) and an artificial gene RNAs for RT-PCR and rRT-PCR quality control assessment. The validations of stability and reproducibility were performed on the panel. Additionally, the panel was implemented to assess the detection capability of Chinese human avian influenza networks. Results The panel has relatively high stability and good reproducibility demonstrated by kappa's tests. In the implementation of panel on Chinese human avian influenza networks, the results suggested that there were a relatively low number of discrepancies for both concise and reproducibility in Chinese avian influenza virus net works. Conclusions A quality control panel of RT-PCR and real-time RT-PCR for avian influenza A (H5N1) surveillance network was developed. An availably statistical data, which are used to assess the detection capability of networks on avian influenza virus (H5N1), can be obtained relatively easily through implementation of the panel on networks. PMID:21406119
Sánchez, Gloria; Elizaquível, Patricia; Aznar, Rosa
The discrimination of infectious and inactivated viruses remains a key obstacle when using quantitative RT-PCR (RT-qPCR) to quantify enteric viruses. In this study, propidium monoazide (PMA) and RNase pretreatments were evaluated for the detection and quantification of infectious hepatitis A virus (HAV). For thermally inactivated HAV, PMA treatment was more effective than RNase treatment for differentiating infectious and inactivated viruses, with HAV titers reduced by more than 2.4 log(10) units. Results showed that combining 50 μM of PMA and RT-qPCR selectively quantify infectious HAV in media suspensions. Therefore, PMA treatment previous to RT-qPCR detection is a promising alternative to assess HAV infectivity.
Grant, Rebecca J; Banyard, Ashley C; Barrett, Tom; Saliki, Jeremiah T; Romero, Carlos H
Real-time RT-PCR (rtRT-PCR) assays for identifying and differentiating infections caused by dolphin morbillivirus (DMV) and porpoise morbillivirus (PMV) were developed by targeting the hypervariable C-terminal domain of the nucleocapsid (N) gene. Total DMV and PMV RNA extracted from infected Vero cells expressing the canine signaling lymphocyte-activation molecule (SLAM) produced positive cycle threshold (C(T)) values after the 17th and 25th cycles, respectively. The assays were then validated using infected cetacean tissue RNA. The assays were specific for either DMV or PMV and did not cross-react with canine distemper virus (CDV), phocid distemper virus (PDV), rinderpest virus (RPV), peste des petits ruminants virus (PPRV) and measles virus (MV). The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was targeted as control for RNA quality, and a consensus GAPDH probe that reacted with 11 different marine mammal species, generating positive C(T) values ranging from the 21st to the 37th cycle was used. The rtRT-PCR assays have advantages over conventional assays in that they are rapid, easier to scale up, and are less prone to cross-contamination and have improved the limit of detection and specificity.
Baud, M; Cabon, J; Salomoni, A; Toffan, A; Panzarin, V; Bigarré, L
The detection of betanodavirus genomic components is a major issue for diagnostics and control of viral nervous necrosis (VNN), a devastating disease affecting fish worldwide. Despite a number of published molecular-based tests, most of them targeting the RNA2 molecule of the virus, diagnostics is still a challenge due to the high genetic diversity within this genus. In the present study, a new one-step real-time RT-PCR (rRT-PCR), targeting RNA1 of most genotypes of betanodaviruses, was proposed and validated. The test detected successfully various isolates of betanodavirus representatives of the four species RGNNV, SJNNV, TPNNV and BFNNV, either produced on cell culture or from clinical samples. It was specific as shown by the absence of signal on samples from healthy sea bass or from field samples of six other fish species without clinical signs of VNN. The assay detected reliably 50-100 copies of plasmids containing the targeted cloned RNA1 region, as well as an infectious dose of virus of 10(2.5)-10(2.85) TCID50/ml. A set of samples was tested by two different laboratories, with similar results, demonstrating the robustness of the test. This is the first one step generic rRT-PCR method for betanodaviruses. It is simple to perform and may be used for first intention diagnostics as well as for confirmation in case of doubtful results obtained with other published tests targeting RNA2.
Cuevas-Romero, Sandra; Blomström, Anne-Lie; Alvarado, Arcelia; Hernández-Jauregui, Pablo; Rivera-Benitez, Francisco; Ramírez-Mendoza, Humberto; Berg, Mikael
In order to provide a rapid and sensitive method for detection of the Porcine rubulavirus La Piedad-Michoacan-Mexico Virus (PoRV-LPMV), we have developed a specific real-time reverse transcriptase polymerase chain reaction assay. The detection of PoRV-LPMV, represents a diagnostic challenge due to the viral RNA being present in very small amounts in tissue samples. In this study, a TaqMan(®) real-time PCR assay was designed based on the phosphoprotein gene of PoRV-LPMV, to allow specific amplification and detection of viral RNA in clinical samples. Assay conditions for the primers and probe were optimized using infected PK15 cells and ten-fold serial dilutions of a plasmid containing the whole P-gene. The sensitivity of the developed TaqMan(®) assay was approximately 10 plasmid copies per reaction, and was shown to be 1000 fold better than a conventional nested RT-PCR. The performance of this real-time RT-PCR method enables studies of various aspects of PoRV-LPMV infection. Finally, the assay detects all current known variants of the virus.
Ben Shabat, M; Meir, R; Haddas, R; Lapin, E; Shkoda, I; Raibstein, I; Perk, S; Davidson, I
Avian influenza viruses (AIVs) of the H9N2 subtype are a major economic problem in the poultry industry in Israel. Most field isolates from the last decade differ significantly from H9N2 isolates from Europe and the USA, rendering published detection methods inadequate. This study aimed to develop a real-time TaqMan((R)) RT-PCR assay, based on a conserved region in the HA9 gene. The assay was validated with viruses representing different genetic subtypes and other common avian pathogens, and was found specific to H9N2. The real-time RT-PCR assay was compared to RT-PCR, which is in routine diagnostic use. Real-time RT-PCR was found to be more sensitive than RT-PCR by 1.5-2.5 orders of magnitude when testing tracheal swabs directly and by 2-3 orders of magnitude allantoic fluid after AIV propagation in embryonated eggs. Sensitivity was quantified by using 10-fold dilutions of the H9-gene amplification fragment, and real-time RT-PCR was found to be 10(4)-fold more sensitive than RT-PCR. Clinical samples, which included tracheal and cloacal swabs, as well as allantoic fluid, were tested by both methods. By real-time RT-PCR 20% more positive H9N2 samples were detected than by RT-PCR. The real-time RT-PCR assay was found suitable for detection and epidemiological survey not only of Israeli H9N2 viruses, but also for isolates from other parts of the world.
Yang, Jin-Guang; Wang, Feng-Long; Chen, De-Xin; Shen, Li-Li; Qian, Yu-Mei; Liang, Zhi-Yong; Zhou, Wen-Chang; Yan, Tai-He
Tobacco mosaic virus (TMV) causes significant losses in many economically important crops. Contaminated soils may play roles as reservoirs and sources of transmission for TMV. In this study we report the development of an immunocapture real-time RT-PCR (IC-real-time RT-PCR) assay for direct detection of TMV in soils without RNA isolation. A series of TMV infected leaf sap dilutions of 1:101, 1:102, 1:103, 1:104, 1:105 and 1:106 (w/v, g/mL) were added to one gram of soil. The reactivity of DAS-ELISA and conventional RT-PCR was in the range of 1:102 and 1:103 dilution in TMV-infested soils, respectively. Meanwhile, the detection limit of IC-real-time RT-PCR sensitivity was up to 1:106 dilution. However, in plant sap infected by TMV, both IC-real-time RT-PCR and real-time RT-PCR were up to 1:106 dilution, DAS-ELISA could detect at least 1:103 dilution. IC-real-time RT-PCR method can use either plant sample extracts or cultivated soils, and show higher sensitivity than RT-PCR and DAS-ELISA for detection of TMV in soils. Therefore, the proposed IC-real-time RT-PCR assay provides an alternative for quick and very sensitive detection of TMV in soils, with the advantage of not requiring a concentration or RNA purification steps while still allowing detection of TMV for disease control. PMID:23211755
Farkas, Tibor; Singh, Amy; Le Guyader, Françoise S; La Rosa, Giuseppina; Saif, Linda; McNeal, Monica
Noroviruses are important causes of acute gastroenteritis and are classified into six genogroups with GI, GII and GIV containing human pathogens. This high genetic diversity represents a significant challenge for diagnostic assay development. Genogroup specific monoplex and multiplex real time RT-PCR assays are widely used for the detection of GI and GII noroviruses. On the other hand, GIV norovirus detection is not part of routine laboratory diagnosis. This study describes the development and evaluation of a one tube, real time RT-PCR assay for the simultaneous detection and quantification of GI, GII and GIV noroviruses, including both GIV.1 (human) and GIV.2 (animal) strains. Assay performance was evaluated on a panel of norovirus positive clinical samples by comparison of monoplex and multiplex standard curves and Ct values. The multiplex assay demonstrated equal sensitivity and specificity to the monoplex assays and was able to detect all GI, GII and GIV noroviruses with Ct values equal to that of the monoplex assays. The multiplex assay described in this study will be instrumental for the better understanding of GIV norovirus epidemiology, including their possible zoonotic nature.
Ling, Hui; Wu, Qibin; Guo, Jinlong; Xu, Liping; Que, Youxiong
The increasingly used real time quantitative reverse transcription-PCR (qRT-PCR) method for gene expression analysis requires one or several reference gene(s) acting as normalization factor(s). In order to facilitate gene expression studies in sugarcane (Saccharum officinarum), a non-model plant with limited genome information, the stability of 13 candidate reference genes was evaluated. The geNorm, NormFinder and deltaCt methods were used for selecting stably expressed internal controls across different tissues and under various experimental treatments. These results revealed that, among these 13 candidate reference genes, GAPDH, eEF-1a and eIF-4α were the most stable and suitable for use as normalization factors across all various experimental samples. In addition, APRT could be a candidate for examining the relationship between gene copy number and transcript levels in sugarcane tissue samples. According to the results evaluated by geNorm, combining CUL and eEF-1α in hormone treatment experiments; CAC and CUL in abiotic stress tests; GAPDH, eEF-1a and CUL in all treatment samples plus CAC, CUL, APRT and TIPS-41 in cultivar tissues as groups for normalization would lead to more accurate and reliable expression quantification in sugarcane. This is the first systematic validation of reference genes for quantification of transcript expression profiles in sugarcane. This study should provide useful information for selecting reference genes for more accurate quantification of gene expression in sugarcane and other plant species. PMID:24823940
Yuan, Wen; Wang, Jing; Xu, Fengjiao; Huang, Bihong; Lian, Yuexiao; Rao, Dan; Yin, Xueqin; Wu, Miaoli; Zhu, Yujun; Zhang, Yu; Huang, Ren; Guo, Pengju
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.
Park, Hyung Seok; Han, Hyun Ju; Lee, Soohyeon; Kim, Gun Min; Park, Seho; Choi, Yeon A; Lee, Jeong Dong; Kim, Gi Moon
Purpose The roles of circulating tumor cells (CTCs) as predictive and prognostic factors, as well as key mediators in the metastatic cascade, have been investigated. This study aimed to validate a method to quantify CTCs in peripheral blood using a real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay for cytokeratin (CK)-19 and to evaluate the utility of this assay in detecting CTCs in breast cancer patients. Materials and Methods Real-time monitoring PCR of fluorescently labeled specific hybridization probes for CK-19 mRNA was established. Peripheral blood samples from 30 healthy donors, 69 patients with early breast cancer, 47 patients with locally advanced breast cancer, and 126 patients with metastatic breast cancer were prospectively obtained and analyzed for CTC detection. Results CK-19 mRNA was not detectable in healthy subjects using the real-time RT-PCR method. The detection rates of CK-19 mRNA in breast cancer patients were 47.8% for early breast cancer (33/69), 46.8% for locally advanced breast cancer (22/47), and 61.1% for metastatic breast cancer (77/129). The detection rate of CK-19-positive CTCs in metastatic disease was slightly higher than early or locally advanced breast cancer; however, the detection rate according to disease burden was not statistically different (p=0.097). The detection rate was higher in patients with pleural metastasis (p=0.045). CTC detection was associated with poor survival (p=0.014). Conclusion A highly specific and sensitive CK-19 mRNA-based method to detect CTCs in peripheral blood in breast cancer patients can be used in further prospective studies to evaluate the predictive and prognostic importance of CTCs. PMID:27873491
Soltan, Mohamed A; Tsai, Yun-Long; Lee, Pei-Yu A; Tsai, Chuan-Fu; Chang, Hsiao-Fen G; Wang, Hwa-Tang T; Wilkes, Rebecca P
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.
Hosmillo, Myra D T; Jeong, Young-Ju; Kim, Hyun-Jeong; Collantes, Therese Marie; Alfajaro, Mia Madel; Park, Jun-Gyu; Kim, Ha-Hyun; Kwon, Hyung-Jun; Park, Su-Jin; Kang, Mun-Il; Park, Sang-Ik; Cho, Kyoung-Oh
Toroviruses (ToVs) are a group of emerging viruses that cause gastroenteritis in domestic animals and humans. Currently, methods such as real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) have not yet been developed for the rapid detection and quantitation of bovine (BToV) and porcine (PToV) toroviruses. Using BToV and PToV RNA standards generated by in vitro transcription, the detection limit of the SYBR Green real-time RT-PCR assay was 2.54 x 10(2) BToV and 2.17 x 10(3) PToV copies/reaction (correlation coefficiency=0.99 and 0.97, respectively), whereas those of RT-PCR and nested PCR were 2.54 x 10(5) and 2.54 x 10(4) (BToV) and 2.17 x 10(7) and 2.17 x 10(5) (PToV) cRNA viral copies/reaction, respectively. Archived diarrhea specimens of calves (n=121) and piglets (n=86) were subjected to RT-PCR, nested PCR and SYBR Green real-time RT-PCR. By conventional RT-PCR, 1 (0.8%) bovine and 7 (8.1%) porcine samples tested positive to BToV and PToV, respectively. With nested PCR, 13 (10.7%) bovine and 17 (19.8%) porcine samples tested positive. SYBR Green real-time RT-PCR assay detected BToV and PToV in 22 of 121 (18.2%) bovine and 31 of 86 (36.0%) porcine samples. These results indicate that SYBR Green real-time RT-PCR (P<0.05) is a more sensitive assay, which can be reproduced as a reliable, sensitive, and rapid tool for the detection and quantitation of toroviruses.
Zampieri, Denise; Nora, Luísa C; Basso, Vanessa; Camassola, Marli; Dillon, Aldo J P
Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a methodology that facilitates the quantification of mRNA expression in a given sample. Analysis of relative gene expression by qRT-PCR requires normalization of the data using a reference gene that is expressed at a similar level in all evaluated conditions. Determining an internal control gene is essential for gene expression studies. Gene expression studies in filamentous fungi frequently use the β-actin gene (actb), β-tubulin, and glyceraldehyde-3-phosphate dehydrogenase as reference genes because they are known to have consistent expression levels. Until now, no study has been performed to select an internal control gene for the filamentous fungal species Penicillium echinulatum. The aim of this study was to evaluate and validate internal control genes to enable the study of gene expression in P. echinulatum using qRT-PCR. P. echinulatum strain S1M29 was grown in conditions to either induce (cellulose and sugar cane bagasse) or repress (glucose) gene expression to analyze 23 candidate normalization genes for stable expression. Two software programs, BestKeeper and geNorm, were used to assess the expression of the candidate normalization genes. The results indicate that the actb reference gene is more stably expressed in P. echinulatum. This is the first report in the literature that determines a normalization gene for this fungus. From the results obtained, we recommend the use of the P. echinulatum actb gene as an endogenous control for gene expression studies of cellulases and hemicellulases by qRT-PCR.
Zhang, Jian; Hu, Yong-hua; Sun, Bo-guang; Xiao, Zhi-zhong; Sun, Li
Disease outbreaks caused by iridoviruses are known to affect farmed flounder (Paralichthys olivaceus) and turbot (Scophthalmus maximus). To facilitate quantitative real time RT-PCR (qRT-PCR) analysis of gene expression in flounder and turbot during viral infection, we in this study examined the potentials of 9 housekeeping genes of flounder and turbot as internal references for qRT-PCR under conditions of experimental infection with megalocytivirus, a member of the Iridoviridae family. The mRNA levels of the 9 housekeeping genes in the brain, gill, heart, intestine, kidney, liver, muscle, and spleen of flounder and turbot were determined by qRT-PCR at 24h and 72h post-viral infection, and the expression stabilities of the genes were determined with geNorm and NormFinder algorithms. The results showed that (i) viral infection induced significant changes in the mRNA levels of the all the examined genes in a manner that was dependent on both tissue type and infection stage; (ii) for a given time point of infection, stability predictions made by the two algorisms were highly consistent for most tissues; (iii) the optimum reference genes differed at different infection time points at least in some tissues; (iv) at both examined time points, no common reference genes were identified across all tissue types. These results indicate that when studying gene expression in flounder and turbot in relation to viral infection, different internal references may have to be used not only for different tissues but also for different infection stages.
Hwang, Seoyeon; Kang, Byunghak; Hong, Jiyoung; Kim, Ahyoun; Kim, Hyejin; Kim, Kisang; Cheon, Doo-Sung
Human enterovirus (EV) 71 is the main etiological agent of hand, foot, and mouth disease (HFMD). It is associated with neurological complications, and caused fatalities during recent outbreaks in the Asia-Pacific region. Infections caused by EV71 could lead to many complications, ranging from brainstem encephalitis to pulmonary oedema, resulting in high mortality. In this study, a duplex real-time RT-PCR assay was developed in order to simultaneously detect pan-EV and EV71. EV71-specific primers and probes were designed based on the highly conserved VP1 region of EV71. Five EV71 strains were detected as positive, and no positive fluorescence signal was observed in the duplex real-time RT-PCR for other viral RNA, which showed 100% specificity for the selected panel, and no cross-reactions were observed in this duplex real-time RT-PCR. The EV71-specific duplex real-time RT-PCR was more sensitive than conventional RT-PCR, and detected viral titers that were 10-fold lower than those measured by the latter. Of the 381 HFMD clinical specimens, 196 (51.4%) cases were pan-EV-positive, of which 170 (86.7%) were EV71-positive when tested by pan-EV and EV71-specific duplex real-time RT-PCR. EV71-specific duplex real-time RT-PCR offers a rapid and sensitive method to detect EV71 from clinical specimens, and will allow quarantine measures to be taken more effectively during outbreaks.
Hanaki, Ken-Ichi; Ike, Fumio; Kajita, Ayako; Yasuno, Wataru; Yanagiba, Misato; Goto, Motoki; Sakai, Kouji; Ami, Yasushi; Kyuwa, Shigeru
A one-step SYBR Green I real-time RT-PCR assay was developed for the detection and quantification of a broad range of murine noroviruses (MNVs). The primer design was based on the multiple sequence alignments of 101 sequences of the open reading frame (ORF)1-ORF2 junction of MNV. The broad reactivity and quantitative capacity of the assay were validated using 7 MNV plasmids. The assay was completed within 1 h, and the reliable detection limit was 10 copies of MNV plasmid or 0.063 median tissue culture infective doses per milliliter of RAW264 cell culture-propagated viruses. The diagnostic performance of the assay was evaluated using 158 mouse fecal samples, 91 of which were confirmed to be positive. The melting curve analysis demonstrated the diversity of MNV in the samples. This is the first report of a broadly reactive one-step SYBR Green I real-time RT-PCR assay for detecting of MNVs. The rapid and sensitive performance of this assay makes it a powerful tool for diagnostic applications.
Mari, Viviana; Losurdo, Michele; Lucente, Maria Stella; Lorusso, Eleonora; Elia, Gabriella; Martella, Vito; Patruno, Giovanni; Buonavoglia, Domenico; Decaro, Nicola
HoBi-like pestiviruses are emerging pestiviruses that infect cattle causing clinical forms overlapping to those induced by bovine viral diarrhea virus (BVDV) 1 and 2. As a consequence of their widespread distribution reported in recent years, molecular tools for rapid discrimination among pestiviruses infecting cattle are needed. The aim of the present study was to develop a multiplex real-time RT-PCR assay, based on the TaqMan technology, for the rapid and unambiguous characterisation of all bovine pestiviruses, including the emerging HoBi-like strains. The assay was found to be sensitive, specific and repeatable, ensuring detection of as few as 10(0)-10(1) viral RNA copies. No cross-reactions between different pestiviral species were observed even in samples artificially contaminated with more than one pestivirus. Analysis of field samples tested positive for BVDV-1, BVDV-2 or HoBi-like virus by a nested PCR protocol revealed that the developed TaqMan assay had equal or higher sensitivity and was able to discriminate correctly the viral species in all tested samples, whereas a real-time RT-PCR assay previously developed for HoBi-like pestivirus detection showed cross-reactivity with few high-titre BVDV-2 samples.
Abera, Tsegalem; Thangavelu, Ardhanary
A two-step SYBR Green I based real time RT-PCR targeting the matrix (M) gene of Peste des petits ruminants virus (PPRV) was developed. The specificity of the assay was assessed against viral nucleic acid extracted from a range of animal viruses of clinical and structural similarities to PPRV including canine distemper virus, measles virus, bluetongue virus and Newcastle disease virus. But none of the viruses and no template control showed an amplification signal. Sensitivity of the same assay was assessed based on plasmid DNA copy number and with respect to infectivity titre. The lower detection limit achieved was 2.88 plasmid DNA copies/μl with corresponding Ct value of 35.93. Based on tissue culture infectivity titre the lower detection limits were 0.0001TCID50/ml and 1TCID50/ml for the SYBR green I based real time RT-PCR and conventional RT-PCR, respectively. The calculated coefficient of variations values for intra- and inter-assay variability were low, ranging from 0.21% to 1.83% and 0.44% to 1.97%, respectively. The performance of newly developed assay was evaluated on a total of 36 clinical samples suspected of PPR and compared with conventional RT-PCR. The SYBR Green I based real time RT-PCR assay detected PPRV in 32 (88.8%) of clinical samples compared to 19 (52.7%) by conventional RT-PCR. Thus, the two-step SYBR Green I based real time RT-PCR assay targeting the M gene of PPRV reported in this study was highly sensitive, specific and reproducible for detection and quantitation of PPRV nucleic acids.
Fu, Wei-Lin; Sun, Sheng-Ren; Fu, Hua-Ying; Chen, Ru-Kai; Su, Jin-Wei; Gao, San-Ji
Sugarcane mosaic disease is caused by the Sugarcane streak mosaic virus (SCSMV; genus Poacevirus, family Potyviridae) which is common in some Asian countries. Here, we established a protocol of a one-step real-time quantitative reverse transcription PCR (real-time qRT-PCR) using the TaqMan probe for the detection of SCSMV in sugarcane. Primers and probes were designed within the conserved region of the SCSMV coat protein (CP) gene sequences. Standard single-stranded RNA (ssRNA) generated by PCR-based gene transcripts of recombinant pGEM-CP plasmid in vitro and total RNA extracted from SCSMV-infected sugarcane were used as templates of qRT-PCR. We further performed a sensitivity assay to show that the detection limit of the assay was 100 copies of ssRNA and 2 pg of total RNA with good reproducibility. The values obtained were approximately 100-fold more sensitive than those of the conventional RT-PCR. A higher incidence (68.6%) of SCSMV infection was detected by qRT-PCR than that (48.6%) with conventional RT-PCR in samples showing mosaic symptoms. SCSMV-free samples were verified by infection with Sugarcane mosaic virus (SCMV) or Sorghum mosaic virus (SrMV) or a combination of both. The developed qRT-PCR assay may become an alternative molecular tool for an economical, rapid, and efficient detection and quantification of SCSMV.
Li, Min; Green, Patrick L
HTLV-1 and HTLV-2 are highly related delta-retroviruses that infect and transform T-lymphocytes, but have distinct pathogenic properties. HTLV replication and survival requires the expression of multiple gene products from an unspliced and a series of highly related alternatively spliced mRNA species. To date, the comparative levels of all known HTLV-1 and HTLV-2 viral mRNAs in different transformed cell lines and at different stages of virus infection have not been assessed. In this study, we compiled a series of oligonucleotide primer pairs and probes to quantify both HTLV-1 and HTLV-2 mRNA species using real-time RT-PCR. The optimized reaction for detection of each mRNA had amplification efficiency greater than 90% with a linear range spanning 25-2.5 x 10(7) copies. The R(2)'s of all standard curves were greater than 0.97. Quantitation of HTLV mRNAs between different cell lines showed variability (gag/pol>or=tax/rex>env>or=accessory proteins), but the overall levels of each mRNA relative to each other within a cell line were similar. These results provide a method to quantify all specific mRNAs from both HTLV-1 and HTLV-2, which can be used to evaluate further viral gene expression and correlate transcript levels to key stages of the virus life cycle and ultimately, pathogenesis.
Luo, Bao-Zheng; Mo, Qiu-Hua; Li, Ru-Shu; Bo, Qing-Ru; Xu, Hai-Nie; Sha, Cai-Hua; Liao, Xiu-Yun
In order to develop a rapid detection kit for novel avian influenza virus (AIV) subtype H7N9, two sets of specific primers and probes were designed based on the nucleotide sequences of hemagglutinin antigen (HA) and neuraminidase antigen (NA) of novel H7N9 virus (2013) available in GenBank to establish the method of TaqMan probe-based multiplex real-time RT-PCR for rapid detection of AIV subtype H7N9. The primer and probe of HA were for all H7 subtype AIVs, while the primer and probe of NA were only for novel N9 subtype AIVs. The results showed that this method had high sensitivity and specificity. This method was applicable to the testing of positive standard sample with a minimum concentration of 10 copies/microL; it not only distinguished H7 subtype from H1, H3, H5, H6, and H9 subtypes, but also distinguished novel N9 subtype from traditional N9 subtype. A total of 2700 samples from Zhuhai, China were tested by this method, and the results were as expected. For the advantages of sensitivity and specificity, the method holds promise for wide application.
Batten, Carrie A; Banyard, Ashley C; King, Donald P; Henstock, Mark R; Edwards, Lorraine; Sanders, Anna; Buczkowski, Hubert; Oura, Chris C L; Barrett, Tom
Peste des petits ruminants virus (PPRV) causes a devastating disease of small ruminants present across much of Africa and Asia. Recent surveillance activities and phylogenetic analyses have suggested that the virus is an emerging problem as it is now being detected in areas previously free of the disease. As such, the virus not only is threatening small ruminant production and agricultural stability in the developing world, but also poses an economic threat to livestock in the European Union (EU) through introduction from European Turkey and North Africa. This report describes the development of a high throughput, rapid, real time RT-PCR method for the sensitive and specific detection of PPRV using robotic RNA extraction. This assay targets the nucleocapsid (N) gene of PPRV and has been shown to detect all four genetic lineages of PPRV in tissues, ocular and nasal swabs and blood samples collected in the field. The lowest detection limit achieved was approximately 10 genome copies/reaction, making this assay an ideal tool for the sensitive and rapid detection of PPRV in diagnostic laboratories.
Efforts to analyze the replicative RNA produced by Maize fine streak virus (MVSF) within maize tissue was complicated by the lack of specificity during cDNA generation using standard reverse transcriptase protocols. Real-time qRT-PCR using cDNA generated by priming with random hexamers does not dist...
Efforts to analyze the replicative RNA produced by Maize fine streak virus (MFSV) within maize tissue was complicated by the lack of specificity during cDNA generation using standard reverse transcriptase protocols. Real-time qRT-PCR using cDNA generated by priming with random hexamers does not dist...
Two TaqMan-based real-time One-Step RT-PCR assays were developed for the rapid and efficient detection of Raspberry bushy dwarf virus (RBDV) and Raspberry leaf mottle virus (RLMV), two of the most common raspberry viruses in North America and Europe. The primers and probes were designed from conser...
Rapid detection of avian influenza virus and identification of the H5 and H7 hemagglutinin subtypes some of which are associated with high pathogenicity in poultry is critical for clinical diagnosis and wild bird monitoring programs. A real-time RT-PCR test for identification of the H7 subtype in N...
Maan, Narender Singh; Belaganahalli, Manjunatha N.; Potgieter, Abraham C.; Kumar, Vinay; Batra, Kanisht; Wright, Isabel M.; Kirkland, Peter D.; Mertens, Peter P. C.
Bluetongue virus is the type species of the genus Orbivirus, family Reoviridae. Bluetongue viruses (BTV) are transmitted between their vertebrate hosts primarily by biting midges (Culicoides spp.) in which they also replicate. Consequently BTV distribution is dependent on the activity, geographic distribution, and seasonal abundance of Culicoides spp. The virus can also be transmitted vertically in vertebrate hosts, and some strains/serotypes can be transmitted horizontally in the absence of insect vectors. The BTV genome is composed of ten linear segments of double-stranded (ds) RNA, numbered in order of decreasing size (Seg-1 to Seg-10). Genome segment 2 (Seg-2) encodes outer-capsid protein VP2, the most variable BTV protein and the primary target for neutralising antibodies. Consequently VP2 (and Seg-2) determine the identity of the twenty seven serotypes and two additional putative BTV serotypes that have been recognised so far. Current BTV vaccines are serotype specific and typing of outbreak strains is required in order to deploy appropriate vaccines. We report development and evaluation of multiple ‘TaqMan’ fluorescence-probe based quantitative real-time type-specific RT-PCR assays targeting Seg-2 of the 27+1 BTV types. The assays were evaluated using orbivirus isolates from the ‘Orbivirus Reference Collection’ (ORC) held at The Pirbright Institute. The assays are BTV-type specific and can be used for rapid, sensitive and reliable detection / identification (typing) of BTV RNA from samples of infected blood, tissues, homogenised Culicoides, or tissue culture supernatants. None of the assays amplified cDNAs from closely related but heterologous orbiviruses, or from uninfected host animals or cell cultures. PMID:27661614
Zhu, Zheng; Fan, Huan; Qi, Xian; Qi, Yuhua; Shi, Zhiyang; Wang, Hua; Cui, Lunbiao; Zhou, Minghao
Most recently a novel avian-origin influenza A (H7N9) virus emerged in China and has been associated with lots of human infection and fatal cases. Genetic analysis of the viral genome revealed that this reassortant virus might be better adapted to humans than other avian influenza viruses. Molecular diagnostic methods are thus urgently needed in public health laboratories. In this study, a SYBR green-based one-step real time reverse transcription-PCR (RT-PCR) was developed to detect the novel H7N9 virus. The primer pairs on the basis of the hemagglutinin and neuraminidase gene sequences of H7N9 viruses amplified subtype-specific fragments with Tm values of 80.77±0.06°C for H7 and 81.20±0.17°C for N9 respectively. The standard curves showed a dynamic linear range across 6 log units of RNA copy number (10(6) to 10(1) copies/ µl) with a detection limit of 10 copies per reaction for both H7 and N9 assays by using serial ten-fold diluted in-vitro transcribed viral RNA. In addition, no cross-reactivity was observed with seasonal H1N1, H1N1 pdm09, H3N2, H5N1 and H9N2 viruses as well as other human respiratory viruses. When the assay was further evaluated in H7N9 virus infected clinical samples, positive amplification signals were obtained in all of the specimens with the accordance between H7 and N9 assays. Therefore, the established SYBR green-based real time RT-PCR assay could provide a rapid, sensitive, specific and reliable alternative approach with lower costs for high throughput screening of suspected samples from humans, animals and environments in first line public health laboratories.
Purcell, Maureen K.; Hart, S. Alexandra; Kurath, Gael; Winton, James R.
The fish rhabdovirus, Infectious hematopoietic necrosis virus (IHNV), is an important pathogen of salmonids. Cell culture assays have traditionally been used to quantify levels of IHNV in samples; however, real-time or quantitative RT-PCR assays have been proposed as a rapid alternative. For viruses having a single-stranded, negative-sense RNA genome, standard qRT-PCR assays do not distinguish between the negative-sense genome and positive-sense RNA species including mRNA and anti-genome. Thus, these methods do not determine viral genome copy number. This study reports development of strand-specific, qRT-PCR assays that use tagged primers for enhancing strand specificity during cDNA synthesis and quantitative PCR. Protocols were developed for positive-strand specific (pss-qRT-PCR) and negative-strand specific (nss-qRT-PCR) assays for IHNV glycoprotein (G) gene sequences. Validation with synthetic RNA transcripts demonstrated the assays could discriminate the correct strand with greater than 1000-fold fidelity. The number of genome copies in livers of IHNV-infected fish determined by nss-qRT-PCR was, on average, 8000-fold greater than the number of infectious units as determined by plaque assay. We also compared the number of genome copies with the quantity of positive-sense RNA and determined that the ratio of positive-sense molecules to negative-sense genome copies was, on average, 2.7:1. Potential future applications of these IHNV strand-specific qRT-PCR assays are discussed.
Matejusova, Iveta; McKay, Paul; McBeath, Alastair J A; Collet, Bertrand; Snow, Michael
A survey was undertaken to determine the potential distribution of viral haemorrhagic septicaemia virus (VHSV) in marine cage-based salmonid farms in Scotland. A rapid, accurate and sensitive quantitative real-time RT-PCR (qRT-PCR) assay was developed, targeting a conserved region of the nucleoprotein (N) gene of the virus. The qRT-PCR assay was shown to be more sensitive than the conventional VHSV RT-PCR. A validation protocol included several different virus isolates as the target and confirmed that the assay could detect all European VHSV genotypes (I, II and III). Both endogenous and exogenous controls were designed to control for integrity of template and distinguish between true VHSV positives and contamination with the positive control material. In total, the universal European VHSV qRT-PCR assay with exogenous positive control was applied to screen 2040 individual Atlantic salmon Salmo salar and 150 individual rainbow trout Oncorhynchus mykiss. No evidence of the presence of VHSV in association with either salmonid species in Scottish marine farms was detected. However, both marine Atlantic salmon and rainbow trout are still considered possible carriers of VHSV, which remains a potential threat to freshwater farming. Therefore, a continued surveillance of these species in marine environment is recommended.
Luchsinger, Vivian; Prades, Yara; Ruiz, Mauricio; Pizarro, Rolando; Rossi, Patricio; Lizama, Luis; Garmendia, María Luisa; Meza, Angela; Larrañaga, Carmen; Avendaño, Luis F
Community-acquired pneumonia (CAP) is the third cause of death worldwide. Viruses are frequently detected in adult CAP. Highly sensitive diagnostic techniques should be used due to poor viral shedding. Different sampling methods can affect viral detection, being necessary to establish the optimal type of sample for identifying respiratory viruses in adults. The detection rates of respiratory viruses by Luminex xTAG® RVP fast assay, real time RT-PCR (rtRT-PCR) (Sacace®), and immunofluorescence assay (IFA) in adult CAP were performed in nasopharyngeal swabs (NPS) and aspirates (NPA) from 179 hospitalized adults. Positivity was 47.5% for Luminex®, 42.5% for rtRT-PCR (P = 0.3), and 2.7% for IFA (2.7%) (P < 0.0). The sensitivity, specificity, and kappa coefficient of xTAG® RVP compared with rtRT-PCR were 84.2%, 79.6%, and 0.62%, respectively. Luminex® and rtRT-PCR detected 65 (58.0%) and 57 (50.9%) viruses in 112 NPA and 35 (34.3%) and 31 (30.4%) in 102 NPS, respectively (P < 0.01). xTAG® RVP is appropriate for detecting respiratory viruses in CAP adults. Both molecular techniques yielded better results with nasopharyngeal aspirate than swabs.
Santiago, Gilberto A; Vergne, Edgardo; Quiles, Yashira; Cosme, Joan; Vazquez, Jesus; Medina, Juan F; Medina, Freddy; Colón, Candimar; Margolis, Harold; Muñoz-Jordán, Jorge L
Dengue is an acute illness caused by the positive-strand RNA dengue virus (DENV). There are four genetically distinct DENVs (DENV-1-4) that cause disease in tropical and subtropical countries. Most patients are viremic when they present with symptoms; therefore, RT-PCR has been increasingly used in dengue diagnosis. The CDC DENV-1-4 RT-PCR Assay has been developed as an in-vitro diagnostic platform and was recently approved by the US Food and Drug Administration (FDA) for detection of dengue in patients with signs or symptoms of mild or severe dengue. The primers and probes of this test have been designed to detect currently circulating strains of DENV-1-4 from around the world at comparable sensitivity. In a retrospective study with 102 dengue cases confirmed by IgM anti-DENV seroconversion in the convalescent sample, the RT-PCR Assay detected DENV RNA in 98.04% of the paired acute samples. Using sequencing as a positive indicator, the RT-PCR Assay had a 97.92% positive agreement in 86 suspected dengue patients with a single acute serum sample. After extensive validations, the RT-PCR Assay performance was highly reproducible when evaluated across three independent testing sites, did not produce false positive results for etiologic agents of other febrile illnesses, and was not affected by pathological levels of potentially interfering biomolecules. These results indicate that the CDC DENV-1-4 RT-PCR Assay provides a reliable diagnostic platform capable for confirming dengue in suspected cases.
Liu, Zongliang; Fu, Yuguang; Ji, Yanhong; Wei, Jianzhong; Cai, Xuepeng; Zhu, Qiyun
Duck Tembusu virus (DTMUV) is a single-stranded positive-sense RNA virus that causes disease to emerge in duck flocks and results in huge economic losses to the duck industry. However, no vaccines and control measures are available in China to date. Development of reliable and fast detection methods is necessary to prevent and control this disease. Therefore, a one-step SYBR Green real-time reverse transcription polymerase chain reaction (RT-PCR) method is established here for DTMUV detection. The results show that the method can specifically detect DTMUV without cross-reactions with selected avian pathogens. The sensitivity of the assay was 1000 times greater than that of a conventional RT-PCR and able to test as few as 20 copies from RNA standard samples. The coefficients of variations of inter- and intra-assay values ranged from 0.09% to 0.36% and 0.1% to 0.23%, respectively. Testing 168 field samples and 96 experimentally infected samples by conventional RT-PCR and the one-step SYBR Green real-time RT-PCR, the positive rates were 35.1% and 73.8% from field samples and 30.2% and 64.6% from infected samples. The one-step SYBR Green real-time RT-PCR developed in this study was shown to be a sensitive, specific, high-throughput, cost-effective, and simple diagnostic tool for the rapid detection and epidemiological surveillance of the emerging DTMUV infection.
Jamnikar Ciglenečki, Urška; Toplak, Ivan
Real-time polymerase chain reaction (real-time PCR) is an accurate, rapid and reliable method that can be used for the detection and also for the quantitation of specific DNA molecules. It can be non-specific, with intercalating dyes (SYBR Green I dye) able to bind to any dsDNA, or specific with a probe (TaqMan), whereby the probe is designed to bind within the amplified PCR fragment. A new real-time reverse transcription and polymerase chain reaction (real time RT-PCR) assay with TaqMan probe for specific detection of acute bee paralysis virus was designed. The assay was optimized to be highly sensitive and analytically specific and tested with a selection of genetically diverse ABPV strains originating from Slovenia, the United Kingdom (UK), Hungary and Germany. The detection limit of the assay and sensitivity comparisons with conventional RT-PCR were analyzed and this assay can detect a minimum of 44 copies of ABPV/reaction and is 230 times more sensitive than conventional RT-PCR. In addition, the assay is highly reproducible, with an average slope of standard curve made of ten-fold dilutions of standard copies/reaction -3.479±0.19 and an average slope of standard curve made of ten-fold dilutions of RNA -3.409±0.18.
Tang, Yi; Lu, Huaguang
Newly emerging avian orthoreovirus (ARV) variants have been continuously detected in Pennsylvania poultry since 2011. In this paper, we report our recent diagnostic assay development of one-step real-time RT-PCR (rRT-PCR) for the rapid and universal detection of all ARVs or reference strains of chicken, pheasant and turkey origins and six σC genotypes of the newly emerging field ARV variants in Pennsylvania (PA) poultry. Primers and probes for the rRT-PCR were designed from the conserved region of the M1 genome segment 5' end based on the whole-genome alignment of various ARV strains, including six field variants or novel strains obtained in PA poultry. The detection limit of the newly developed rRT-PCR for ARV was as low as 10 copies/reaction of viral RNA, and 10(0.50)-10(0.88) tissue culture infectious dose (TCID50)/100 μL of viruses. This new rRT-PCR detected all six σC genotypes from the 66 ARV field variant strains and reference strains tested in this study. There were no cross-reactions with other avian viruses. Reproducibility of the assay was confirmed by intra- and inter-assay tests with variability from 0.12% to 2.19%. Sensitivity and specificity of this new rRT-PCR for ARV were achieved at 100% and 88%, respectively, in comparison with virus isolation as the "gold standard" in testing poultry tissue specimen.
Zhou, Gang; Chiu, David; Qin, Dajiang; Niu, Lizhi; Cai, Jinlei; He, Lihua; Huang, Wenhao; Xu, Kecheng
Cell adhesion molecule CD44v6 has been found to be associated with the progression and metastasis of numerous cancers. In this study, a novel duplex real-time quantitative reverse-transcription PCR (qRT-PCR) assay was developed to quantitatively detect the CD44v6 gene expression in pancreatic cancer patients. The primers and probes of CD44v6 and β-actin genes were designed and standard curve of the duplex qRT-PCR was constructed by optimizing the reaction conditions. The specificity and reproducibility of this assay were satisfactory and the detection limit was 100 copies, which was 10 times more sensitive than the conventional RT-PCR assay. This assay was also used to detect the expression levels of CD44v6 messenger RNA in peripheral blood mononuclear cell in 37 pancreatic cancer patients and 12 healthy people. The results showed that 37 clinical samples were tested positive by the duplex qRT-PCR compared with only 30 by the conventional RT-PCR. The levels of CD44v6 expression showed significant correlation with sex, tumor size, tumor differentiation, clinical stage, lymph node, and liver metastasis (P < 0.05). Compared with the control group, CD44v6 levels in patients prior and 10 days post cryosurgery were significantly increased (P < 0.05) but had no significant change in those 1 month post cryosurgery (P > 0.05). The duplex qRT-PCR assay may provide a useful tool for the evaluation of prognosis and curative effect of pancreatic cancer.
Tang, Joe; Khan, Subuhi; Delmiglio, Catia; Ward, Lisa I
A real-time TaqMan RT-PCR assay was developed for the rapid and sensitive detection of Tomato ringspot virus (ToRSV), an important plant virus which infects a wide range of fruit and ornamental crops. Primers and a probe were designed based on the highly conserved 3'-untranslated region (UTR) sequences of ToRSV, to amplify a 182bp fragment of this region of RNA-1 and RNA-2. The assay was demonstrated to reliably amplify all ToRSV isolates tested. The detection limit was estimated to be about 12 copies of the ToRSV target region. No amplification was observed from the RNA of other nepoviruses or healthy host species. A comparison with a published conventional RT-PCR and a SYBR-based qRT-PCR indicated that both of the published assays lacked reliability and sensitivity, as neither were able to amplify all ToRSV isolates tested, and both were approximately 1000 times less sensitive than the novel TaqMan real-time assay. This TaqMan real-time assay was tested using four different reagent kits and was shown to be robust and stable, with no significant differences in sensitivity between kits. It is expected that the implementation of this TaqMan real-time RT-PCR assay will facilitate efficient phytosanitary certification of nursery stock requiring testing for ToRSV by regulatory agencies, and will also have wider uses for the general detection of ToRSV in a range of hosts.
Wadhwa, Ashutosh; Wilkins, Kimberly; Gao, Jinxin; Condori Condori, Rene Edgar; Gigante, Crystal M.; Zhao, Hui; Ma, Xiaoyue; Ellison, James A.; Greenberg, Lauren; Velasco-Villa, Andres; Orciari, Lillian
Rabies, resulting from infection by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high
Feng, Yufei; Yang, Tao; Xu, Qingyuan; Sun, Encheng; Li, Junping; Lv, Shuang; Wang, Haixiu; Zhang, Qin; Zhang, Jikai; Wu, Donglai
Bluetongue virus (BTV) is the etiological agent of bluetongue (BT) disease, a noncontagious insect-transmitted disease of international importance. To date, 26 BTV serotypes have been recognized worldwide. Methods to discriminate BTV serotypes in clinical samples are essential to epidemiological surveillance efforts and BTV vaccination programs. The BTV VP2 major outer capsid protein, encoded by genomic segment 2 (Seg-2), is the most highly variable BTV protein and is the primary determinant of the virus serotype. Here, we report the development of rapid and reliable real-time RT-PCR assays to detect and discriminate 22 BTV serotypes on the basis of VP2-encoding genomic sequences. Serotype-specific primers and probes detected only the targeted BTV serotype and displayed no cross-amplification of off-target BTV serotypes or other closely related Reoviridae and Bunyaviridae family members. The real-time RT-PCR assays developed were highly sensitive, and the majority of serotype-specific reactions could detect template when present at ≥10 copies. These BTV serotype-specific real-time RT-PCR assays represent a rapid, sensitive, and reliable method for the identification, differentiation and quantification of 22 BTV serotypes.
González-Aguilera, Karla L.; Saad, Carolina F.; Chávez Montes, Ricardo A.; Alves-Ferreira, Marcio; de Folter, Stefan
Quantitative real-time RT-PCR (qRT-PCR) has become one of the most widely used methods for accurate quantification of gene expression. Since there are no universal reference genes for normalization, the optimal strategy to normalize raw qRT-PCR data is to perform an initial comparison of a set of independent reference genes to assess the most stable ones in each biological model. Normalization of a qRT-PCR experiment helps to ensure that the results are both statistically significant and biologically meaningful. Tomato is the model of choice to study fleshy fruit development. The miniature tomato (Solanum lycopersicum L.) cultivar Micro-Tom (MT) is considered a model system for tomato genetics and functional genomics. A new genotype, containing the Rg1 allele, improves tomato in vitro regeneration. In this work, we evaluated the expression stability of four tomato reference genes, namely CAC, SAND, Expressed, and ACTIN2. We showed that the genes CAC and Exp are the best reference genes of the four we tested during fruit development in the MT-Rg1 genotype. Furthermore, we validated the reference genes by showing that the expression profiles of the transcription factors FRUITFULL1 and APETALA2c during fruit development are comparable to previous reports using other tomato cultivars. PMID:27679646
Fowler, Veronica L; Ransburgh, Russell H; Poulsen, Elizabeth G; Wadsworth, Jemma; King, Donald P; Mioulet, Valerie; Knowles, Nick J; Williamson, Susanna; Liu, Xuming; Anderson, Gary A; Fang, Ying; Bai, Jianfa
Seneca Valley virus (SVV) can cause vesicular disease that is clinically indistinguishable from foot-and-mouth disease, vesicular stomatitis and swine vesicular disease. SVV-associated disease has been identified in pigs in several countries, namely USA, Canada, Brazil and China. Diagnostic tests are required to reliably detect this emerging virus, and this report describes the development and evaluation of a novel real-time reverse-transcription (RT) PCR assay (rRT-PCR), targeting the viral polymerase gene (3D) of SVV. This new assay detected all historical and contemporary SVV-1 isolates examined (n=8), while no cross-reactivity was observed with nucleic acid template prepared from other vesicular disease viruses or common swine pathogens. The analytical sensitivity of the rRT-PCR was 0.79 TCID50/ml and the limit of detect was equivalent using two different RT-PCR master-mixes. The performance of the test was further evaluated using pig nasal (n=25) and rectal swab samples (n=25), where concordant results compared to virus sequencing were generated for 43/50 samples. The availability of this assay, will enable laboratories to rapidly detect SVV in cases of vesicular disease in pigs, negated for notifiable diseases, and could enable existing knowledge gaps to be investigated surrounding the natural epidemiology of SVV.
Fowler, Veronica L; Ransburgh, Russell H; Poulsen, Elizabeth G; Wadsworth, Jemma; King, Donald P; Mioulet, Valerie; Knowles, Nick J; Williamson, Susanna; Liu, Xuming; Anderson, Gary A; Fang, Ying; Bai, Jianfa
Seneca Valley virus 1 (SVV-1) can cause vesicular disease that is clinically indistinguishable from foot-and-mouth disease, vesicular stomatitis and swine vesicular disease. SVV-1-associated disease has been identified in pigs in several countries, namely USA, Canada, Brazil and China. Diagnostic tests are required to reliably detect this emerging virus, and this report describes the development and evaluation of a novel real-time (r) reverse-transcription (RT) PCR assay (rRT-PCR), targeting the viral polymerase gene (3D) of SVV-1. This new assay detected all historical and contemporary SVV-1 isolates examined (n=8), while no cross-reactivity was observed with nucleic acid templates prepared from other vesicular disease viruses or common swine pathogens. The analytical sensitivity of the rRT-PCR was 0.79 TCID50/ml and the limit of detection was equivalent using two different rRT-PCR master-mixes. The performance of the test was further evaluated using pig nasal (n=25) and rectal swab samples (n=25), where concordant results compared to virus sequencing were generated for 43/50 samples. The availability of this assay, will enable laboratories to rapidly detect SVV-1 in cases of vesicular disease in pigs, negated for notifiable diseases, and could enable existing knowledge gaps to be investigated surrounding the natural epidemiology of SVV-1.
Zhou, Gang; Chiu, David; Qin, Dajiang; Niu, Lizhi; Cai, Jinlei; He, Lihua; Huang, Wenhao; Xu, Kecheng
The cell adhesion molecules CD44v6 and integrin-β1 are associated with the progression and metastasis of cancer. A novel triplex real-time reverse transcription polymerase chain reaction (qRT-PCR) assay was developed to quantify CD44v6 and integrin-β1 gene expression in peripheral blood mononuclear cells from 30 pancreatic cancer (PC) patients and 12 healthy individuals. The standard curve of the triplex qRT-PCR was constructed by optimizing the reaction condition and the amplification efficiency was 102.5, 101.1, and 100.6 % for CD44v6, integrin-β1 and endogenous gene (β-actin) amplification. Nonspecific bands were not observed from the triplex qRT-PCR amplification and the detection limit of this assay was 100 copies. Expression levels of CD44v6 and integrin-β1 gene were significantly lower in healthy individuals than PC patients (P<0.05). CD44v6 and integrin-β1 gene expression were not associated with the sex, age, and tumor position in PC (P>0.05). CD44v6 gene expression was significantly associated with clinical stage, liver metastasis, and tumor size (P<0.05). Integrin-β1 gene expression was significantly associated with clinical stage and liver metastasis (P<0.05). This triplex qRT-PCR assay may provide a useful tool for diagnosis, prognosis, and therapeutic evaluation in PC.
Donaldson, K.A.; Griffin, Dale W.; Paul, J.H.
A method was developed for the quantitative detection of pathogenic human enteroviruses from surface waters in the Florida Keys using Taqman (R) one-step Reverse transcription (RT)-PCR with the Model 7700 ABI Prism (R) Sequence Detection System. Viruses were directly extracted from unconcentrated grab samples of seawater, from seawater concentrated by vortex flow filtration using a 100kD filter and from sponge tissue. Total RNA was extracted from the samples, purified and concentrated using spin-column chromatography. A 192-196 base pair portion of the 5??? untranscribed region was amplified from these extracts. Enterovirus concentrations were estimated using real-time RT-PCR technology. Nine of 15 sample sites or 60% were positive for the presence of pathogenic human enteroviruses. Considering only near-shore sites, 69% were positive with viral concentrations ranging from 9.3viruses/ml to 83viruses/g of sponge tissue (uncorrected for extraction efficiency). Certain amplicons were selected for cloning and sequencing for identification. Three strains of waterborne enteroviruses were identified as Coxsackievirus A9, Coxsackievirus A16, and Poliovirus Sabin type 1. Time and cost efficiency of this one-step real-time RT-PCR methodology makes this an ideal technique to detect, quantitate and identify pathogenic enteroviruses in recreational waters. Copyright ?? 2002 Elsevier Science Ltd.
Wang, Ying; Chen, Yajuan; Ding, Liping; Zhang, Jiewei; Wei, Jianhua
The vertical segments of Populus stems are an ideal experimental system for analyzing the gene expression patterns involved in primary and secondary growth during wood formation. Suitable internal control genes are indispensable to quantitative real time PCR (qRT-PCR) assays of gene expression. In this study, the expression stability of eight candidate reference genes was evaluated in a series of vertical stem segments of Populus tomentosa. Analysis through software packages geNorm, NormFinder and BestKeeper showed that genes ribosomal protein (RP) and tubulin beta (TUBB) were the most unstable across the developmental stages of P. tomentosa stems, and the combination of the three reference genes, eukaryotic translation initiation factor 5A (eIF5A), Actin (ACT6) and elongation factor 1-beta (EF1-beta) can provide accurate and reliable normalization of qRT–PCR analysis for target gene expression in stem segments undergoing primary and secondary growth in P. tomentosa. These results provide crucial information for transcriptional analysis in the P. tomentosa stem, which may help to improve the quality of gene expression data in these vertical stem segments, which constitute an excellent plant system for the study of wood formation. PMID:27300480
Whiley, David M; Bialasiewicz, Seweryn; Bletchly, Cheryl; Faux, Cassandra E; Harrower, Bruce; Gould, Allan R; Lambert, Stephen B; Nimmo, Graeme R; Nissen, Michael D; Sloots, Theo P
Accurate and rapid diagnosis of novel influenza A(H1N1) infection is critical for minimising further spread through timely implementation of antiviral treatment and other public health based measures. In this study we developed two TaqMan-based reverse transcription PCR (RT-PCR) methods for the detection of novel influenza A(H1N1) virus targeting the haemagglutinin and neuraminidase genes. The assays were validated using 152 clinical respiratory samples, including 61 Influenza A positive samples, collected in Queenland, Australia during the years 2008 to 2009 and a further 12 seasonal H1N1 and H3N2 influenza A isolates collected from years 2000 to 2002. A wildtype swine H1N1 isolate was also tested. RNA from an influenza A(H1N1) virus isolate (Auckland, 2009) was used as a positive control. Overall, the results showed that the RT-PCR methods were suitable for sensitive and specific detection of novel influenza A(H1N1) RNA in human samples.
Kis, Z; Jones, J; Creanga, A; Ferdinand, K; Inui, K; Gerloff, N; Davis, C T; Nguyen, T; Donis, R O
Continued circulation and geographical expansion of highly pathogenic avian influenza H5N1 virus have led to the emergence of numerous clades in Vietnam. Although viral RNA sequencing and phylogenetic analysis are the gold standard for H5N1 HA clade designation, limited sequencing capacity in many laboratories precludes rapid H5N1 clade identification and detection of novel viruses. Therefore, a Taqman real-time RT-PCR assay for rapid differentiation of the four major H5N1 clades detected in Vietnam was developed. Using HA sequence alignments of clades 1.1, 220.127.116.11, 2.3.4, and 7 viruses, primers and FAM-labeled probes were designed to target conserved regions characteristic of each clade. The assay was optimized and evaluated using circulating clades of H5N1 collected in Vietnam from 2007 to 2012 and shown to be both sensitive and specific for the differentiation of the four H5N1 clades. The assay provides a useful tool for screening of large specimen collections for HA gene sequencing and phylogenetic analysis and for the rapid identification of molecular clade signatures to support outbreak investigations and surveillance activities. Finally, this assay may be useful to monitor for the emergence of novel or variant clades of H5N1 in Vietnam in the future or in other countries where these particular clades may circulate.
Chen, Huixin; Parimelalagan, Mariya; Lai, Yee Ling; Lee, Kim Sung; Koay, Evelyn Siew-Chuan; Hapuarachchi, Hapuarachchige C; Ng, Lee Ching; Ho, Phui San; Chu, Justin Jang Hann
Chikungunya virus (CHIKV) and dengue virus (DENV) have emerged as the two most important arbovirus diseases of global health significance. Similar clinical manifestations, transmission vectors, geographical distribution, and seasonal correlation often result in misdiagnosis of chikungunya infections as dengue cases and vice versa. In this study, we developed a rapid and accurate laboratory confirmative method to simultaneously detect, quantify, and differentiate DENV serotypes 1, 2, 3, and 4 and CHIKV. This SYBR Green I-based one-step multiplex real-time RT-PCR assay is highly sensitive and specific for CHIKV and DENV. Melting temperature analysis of PCR amplicons was used to serotype DENV and to differentiate from CHIKV. The detection limit of the assay was 20, 10, 50, 5, and 10 RNA copies/reaction for DENV-1, DENV-2, DENV-3, DENV-4, and CHIKV, respectively. Our assay did not cross-react with a panel of viruses that included other flaviviruses, alphaviruses, influenza viruses, human enteroviruses, and human coronaviruses. The feasibility of using this assay for clinical diagnosis was evaluated in DENV- and CHIKV-positive patient sera. Accordingly, the assay sensitivity for DENV-1, DENV-2, DENV-3, DENV-4, and CHIKV was 89.66%, 96.67%, 96.67%, 94.12%, and 95.74%, respectively, with 100% specificity. These findings confirmed the potential of our assay to be used as a rapid test for simultaneous detection and serotyping of DENV and CHIKV in clinical samples.
Jor, Evert; Myrmel, Mette; Jonassen, Christine M
A novel SYBR Green based real-time RT-PCR assay for detection of genogroup III bovine noroviruses (BoNoV) was developed and the assay applied to 419 faecal samples from calves with and without diarrhoea. The samples were obtained from 190 Norwegian dairy and beef herds. BoNoV was detected in 49.6% of the samples from 61.1% of the herds indicating that BoNoV is ubiquitous in Norway. The overall prevalence was not significantly different in diarrhoea and non-diarrhoea samples. Analyses of polymerase gene sequences revealed both genotype III/1 and III/2 with genotype III/2 (Newbury2-like) being the most prevalent. Detected capsid sequences were restricted to Newbury2-like and the chimeric Bo/Thirsk10/00/UK strain. The RNA polymerase genotypes of the circulating BoNoVs in Norway were predicted by melting temperature analysis. Additional data from a challenge experiment suggest that a high proportion of young calves are shedding low levels of BoNoV for a prolonged time after recovering from the associated diarrhoea. The findings may explain some of the discrepancies in detection rates from previous studies and explain why some studies have failed to detect significant prevalence differences between calves with and without diarrhoea. It may also shed new light on some epidemiological aspects of norovirus infections.
Nagy, Alexander; Vostinakova, Veronika; Pirchanova, Zuzana; Cernikova, Lenka; Dirbakova, Zuzana; Mojzis, Miroslav; Jirincova, Helena; Havlickova, Martina; Dan, Adam; Ursu, Krisztina; Vilcek, Stefan; Hornickova, Jitka
The objective of our study was to develop and evaluate a TaqMan real-time RT-PCR (RRT-PCR) assay for universal detection of influenza A (IA) viruses. The primers and LNA-modified octanucleotide probe were selected to correspond to extremely conserved regions of the membrane protein (MP) segment identified by a comprehensive bioinformatics analysis including 10,405 IA viruses MP sequences, i.e., all of the sequences of the Influenza Virus Sequence database collected as of August 20, 2009. The RRT-PCR has a detection limit of approximately five copies of target RNA/reaction and excellent reaction parameters tested in four IA viruses reference laboratories. The inclusivity of the assay was estimated at both the bioinformatic and the experimental level. Our results predicted that this RRT-PCR assay was able to detect 99.5% of known human IA virus strains, 99.84% of pandemic influenza A (H1N1) strains, 99.75% of avian strains, 98.89% of swine strains, 98.15% of equine strains, and 100% of influenza A viruses of other origin.
Sun, Bo-Guang; Hu, Yong-Hua
Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) has been used extensively for studying gene expression in diverse organisms including fish. In this study, with an aim to identify reliable reference genes for qRT-PCR in red drum (Sciaenops ocellatus), an economic fish species, we determined the expression stability of seven housekeeping genes in healthy and bacterium-infected red drum. Each of the selected candidate genes was amplified by qRT-PCR from the brain, gill, heart, intestine, kidney, liver, muscle, and spleen of red drum infected with or without a bacterial pathogen for 12 and 48 h. The mRNA levels of the genes were analyzed with the geNorm and NormFinder algorithms. The results showed that in the absence of bacterial infection, translation initiation factor 3, NADH dehydrogenase 1, and QM-like protein may be used together as internal references across the eight examined tissues. Bacterial infection caused variations in the rankings of the most stable genes in a tissue-dependent manner. For all tissues, two genes sufficed for reliable normalization at both 12 and 48 h post-infection. However, the optimal gene pairs differed among tissues and, for four of the examined eight tissues, between infection points. These results indicate that when studying gene expression in red drum under conditions of bacterial infection, the optimal reference genes should be selected on the basis of tissue type and, for accurate normalization, infection stage.
Osman, Fatima; Rowhani, Adib
An extraction technique for reverse transcription-PCR (RT-PCR) detection of plant pathogens including viruses, bacteria and phytoplasma is described. The total nucleic acid of these plant pathogens was obtained by direct spotting of crude sap derived from infected leaf, petiole or cambial tissue onto two different types of membranes, positively charged Hybond N(+) Nylon and FTA membranes, and processed for use in PCR. Thirteen different plant viruses, Xylella fastidiosa (causal agent of Pierce's disease) and phytoplasmas were included in the experiment. A thermal treatment (95 degrees C for 10 min) of the Hybond N(+) Nylon discs in a buffered solution improved the detection, but for FTA membrane discs the thermal treatment was not required and the discs were directly placed in the PCR reaction cocktail. Specific amplification of genomic or ribosomal RNA fragments of these pathogens was obtained by one-step RT-PCR except for X. fastidiosa in which a fragment of the genomic DNA was used for amplification. The same sample preparation methods also worked well for real-time RT-PCR (TaqMan). The sample preparation techniques reported here could be used to store samples for future PCR test or for long distance shipment to a detection laboratory.
A SYBR Green-based real-time RT-PCR assay for simple and rapid detection and differentiation of highly pathogenic and classical type 2 porcine reproductive and respiratory syndrome virus circulating in China.
Chai, Zheng; Ma, Wenjun; Fu, Fang; Lang, Yuekun; Wang, Wei; Tong, Guangzhi; Liu, Qinfang; Cai, Xuehui; Li, Xi
SYBR Green coupled to melting curve analysis has been suggested to detect RNA viruses showing high genomic variability. Here, a SYBR Green-based real-time RT-PCR assay was developed for simultaneous detection and differentiation of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) and classical type 2 PRRSV (C-PRRSV). The different strains were identified by their distinctive melting temperatures: 82.98 ± 0.25 °C and 85.95 ± 0.24 °C for HP-PRRSVs or 82.74 ± 0.26 °C for C-PRRSVs. Specificity was tested using nine other viral and bacterial pathogens of swine. The detection limit was 1 TCID(50) for HP- or C-PRRSV. Furthermore, the detection results for samples from an animal trial with HP- or C-PRRSV infections showed that the SYBR Green-based real-time RT-PCR was more sensitive than the conventional RT-PCR. Additionally, an analysis of 319 field samples from North China, Central China and Northeast China showed that HP- and C-PRRSVs co-circulated in pig herds. Thus, the SYBR Green-based real-time RT-PCR, which can be performed within one hour, is a rapid, sensitive and low-cost diagnostic tool for rapid differential detection and routine surveillance of HP- and classical type 2 PRRSVs in China.
Callison, Scott A; Hilt, Deborah A; Boynton, Tye O; Sample, Brenda F; Robison, Robert; Swayne, David E; Jackwood, Mark W
It is important to rapidly differentiate infectious bronchitis virus (IBV) from disease agents like highly pathogenic avian influenza virus and exotic Newcastle disease virus, which can be extremely similar in the early stages of their pathogenesis. In this study, we report the development and testing of a real-time RT-PCR assay using a Taqman-labeled probe for early and rapid detection of IBV. The assay amplifies a 143-bp product in the 5'-UTR of the IBV genome and has a limit of detection and quantification of 100 template copies per reaction. All 15 strains of IBV tested as well as two Turkey coronavirus strains were amplified, whereas none of the other pathogens examined, tested positive. Evaluation of the assay was completed with 1329 tracheal swab samples. A total of 680 samples collected from IBV antibody negative birds were negative for IBV by the real-time RT-PCR assay. We tested 229 tracheal swabs submitted to two different diagnostic laboratories and found 79.04% of the tracheal swabs positive for IBV by real-time RT-PCR, whereas only 27.51% of the samples were positive by virus isolation, which is the reference standard test. We also collected a total of 120 tracheal swabs at six different time points from birds experimentally infected with different dosages of IBV and found that, independent of the dose given, the viral load in the trachea plateau at 5 days post-inoculation. In addition, an inverse relationship between the dose of virus given and the viral load at 14 days post-inoculation was observed. Finally, we tested 300 total tracheal swab samples, from a flock of commercial broilers spray vaccinated for IBV in the field. The percentage of birds infected with the IBV vaccine at 3, 7, and 14 days post-vaccination was 58%, 65%, and 83%, respectively, indicating that only slightly more than half the birds were initially infected then the vaccine was subsequently transmitted to other birds in the flock. This observation is significant because
Picard-Meyer, Evelyne; Peytavin de Garam, Carine; Schereffer, Jean Luc; Marchal, Clotilde; Robardet, Emmanuelle; Cliquet, Florence
This study evaluates the performance of five two-step SYBR Green RT-qPCR kits and five one-step SYBR Green qRT-PCR kits using real-time PCR assays. Two real-time thermocyclers showing different throughput capacities were used. The analysed performance evaluation criteria included the generation of standard curve, reaction efficiency, analytical sensitivity, intra- and interassay repeatability as well as the costs and the practicability of kits, and thermocycling times. We found that the optimised one-step PCR assays had a higher detection sensitivity than the optimised two-step assays regardless of the machine used, while no difference was detected in reaction efficiency, R (2) values, and intra- and interreproducibility between the two methods. The limit of detection at the 95% confidence level varied between 15 to 981 copies/µL and 41 to 171 for one-step kits and two-step kits, respectively. Of the ten kits tested, the most efficient kit was the Quantitect SYBR Green qRT-PCR with a limit of detection at 95% of confidence of 20 and 22 copies/µL on the thermocyclers Rotor gene Q MDx and MX3005P, respectively. The study demonstrated the pivotal influence of the thermocycler on PCR performance for the detection of rabies RNA, as well as that of the master mixes.
Monavari, S H R; Mollaie, H R; Fazlalipour, M
Every year, seasonal epidemics of influenza viruses are causing considerable morbidity and mortality worldwide. Also infrequent novel and rearranged strains of influenza viruses have caused quick, acute universal pandemics resulting in millions of mortalities. The usage of efficient and accurate detection is superior for infection control, effective treatment, and epidemiological supervision. Therefore, evaluation of useful real-time PCR molecular tests for the detection of pandemic viruses is important before the next wave of the pandemic. A novel quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) assay with specific primers was used successfully for detection and monitoring of the influenza A, B, and swine influenza. The newly designed primers target highly conserved regions in influenza viruses. Our qRT-PCR assay is highly specific for detecting influenza A, B, and swine influenza viruses. The cutoff CT value was determined <38 for domestic human diagnostic test, under conditions of FDA emergency, and the reaction efficiency of the InfA, swInfA, and InfB assays were thereby estimated to be 97.9 % (R2 = 0.998), 98.3 % (R2 = 0.986), and 99.5 % (R2 = 0.995), respectively. Interestingly, based on our finding, there is no cross reactivity of detecting other viruses.
Jiménez-Clavero, Miguel Angel; Agüero, Montserrat; Rojo, Gema; Gómez-Tejedor, Concepción
West Nile virus represents an emerging threat for animal and human health worldwide. This virus exhibits a marked genetic variation, with at least 2 distinct evolutionary lineages. Lineage 1 has been recognized in Africa, Asia, Europe, Oceania, and more recently in the Americas, whereas lineage 2 is restricted to Africa. Perhaps for this reason, the available real-time RT-PCR methods for detecting West Nile virus genome have mainly focused on lineage 1. However, both viruses may potentially be spread beyond their endemic areas by migratory birds. This report describes a new real-time reverse transcription-PCR (RT-PCR) method based on a 5'-Taq nuclease-3' minor groove binder DNA probe (TaqMan MGB) that allows the detection of a wide range of West Nile virus isolates, including both lineages 1 and 2. This method was able to detect West Nile viruses from different origins (North and Central Africa, Middle East, Europe, and North America), whereas other flaviviruses (Usutu, Dengue, Yellow fever) analyzed in parallel remained negative. The sensitivity achieved by this assay was 10(-2)-10(-3) pfu/tube. This method, which can be performed in 96-well format, could be suitable for the large-scale surveillance of West Nile virus in areas where both lineages can potentially spread.
Hanon, J-B; Van der Stede, Y; Antonissen, A; Mullender, C; Tignon, M; van den Berg, T; Caij, B
Control of bovine viral diarrhoea (BVD) in Belgium is currently implemented on a voluntary basis at herd level and mainly relies on detection and culling of persistently infected (PI) animals. The present field study was conducted during the winter of 2010/2011 to assess the performances of diagnostic assays used in the testing scheme for BVD as proposed by the two Belgian regional laboratories. Individual blood samples were collected from 4972 animals, and individual samples from the same herd were pooled (maximum of 30 individual samples per pool) and screened for the presence of Bovine Viral Diarrhoea Virus (BVDV)-specific RNA using a commercial real-time RT-PCR test (ADIAGENE). Individual samples from positive pools were then tested in parallel with the same RT-PCR test and with an antigen-capture ELISA test (IDEXX) to detect viremic animals. This study demonstrated that individual results differed according to the type of assay used (P < 0.001): 140 animals (2.8%) were positive by RT-PCR and 72 (1.4%) by antigen-ELISA. A second blood sample was taken 40 days later from 74 PCR positive animals to detect persistent viremia: 17 (23%) of these were still PCR positive and considered to be PI and the 57 that no longer tested positive were assumed to be transiently infected (TI) animals. All PI animals were positive also by antigen-ELISA at both time points. Among TI animals, 10 (16%) were positive by antigen-ELISA at the first but none at the second sampling. A highly significant difference in cycle threshold (Ct ) values obtained by RT-PCR was observed between PI and TI animals. ROC analysis was performed to establish thresholds to confirm with high probability that an animal is PI, based on the result of RT-PCR test performed on a single individual blood sample.
Zeynalova, Shalala; Guliyev, Fizuli; Vatani, Mahira; Abbasov, Bahruz
The Azerbaijan State Veterinary Control Service (SVCS) has conducted active serological surveillance for avian influenza (AI) in poultry since 2006, when the first outbreak of AI H5N1 occurred in Azerbaijan. Samples are collected from September to May annually and tested using a hemagglutination inhibition (HI) assay to detect antibodies against H5 AI viruses. HI testing is also performed for Newcastle disease virus (NDV) upon request, but since this method cannot distinguish between natural infections and immune responses to vaccination, all positive results require follow-up epidemiological investigations. Furthermore, blood collection for the surveillance program is time-intensive and can be stressful to birds. In order to improve the national surveillance program, alternative sampling and testing methodologies were applied among a population of birds in the Barda region and compared with results of the national surveillance program. Tracheal and cloacal swabs were collected instead of blood. Rather than testing individual samples, RNA was pooled to conserve resources and time, and pools were tested by real-time reverse transcription polymerase chain reaction (rRT-PCR). Environmental sampling at a live bird market was also introduced as another surveillance mechanism. A total of 1,030 swabs were collected, comprising tracheal, and cloacal samples from 441 birds and 148 environmental surface samples from farms or the live bird market. During the same time, 3,890 blood samples were collected nationally for the surveillance program; 400 of these samples originated in the Barda region. Birds sampled for rRT-PCR were likely different than those tested as part of national surveillance. All swab samples tested negative by rRT-PCR for both AI and NDV. All blood samples tested negative for H5 by HI, while 6.2% of all samples and 5% of the Barda samples tested positive for exposure to NDV. Follow-up investigations found that positive samples were from birds vaccinated in
Volle, Romain; Nourrisson, Céline; Mirand, Audrey; Regagnon, Christel; Chambon, Martine; Henquell, Cécile; Bailly, Jean-Luc; Peigue-Lafeuille, Hélène; Archimbaud, Christine
Human enteroviruses are the most frequent cause of aseptic meningitis and are involved in other neurological infections. Qualitative detection of enterovirus genomes in cerebrospinal fluid is a prerequisite in diagnosing neurological diseases. The pathogenesis of these infections is not well understood and research in this domain would benefit from the availability of a quantitative technique to determine viral load in clinical specimens. This study describes the development of a real-time RT-qPCR assay using hydrolysis TaqMan probe and a competitive RNA internal control. The assay has high specificity and can be used for a large sample of distinct enterovirus strains and serotypes. The reproducible limit of detection was estimated at 1875 copies/ml of quantitative standards composed of RNA transcripts obtained from a cloned echovirus 30 genome. Technical performance was unaffected by the introduction of a competitive RNA internal control before RNA extraction. The mean enterovirus RNA concentration in an evaluation series of 15 archived cerebrospinal fluid specimens was determined at 4.78 log(10)copies/ml for the overall sample. The sensitivity and reproducibility of the real time RT-qPCR assay used in combination with the internal control to monitor the overall specimen process make it a valuable tool with applied research into enterovirus infections.
Cabrerizo, María; Calvo, Cristina; Rabella, Nuria; Muñoz-Almagro, Carmen; del Amo, Eva; Pérez-Ruiz, Mercedes; Sanbonmatsu-Gámez, Sara; Moreno-Docón, Antonio; Otero, Almudena; Trallero, Gloria
Human enteroviruses (EVs) and parechoviruses (HPeVs) are important etiological agents causing infections such as meningitis, encephalitis and sepsis-like disease in neonates and young children. We have developed a real-time RT-PCR for simultaneous detection of EV and HPeV in clinical samples. Primers and probe sets were designed from the conserved 5'-noncoding region of the genomes. The sensitivity, specificity and reproducibility of the technique were measured using a set of 25 EV and 6 HPeV types. All EVs but no HPeVs were detected with the EV primers-probe set. The HPeV primers-probe set detected only the 6 HPeV types. The lower detection limit was found to be 4 and 40CCID50/ml for HPeV and EV respectively, demonstrating high sensitivity of the technique for both viruses. The threshold cycle values were highly reproducible on repeat testing of positive controls among assay runs. The assay was evaluated in 53 clinical samples of suspected meningitis, sepsis or febrile syndromes from children under 3 years. In 11 of these (21%) EVs were detected, while 4, i.e. 7.5%, were HPeV positive. Molecular typing was carried out for 73% of the viruses. In summary, the RT-PCR method developed demonstrated effectively both EV and HPeV detection, which can cause similar clinical symptoms in infants.
Wen, Guoyuan; Zhang, Tao; Yang, Jun; Luo, Qingping; Liao, Yonghong; Hu, Zhibin; Zhang, Rongrong; Wang, Hongling; Ai, Diyun; Luo, Ling; Song, Nianhua; Shao, Huabin
A one-step real-time RT-PCR assay using a minor groove binding probe was developed for the specific detection of Chinese wild-type classical swine fever virus (CSFV). The assay detected wild-type CSFV strains representing different genotypes, but did not amplify viral RNA from the Hog Cholera Lipinized Virus (HCLV) vaccine-strain and other porcine viruses. The assay had a detection limit of 10 copies/reaction or 3.0 median tissue culture infective dose/reaction. In comparison to the sequencing nested RT-PCR assay, the sensitivity and specificity of the assay were 98.3% and 94.3%, respectively, when testing 515 veterinary samples. Wild-type CSFV RNA was detected in nasal swabs 2-4 days before detection in serum samples from pigs exposed to infection by contact, and 2-4 days prior to the onset of clinical disease. HCLV RNA remained undetectable in nasal swabs and serum samples from vaccinated pigs. In conclusion, the novel assay described in this study provides a rapid and sensitive method for differentiating between wild-type and the HCLV-strain of CSFV. It could be used for monitoring in CSF outbreak areas or as a screening method for CSFV eradication strategies.
Widén, F; Everett, H; Blome, S; Fernandez Pinero, J; Uttenthal, A; Cortey, M; von Rosen, T; Tignon, M; Liu, L
Classical swine fever is one of the most important infectious diseases for the pig industry worldwide due to its economic impact. Vaccination is an effective means to control disease, however within the EU its regular use is banned owing to the inability to differentiate infected and vaccinated animals, the so called DIVA principle. This inability complicates monitoring of disease and stops international trade thereby limiting use of the vaccine in many regions. The C-strain vaccine is safe to use and gives good protection. It is licensed for emergency vaccination in the EU in event of an outbreak. Two genetic assays that can distinguish between wild type virus and C-strain vaccines have recently been developed. Here the results from a comparison of these two real-time RT-PCR assays in an interlaboratory exercise are presented. Both assays showed similar performance.
Babu, Binoy; Jeyaprakash, Ayyamperumal; Jones, Debra; Schubert, Timothy S; Baker, Carlye; Washburn, Brian K; Miller, Steven H; Poduch, Kristina; Knox, Gary W; Ochoa-Corona, Francisco M; Paret, Mathews L
Rose rosette virus (RRV), belonging to the genus Emaravirus, is a highly destructive pathogen that causes rose rosette disease. The disease is a major concern for the rose industry in the U.S. due to the lack of highly sensitive methods for early detection of RRV. This is critical, as early identification of the infected plants and eradication is necessary in minimizing the risks associated with the spread of the disease. A highly reliable, specific and sensitive detection assay is thus required to test and confirm the presence of RRV in suspected plant samples. In this study a TaqMan real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of RRV from infected roses, utilizing multiple gene targets. Four pairs of primers and probes; two of them (RRV_2-1 and RRV_2-2) based on the consensus sequences of the glycoprotein gene (RNA2) and the other two (RRV_3-2 and RRV_3-5) based on the nucleocapsid gene (RNA3) were designed. The specificity of the primers and probes was evaluated against other representative viruses infecting roses, belonging to the genera Alfamovirus, Cucumovirus, Ilarvirus, Nepovirus, Tobamovirus, and Tospovirus and one Emaravirus (Wheat mosaic virus). Dilution assays using the in vitro transcripts (spiked with total RNA from healthy plants, and non-spiked) showed that all the primers and probes are highly sensitive in consistently detecting RRV with a detection limit of 1 fg. Testing of the infected plants over a period of time (three times in monthly intervals) indicated high reproducibility, with the primer/probe RRV_3-5 showing 100% positive detection, while RRV_2-1, RRV_2-2 and RRV_3-2 showed 90% positive detection. The developed real-time RT-PCR assay is reliable, highly sensitive, and can be easily used in diagnostic laboratories for testing and confirmation of RRV.
Suwannakarn, Kamol; Payungporn, Sunchai; Chieochansin, Thaweesak; Samransamruajkit, Rujipat; Amonsin, Alongkorn; Songserm, Thaweesak; Chaisingh, Arunee; Chamnanpood, Pornchai; Chutinimitkul, Salin; Theamboonlers, Apiradee; Poovorawan, Yong
In this study, a specific and sensitive one-step multiplex real-time RT-PCR was developed in two assays by using primers and a number of specific locked nucleic acid (LNA)-mediated TaqMan probes which increase the thermal stability of oligonucleotides. The first assay consisted of primers and probes specific to the matrix (M1) gene of influenza A virus, matrix (M1) gene of influenza B virus and GAPDH gene of host cells for typing of influenza virus and verification by an internal control, respectively. The other assay employed primers and probes specific to the hemagglutinin gene of H1, H3 and H5 subtypes in order to identify the three most prominent subtypes of influenza A capable of infecting humans. The specificity results did not produce any cross reactivity with other respiratory viruses or other subtypes of influenza A viruses (H2, H4 and H6-H15), indicating the high specificity of the primers and probes used. The sensitivity of the assays which depend on the type or subtype being detected was approximately 10 to 10(3)copies/microl that depended on the types or subtypes being detected. Furthermore, the assays demonstrated 100% concordance with 35 specimens infected with influenza A viruses and 34 specimens infected with other respiratory viruses, which were identified by direct nucleotide sequencing. In conclusion, the multiplex real-time RT-PCR assays have proven advantageous in terms of rapidity, specificity and sensitivity for human specimens and thus present a feasible and attractive method for large-scale detection aimed at controlling influenza outbreaks.
Lopez-Vazquez, C; Bandín, I; Dopazo, C P
In the present study, 2 systems of real-time RT-PCR-one based on SYBR Green and the other on TaqMan-were designed to detect strains from any genotype of viral haemorrhagic septicaemia virus (VHSV), with high sensitivity and repeatability/reproducibility. In addition, the method was optimized for quantitative purposes (qRT-PCR), and standard curves with different types of reference templates were constructed and compared. Specificity was tested against 26 isolates from 4 genotypes. The sensitivity of the procedures was first tested against cell culture isolation, obtaining a limit of detection (LD) of 100 TCID50 ml-1 (100-fold below the LD using cell culture), at a threshold cycle value (Ct) of 36. Sensitivity was also evaluated using RNA from crude (LD = 1 fg; 160 genome copies) and purified virus (100 ag; 16 copies), plasmid DNA (2 copies) and RNA transcript (15 copies). No differences between both chemistries were observed in sensitivity and dynamic range. To evaluate repeatability and reproducibility, all experiments were performed in triplicate and on 3 different days, by workers with different levels of experience, obtaining Ct values with coefficients of variation always <5. This fact, together with the high efficiency and R2 values of the standard curves, encouraged us to analyse the reliability of the method for viral quantification. The results not only demonstrated that the procedure can be used for detection, identification and quantification of this virus, but also demonstrated a clear correlation between the regression lines obtained with different standards, which will help scientists to compare sensitivity results between different studies.
Haines, Felicity J; Hofmann, Martin A; King, Donald P; Drew, Trevor W; Crooke, Helen R
A single-step, multiplex, real-time polymerase chain reaction (RT-PCR) was developed for the simultaneous and differential laboratory diagnosis of Classical swine fever virus (CSFV) and African swine fever virus (ASFV) alongside an exogenous internal control RNA (IC-RNA). Combining a single extraction methodology and primer and probe sets for detection of the three target nucleic acids CSFV, ASFV and IC-RNA, had no effect on the analytical sensitivity of the assay and the new triplex RT-PCR was comparable to standard PCR techniques for CSFV and ASFV diagnosis. After optimisation the assay had a detection limit of 5 CSFV genome copies and 22 ASFV genome copies. Analytical specificity of the triplex assay was validated using a panel of viruses representing 9 of the 11 CSFV subgenotypes, at least 8 of the 22 ASFV genotypes as well as non-CSFV pestiviruses. Positive and negative clinical samples from animals infected experimentally, due to field exposure or collected from the UK which is free from both swine diseases, were used to evaluate the diagnostic sensitivity and specificity for detection of both viruses. The diagnostic sensitivity was 100% for both viruses whilst diagnostic specificity estimates were 100% for CSFV detection and 97.3% for ASFV detection. The inclusion of a heterologous internal control allowed identification of false negative results, which occurred at a higher level than expected. The triplex assay described here offers a valuable new tool for the differential detection of the causative viruses of two clinically indistinguishable porcine diseases, whose geographical occurrence is increasingly overlapping.
Haines, Felicity J.; Hofmann, Martin A.; King, Donald P.; Drew, Trevor W.; Crooke, Helen R.
A single-step, multiplex, real-time polymerase chain reaction (RT-PCR) was developed for the simultaneous and differential laboratory diagnosis of Classical swine fever virus (CSFV) and African swine fever virus (ASFV) alongside an exogenous internal control RNA (IC-RNA). Combining a single extraction methodology and primer and probe sets for detection of the three target nucleic acids CSFV, ASFV and IC-RNA, had no effect on the analytical sensitivity of the assay and the new triplex RT-PCR was comparable to standard PCR techniques for CSFV and ASFV diagnosis. After optimisation the assay had a detection limit of 5 CSFV genome copies and 22 ASFV genome copies. Analytical specificity of the triplex assay was validated using a panel of viruses representing 9 of the 11 CSFV subgenotypes, at least 8 of the 22 ASFV genotypes as well as non-CSFV pestiviruses. Positive and negative clinical samples from animals infected experimentally, due to field exposure or collected from the UK which is free from both swine diseases, were used to evaluate the diagnostic sensitivity and specificity for detection of both viruses. The diagnostic sensitivity was 100% for both viruses whilst diagnostic specificity estimates were 100% for CSFV detection and 97.3% for ASFV detection. The inclusion of a heterologous internal control allowed identification of false negative results, which occurred at a higher level than expected. The triplex assay described here offers a valuable new tool for the differential detection of the causative viruses of two clinically indistinguishable porcine diseases, whose geographical occurrence is increasingly overlapping. PMID:23923045
Gruselle, Olivier; Coche, Thierry; Louahed, Jamila
Melanoma antigen A3 (MAGE-A3) is a member of the MAGE family of tumor antigens and a relevant candidate for use in cancer immunotherapy. However, not all tumors express MAGE-A3, and closely related members of the MAGE family can be co-expressed with MAGE-A3 in the same tumor. Therefore, in the frame of MAGE-A3 clinical trials, it appeared necessary to evaluate tumors for MAGE-A3 expression with a highly specific quantitative assay to select patients who are eligible for anti-MAGE-A3 immunotherapy treatment. Herein, we describe the development and validation of a quantitative real-time RT-PCR (RT-qPCR) assay for the determination of MAGEA3 gene expression in tumor tissues. In the early phases of development, the designed primers and probe were not able to distinguish between MAGE-A3 and MAGE-A6. To ensure the specificity for MAGE-A3 over MAGE-A6, our strategy was to use a 5'-nuclease probe (or hydrolysis probe). The final assay was shown to be specific and linear within the analytical range, with an acceptable CV for repeatability and intermediate precision. When compared with a reference semiquantitative RT-PCR assay, the two methods were in good agreement, with only 4.23% of the samples giving discordant results. In conclusion, we have developed a MAGE-A3-specific RT-qPCR assay, compatible with a high-throughput setting for the estimation of MAGEA3 gene expression in present and future clinical trials.
Mishima, Takuya; Mizuguchi, Yoshiaki; Kawahigashi, Yutaka; Takizawa, Takami; Takizawa, Toshihiro
More than 700 microRNAs (miRNAs) have been cloned, and the functions of these molecules in developmental timing, cell proliferation, and cancer have been investigated widely. MiRNAs are analyzed with Northern blot and sequential colony evaluation; however, reverse transcription-polymerase chain reaction (RT-PCR)-based miRNA assay remains to be developed. In this report, we describe improved real-time RT-PCR methods using specific or non-specific RT primer for the semi-quantitative analysis of miRNA expression. The use of the new methods in a model study revealed differential expression of miRNA-1 (miR-1) and miR-124 in mouse organs. Specifically, our methods revealed that miR-124 concentrations in the mouse central nervous system (CNS; cerebral cortex, cerebellum, and spinal cord) were more than 100 times those in other organs. By contrast, miR-1 expression in the CNS was 100-1000 times lower than that in skeletal muscle and heart. Furthermore, we revealed anatomically regional differences in miR-124 expression within the CNS: expression ratios versus the cerebral cortex were 60.7% for the cerebellum and 35.4% for the spinal cord. These results suggest that our RT-PCR-based methods would be a powerful tool for studies of miRNA expression that is associated with various neural events.
Sehata, Go; Sato, Hiroaki; Ito, Toshihiro; Imaizumi, Yoshitaka; Noro, Taichi; Oishi, Eiji
We used real-time RT-PCR and virus titration to examine canine distemper virus (CDV) kinetics in peripheral blood and rectal and nasal secretions from 12 experimentally infected dogs. Real-time RT-PCR proved extremely sensitive, and the correlation between the two methods for rectal and nasal (r=0.78, 0.80) samples on the peak day of viral RNA was good. Although the dogs showed diverse symptoms, viral RNA kinetics were similar; the peak of viral RNA in the symptomatic dogs was consistent with the onset of symptoms. These results indicate that real-time RT-PCR is sufficiently sensitive to monitor CDV replication in experimentally infected dogs regardless of the degree of clinical manifestation and suggest that the peak of viral RNA reflects active CDV replication.
Hull, Rene; Nattanmai, Seela; Kramer, Laura D.; Bernard, Kristen A.; Tavakoli, Norma P.
A duplex TaqMan real-time RT-PCR assay was developed for the detection of St. Louis encephalitis virus (SLEV) and Eastern equine encephalitis virus (EEEV), for use in human and vector surveillance. The respective targets selected for the assay were the conserved NS5 and E1 genes of the two viruses. Due to the insufficient number of NS5 sequences from SLEV strains in the GenBank database, we determined the sequence of an approximately 1-kb region for each of 25 strains of SLEV in order to select primers and probes in a conserved region. Our assay has a sensitivity of 5 gene copies/reaction for EEEV and 10 gene copies/reaction for SLEV, and it’s performance is linear over at least 6 log10 gene copies. The assay is specific and detected all strains of SLEV (69) and EEEV (12) that were tested. An internal control ensures detection of efficient nucleic acid extraction and possible PCR inhibition. PMID:18715737
Wernike, Kerstin; Beer, Martin; Hoffmann, Bernd
High sensitivity, minor risk of cross-contamination and in particular the rapid reaction time make quantitative real-time polymerase chain reaction (qPCR) assays well suited for outbreak investigations as well as for monitoring epidemics of pathogens. In this study qPCR assays for three highly contagious animal diseases, namely foot-and-mouth-disease (FMD), influenza A (IA) and classical swine fever (CSF) have been developed. Furthermore, an amplification control targeting 18S ribosomal RNA was included. Each assay was validated with samples from infected animals using three different standard qPCR-machines in two thermal profiles: one standard and one high-speed approach, respectively. The high-speed PCR assays allowed the reliable diagnosis of FMD, influenza A and CSF in less than 28 min with an analytical sensitivity of at least 200 genome copies/μl in every case, with slight differences regarding reaction time and sensitivity for the individual PCR-cycler instruments. Therefore, the newly established rapid RT-PCR systems will be a valuable method for the monitoring and control of these three important viruses and will be a robust option for the development of novel molecular pen-side tests.
Foord, Adam J; Selleck, Paul; Colling, Axel; Klippel, Jessica; Middleton, Deborah; Heine, Hans G
Equine influenza (EI) virus (H3N8) was identified in the Australian horse population for the first time in August 2007. The principal molecular diagnostic tool used for detection was a TaqMan real-time reverse transcription-polymerase chain reactions (RT-PCR) assay specific for the matrix (MA) gene of influenza virus type A (IVA). As this assay is not specific for EI, we developed a new EI H3-specific TaqMan assay targeting the haemagglutinin (HA) gene of all recent EI H3 strains. The IVA and the EI H3 TaqMan assays were assessed using in vitro transcribed RNA template, virus culture, diagnostic samples from the outbreak and samples from experimentally infected horses. The EI H3 TaqMan assay had a higher diagnostic sensitivity (DSe) when compared to the IVA TaqMan assay and also when using a conventional PCR for EI H3 as a standard of comparison. The performance of both TaqMan assays was compared with an antigen detection ELISA and virus isolation using nasal swabs collected daily from horses experimentally infected with the outbreak strain A/equine/Sydney/2888-8/2007. The EI H3 TaqMan assay was the most sensitive of the assays, able to detect EI from day 1 or 2 post-challenge, as early as virus isolation, and before clinical signs of disease were observed.
Gray-Mitsumune, M; Abe, H; Takahashi, J; Sundberg, B; Mellerowicz, E J
We explore a rapid in situ RT-PCR protocol for gene expression studies in woody stem tissues. In situ RT-PCR was performed using fluorescent dye-conjugated nucleic acid and the fluorescence signals derived from target RNAs were detected using confocal laser scanning microscopy. The signal to background ratio was greatly enhanced by performing two rounds of PCR reactions, first without the fluorescent dye and second with the dye. Using this protocol, we obtained strong gene-specific signals in secondary stem tissues. The signals were PCR-dependent, as shown by the lack of cytoplasmic signals in the tissue sections in which either DNA polymerase or primers were omitted from PCR reactions, and were RNA-dependent, as shown by great reduction of cytoplasmic signals when sections were treated with RNase before RT reactions. To verify our protocol, transcript localization of the rbcS gene was examined in secondary stems of hybrid aspen ( Populus tremula L. x tremuloides Michx.) and compared to the chlorophyll autofluorescence signal. The in situ RT-PCR signals form the rbcS gene and chlorophyll autofluorescence co-localized in the same cell types. The signal was also confirmed by Northern blot analysis of isolated RNA from the cambium and developing xylem, thus confirming the validity of the protocol. Some difficulties of in situ transcript localization and the interpretation of the signal distribution in the secondary tissues are discussed.
The probability of detecting influenza A virus (IAV) in oral fluid (OF) specimens was calculated for each of 13 real-time, reverse transcription polymerase chain reaction (rRT-PCR) and 7 virus isolation (VI) assays. To conduct the study, OF was inoculated with H1N1 or H3N2 IAV and serially 10-fold d...
Asian prunus viruses (APV 1, APV 2 and APV 3) and Plum bark necrosis stem pitting associated virus (PBNSPaV) are two recently described viruses infecting Prunus spp., and Peach latent mosaic viroid (PLMVd) is a viroid that infects the same species. A single-tube multiplex, TaqMan real-time RT-PCR as...
Otis, Jessica P; Ackermann, Laynez W; Denning, Gerene M; Carey, Hannah V
Previous work has suggested that central and peripheral opioid signaling are involved in regulating torpor behavior and tissue protection associated with the hibernation phenotype. We used quantitative real-time PCR (qRT-PCR) to measure mRNA levels of opioid peptide precursors and receptors in the brain and heart of summer ground squirrels (Ictidomys tridecemlineatus) and winter hibernating squirrels in the torpid or interbout arousal states. The use of appropriate reference genes for normalization of qRT-PCR gene expression data can have profound effects on the analysis and interpretation of results. This may be particularly important when experimental subjects, such as hibernating animals, undergo significant morphological and/or functional changes during the study. Therefore, an additional goal of this study was to identify stable reference genes for use in qRT-PCR studies of the 13-lined ground squirrel. Expression levels of 10 potential reference genes were measured in the small intestine, liver, brain, and heart, and the optimal combinations of the most stable reference genes were identified by the GeNorm Excel applet. Based on this analysis, we provide recommendations for reference genes to use in each tissue that would be suitable for comparative studies among different activity states. When appropriate normalization of mRNA levels was used, there were no changes in opioid-related genes in heart among the three activity states; in brain, DOR expression was highest during torpor, lowest in interbout arousal and intermediate in summer. The results support the idea that changes in DOR expression may regulate the level of neuronal activity in brain during the annual hibernation cycle and may contribute to hibernation-associated tissue protection.
Yang, Qi; Zou, Bo; Ren, Weibo; Ding, Yong; Wang, Zhen; Wang, Ruigang; Wang, Kai; Hou, Xiangyang
Stipa grandis P. Smirn. is a dominant plant species in the typical steppe of the Xilingole Plateau of Inner Mongolia. Selection of suitable reference genes for the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) is important for gene expression analysis and research into the molecular mechanisms underlying the stress responses of S. grandis. In the present study, 15 candidate reference genes (EF1 beta, ACT, GAPDH, SamDC, CUL4, CAP, SNF2, SKIP1, SKIP5, SKIP11, UBC2, UBC15, UBC17, UCH, and HERC2) were evaluated for their stability as potential reference genes for qRT-PCR under different stresses. Four algorithms were used: GeNorm, NormFinder, BestKeeper, and RefFinder. The results showed that the most stable reference genes were different under different stress conditions: EF1beta and UBC15 during drought and salt stresses; ACT and GAPDH under heat stress; SKIP5 and UBC17 under cold stress; UBC15 and HERC2 under high pH stress; UBC2 and UBC15 under wounding stress; EF1beta and UBC17 under jasmonic acid treatment; UBC15 and CUL4 under abscisic acid treatment; and HERC2 and UBC17 under salicylic acid treatment. EF1beta and HERC2 were the most suitable genes for the global analysis of all samples. Furthermore, six target genes, SgPOD, SgPAL, SgLEA, SgLOX, SgHSP90 and SgPR1, were selected to validate the most and least stable reference genes under different treatments. Our results provide guidelines for reference gene selection for more accurate qRT-PCR quantification and will promote studies of gene expression in S. grandis subjected to environmental stress. PMID:28056110
Zhao, Ye; Chen, Muyan; Wang, Tianming; Sun, Lina; Xu, Dongxue; Yang, Hongsheng
Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a technique that is widely used for gene expression analysis, and its accuracy depends on the expression stability of the internal reference genes used as normalization factors. However, many applications of qRT-PCR used housekeeping genes as internal controls without validation. In this study, the expression stability of eight candidate reference genes in three tissues (intestine, respiratory tree, and muscle) of the sea cucumber Apostichopus japonicus was assessed during normal growth and aestivation using the geNorm, NormFinder, delta CT, and RefFinder algorithms. The results indicate that the reference genes exhibited significantly different expression patterns among the three tissues during aestivation. In general, the β-tubulin (TUBB) gene was relatively stable in the intestine and respiratory tree tissues. The optimal reference gene combination for intestine was 40S ribosomal protein S18 (RPS18), TUBB, and NADH dehydrogenase (NADH); for respiratory tree, it was β-actin (ACTB), TUBB, and succinate dehydrogenase cytochrome B small subunit (SDHC); and for muscle it was α-tubulin (TUBA) and NADH dehydrogenase [ubiquinone] 1 α subcomplex subunit 13 (NDUFA13). These combinations of internal control genes should be considered for use in further studies of gene expression in A. japonicus during aestivation.
Kim, K; Katayama, H; Kitajima, M; Tohya, Y; Ohgaki, S
A method was developed for discriminating damaged viruses or naked viral RNA from intact viruses by ethidium monoazide (EMA) treatment before RT-PCR. The applied EMA treatment consisted of three steps: (1) EMA dose, (2) exposure to light, and (3) additional purification by spin-column gel filtration. Approximately 4-log reduction in viral RNA concentration was observed by adding a dose of 10 μg/mL-EMA with 300 s of light irradiation. Although residual EMA can be an inhibitor of RT-PCR, its effect was reduced by spin-column gel filtration or a QIAamp® Viral RNA Mini Kit. EMA-RT-PCR was applied to the thermally treated PV1. Results of EMA-RT-PCR were similar to the plaque assay when PV1 was thermally inactivated. Although this is a preliminary study investigating applicability of the EMA-RT-PCR method for RNA viruses, the results suggest that the method is potentially applicable for the selective detection of epidemiologically important enteric viruses in water such as enteroviruses and noroviruses.
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
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
Liu, L; Benyeda, Z; Zohari, S; Yacoub, A; Isaksson, M; Leijon, M; LeBlanc, N; Benyeda, J; Belák, S
Newcastle disease virus (NDV), also known as virulent forms of avian paramyxovirus serotype 1 (AMPV-1), is the causative agent of Newcastle disease affecting many species of birds and causing heavy losses to the poultry industry worldwide. Early, rapid and sensitive detection of the viruses or the viral nucleic acids is very important for disease diagnosis and control. This study aimed to evaluate sample preparation under field conditions and the application of a real-time RT-PCR method in the portable T-COR4 platform for the rapid, on-site detection of NDV on a farm. In the laboratory setting, the portable real-time RT-PCR assay had a similar performance compared with that obtained with a larger, stationary Rotor Gene real-time thermocycler. In the field conditions, viral nucleic acids were manually extracted just outside of animal units with minimal equipment and real-time RT-PCR detection was performed with the portable thermocycler T-COR4 placed in a nearby room. The portable assay at the farm detected viral RNA in 15 samples and reached an agreement of 83% (39/47) when the same RNA preparations were tested in the Rotor Gene thermocycler under the laboratory setting. The results demonstrated the feasibility of performing field detection but also the need to improve and further simplify sample preparation procedures.
Gautam, Rashi; Esona, Mathew D; Mijatovic-Rustempasic, Slavica; Ian Tam, Ka; Gentsch, Jon R; Bowen, Michael D
Group A rotaviruses (RVA) are the leading cause of severe diarrhea in young children worldwide. Two live-attenuated RVA vaccines, Rotarix(®) and RotaTeq(®) are recommended by World Health Organization (WHO) for routine immunization of all infants. Rotarix(®) and RotaTeq(®) vaccines have substantially reduced RVA associated mortality but occasionally have been associated with acute gastroenteritis (AGE) cases identified in vaccinees and their contacts. High-throughput assays are needed to monitor the prevalence of vaccine strains in AGE cases and emergence of new vaccine-derived strains following RVA vaccine introduction. In this study, we have developed quantitative real-time RT-PCR (qRT-PCR) assays for detection of Rotarix(®) and RotaTeq(®) vaccine components in stool samples. Real-time RT-PCR assays were designed for vaccine specific targets in the genomes of Rotarix(®) (NSP2, VP4) and RotaTeq(®) (VP6, VP3-WC3, VP3-human) and validated on sequence confirmed stool samples containing vaccine strains, wild-type RVA strains, and RVA-negative stools. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Rotarix(®) NSP2 and VP4 qRT-PCR assays exhibited 92-100% sensitivity, 99-100% specificity, 94-105% efficiency, and a limit of detection of 2-3 copies per reaction. RotaTeq(®) VP6, VP3-WC3, and VP3-human qRT-PCR assays displayed 100% sensitivity, 94-100% specificity, 91-102% efficiency and limits of detection of 1 copy, 2 copies, and 140 copies, respectively. These assays permit rapid identification of Rotarix(®) and RotaTeq(®) vaccine components in stool samples from clinical and surveillance studies and will be helpful in determining the frequency of vaccine strain-associated AGE.
Comparison of conventional RT-PCR, reverse-transcription loop-mediated isothermal amplification, and SYBR green I-based real-time RT-PCR in the rapid detection of bovine viral diarrhea virus nucleotide in contaminated commercial bovine sera batches.
Zhang, Shu-Qin; Tan, Bin; Li, Peng; Wang, Feng-Xue; Guo, Li; Yang, Yong; Sun, Na; Zhu, Hong-Wei; Wen, Yong-Jun; Cheng, Shi-Peng
Bovine viral diarrhea virus (BVDV) can contaminate biological products produced in bovine or porcine cells or manufactured using bovine sera. A rapid, specific, sensitive, and practical method of detecting BVDV in bio-products is needed. The purpose of this study was to compare three assays with respect to their ability to accurately detect BVDV in biological samples, namely reverse-transcription loop-mediated isothermal amplification (RT-LAMP), SYBR green I-based real-time RT-PCR, and conventional RT-PCR. All assays detected BVDV nucleotide and differentiated between BVDV-free and -contaminated bovine sera successfully. In addition, the results were specific to BVDV: the amplification of samples containing the closely related classical swine fever virus or other pathogenic bovine viruses yielded negative results. The lowest detection threshold, 10(1) copies, was displayed by the SYBR green I-based real-time RT-PCR and RT-LAMP assay. This assay was also the most effective in the detection of BVDV contamination in a set of commercially available bovine sera. The field conditions suggest that RT-LAMP is specific and sensitive to detecting BVDV in biological samples and may be used for quality control of biomaterials.
Murphy, Patricia G; Henderson, Dorian T; Adams, Melissa D; Horlick, Elizabeth A; Dixon, Eric P; King, Lorraine M; Avissar, Patricia L; Brown, Charlotte A; Fischer, Timothy J; Malinowski, Douglas P
This study was performed to demonstrate that RNA isolated from cell lines and cervical cytology specimens stored in SurePath preservative fluid would be functional in real-time RT-PCR assays. RNA was isolated from cervical cell lines or cytology samples stored in SurePath preservative at room temperature for 2-5 weeks using five commercially available RNA purification kits, three of which contain proteinases. The quality of the RNA was assessed by real time RT-PCR amplification of GAPDH, GUSB, U1A, HPV 16 and 18 E6 mRNAs. RNA was isolated successfully from cells that were stored in SurePath preservative fluid with only the three protocols that contained proteinases. GAPDH was amplified in 98-100% of the samples, GUSB in 90-98%, and the least abundant transcript, U1A, was amplified in 81-96% of the samples. HPV 16 and 18 E6 transcripts were detected in 56% of high grade, 39% of low grade and 2% of normal samples, with a concordance between DNA genotype and E6 mRNA expression of 97%. We demonstrated that RNA can be extracted from cervical cell lines and cytology specimens stored in BD SurePath preservative fluid with three different procedures that all contain proteinases. This RNA is suitable for real-time RT-PCR applications.
Lim, Stephanie M; Koraka, Penelope; Osterhaus, Albert D M E; Martina, Byron E E
Studying the tropism and replication kinetics of West Nile virus (WNV) in different cell types in vitro and in tissues in animal models is important for understanding its pathogenesis. As detection of the negative strand viral RNA is a more reliable indicator of active replication for single-stranded positive-sense RNA viruses, the specificity of qRT-PCR assays currently used for the detection of WNV positive and negative strand RNA was reassessed. It was shown that self- and falsely-primed cDNA was generated during the reverse transcription step in an assay employing unmodified primers and several reverse transcriptases. As a result, a qRT-PCR assay using the thermostable rTth in combination with tagged primers was developed, which greatly improved strand specificity by circumventing the events of self- and false-priming. The reliability of the assay was then addressed in vitro using BV-2 microglia cells as well as in C57/BL6 mice. It was possible to follow the kinetics of positive and negative-strand RNA synthesis both in vitro and in vivo; however, the sensitivity of the assay will need to be optimized in order to detect and quantify negative-strand RNA synthesis in the very early stages of infection. Overall, the strand-specific qRT-PCR assay developed in this study is an effective tool to quantify WNV RNA, reassess viral replication, and study tropism of WNV in the context of WNV pathogenesis.
Phelps, Nicholas B D; Patnayak, Devi P; Jiang, Yin; Goyal, Sagar M
Viral hemorrhagic septicemia virus (VHSV) is a highly contagious and pathogenic virus of fish. The virus infects more than 70 fish species worldwide, in both fresh and salt water. A new viral strain (VHSV-IVb) has proven both virulent and persistent, spreading throughout the Great Lakes of North America and to inland water bodies in the region. To better understand the geographic distribution of the virus, we used a modified real-time reverse transcription polymerase chain reaction (rRT-PCR) assay for high-throughput testing of fish for VHSV. The assay was shown to be twice as sensitive as the gold standard, virus isolation, and did not cross react with other viruses found in fish. In addition, the diagnostic turnaround time was reduced from 28 to 30 d for virus isolation to 2-4 d for rRT-PCR. To demonstrate the usefulness of the rRT-PCR assay, 115 high-priority water bodies in Minnesota were tested by both methods from April 2010 to June 2011. All survey sites tested negative for VHSV by both methods. The survey results have informed fisheries managers on the absence of VHSV in Minnesota and have better prepared them for the eventual arrival of the disease. In addition, the results demonstrate the value of this rRT-PCR as a surveillance tool to rapidly identify an outbreak so that it can be controlled in a timely manner.
Zhao, Jian-Jun; Cheng, Dan; Li, Na; Sun, Yuan; Shi, Zixue; Zhu, Qing-Hu; Tu, Changchun; Tong, Guang-Zhi; Qiu, Hua-Ji
Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), one of OIE listed diseases. Most of the currently available detection methods do not allow discrimination between wild-type CSF viruses and the vaccine strains. This study was designed to develop a multiplex real-time RT-PCR for the quantitative and differential detection of wild-type viruses and C-strain vaccine widely used in China. CSFV specific primers and two differently labeled TaqMan probes for the differentiation of wild-type viruses from C-strain vaccine were designed in the 5'-untranslated region of the viral genome of CSFV. The two TaqMan probes specifically hybridize wild-type viruses of different subgroups and C-strain vaccine, respectively, in the multiplex real-time RT-PCR, with no cross-reaction to a number of non-CSFV porcine viruses. The sensitivity of the assay for detecting wild-type and C-strain-type vaccine viruses was determined to be 41.8 and 81.5copies/microL viral RNA, respectively. Completely correct differentiation of wild-type viruses from C-strain vaccine was achieved when testing reference strains and characterized field isolates of CSFV in China. The multiplex real-time RT-PCR was able to detect the viral RNA in the whole blood samples of experimentally infected pigs as early as 2 days post-infection, 3 to 4 days prior to the onset of clinical signs in co-housed pigs. The agreements between the multiplex real-time RT-PCR and a multiplex RT-nested PCR for detection of wild-type and C-strain-type viruses were 96.9% and 100%, respectively, when detecting 106 different field samples. There is a positive correlation between the titers of C-strain vaccines titrated in rabbits and RNA copies quantitated by the multiplex real-time RT-PCR. The novel assay described here is rapid and sensitive, and is useful for differentiating field strains and C-strain of CSFV in China.
Vibrio parahaemolyticus is a significant human pathogen capable of causing foodborne gastroenteritis associated with the consumption of contaminated raw or undercooked seafood. Quantitative RT-PCR (qRT-PCR) is a useful tool for studying gene expression in V. parahaemolyticus to characterize the viru...
Mulholland, Catherine; McMenamy, Michael J; Hoffmann, Bernd; Earley, Bernadette; Markey, Bryan; Cassidy, Joseph; Allan, Gordon; Welsh, Michael D; McKillen, John
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.
Acevedo, Ana M; Perera, Carmen L; Vega, Armando; Ríos, Liliam; Coronado, Liani; Relova, Damarys; Frías, Maria T; Ganges, Llilianne; Núñez, José I; Pérez, Lester J
Infectious bronchitis is a highly contagious viral disease of poultry caused by infectious bronchitis virus (IBV) and is considered one of the most economically important viral diseases of chickens. Control of IBV has been attempted using live attenuated and inactivated vaccines. Live attenuated vaccines of the Massachusetts (Mass.) serotype are the most commonly used for this purpose. Due to the continuous emergence of new variants of the infectious bronchitis virus, the identification of the type of IBV causing an outbreak in commercial poultry is important in the selection of the appropriate vaccine(s) capable of inducing a protective immune response. The present work was aimed at developing and evaluating a duplex SYBR Green I-based real-time RT-PCR (rRT-PCR) assay for the simultaneous detection and differentiation of Mass. and non-Mass. serotypes of IBV. The duplex rRT-PCR yielded curves of amplification with two specific melting curves (Tm1 = 83 °C ± 0.5 °C and Tm2 = 87 °C ± 0.5 °C) and only one specific melting peak (Tm = 87 °C ± 0.5 °C) when the IBV Mass. serotype and IBV non-Mass. serotype strains were evaluated, respectively. The detection limit of the assay was 8.2 gene copies/μL based on in vitro transcribed RNA and 0.1 EID50/mL. The assay was able to detect all the IBV strains assessed and discriminated well among the IBV Mass. and the IBV non-Mass. serotypes strains. In addition, amplification curves were not obtained with any of the other viruses tested. From the 300 field samples tested, the duplex rRT-PCR yielded a total of 80 samples that were positive for IBV (26.67%), 73 samples identified as the IBV Mass. serotype and seven samples as identified as the IBV non-Mass. serotype. A comparison of the performance of test as assessed with field samples revealed that the duplex rRT-PCR detected a higher number of IBV-positive samples than when conventional RT-PCR or virus isolation tests were used. The duplex rRT-PCR presented here is a
Agarwal, Ankita; Singh, Anil K; Sharma, Shashi; Soni, Manisha; Thakur, Ashish K; Gopalan, N; Parida, M M; Rao, P V L; Dash, Paban K
Chikungunya has emerged as one of the most important arboviral infection of global significance. Expansion of Chikungunya virus endemic areas can be ascribed to naive population, increasing vector population and adaptability of virus to new vector. In this study, a SYBR Green I based quantitative RT-PCR assay was developed. The assay was found to be 10-fold more sensitive than conventional RT-PCR and no cross reactivity was observed with related alphaviruses and flaviviruses. The detection efficiency of the assay was impervious to mosquitoes of different pool sizes. Vector surveillance has resulted in detection of CHIKV RNA in Aedes aegypti, confirming its vectorial potential for CHIKV in northern India. The assessment of the assay was further carried out by studying the competence of Indian Ae. aegypti for CHIKV, which revealed 100% infection rate and dissemination rate with 60% transmission rate. The replication kinetics of CHIKV in different anatomical sites of Ae. aegypti revealed highest titre at day 6 post infection in midgut and at day 10 post infection in saliva, legs and wings. The implementation of the assay in detecting lower viral load makes it a remarkable tool for surveillance of virus activity in mosquitoes.
Reid, Scott M; Mioulet, Valerie; Knowles, Nick J; Shirazi, Nazeem; Belsham, Graham J; King, Donald P
Rapid and accurate diagnosis is essential for effective control of foot-and-mouth disease (FMD). In countries where FMD is endemic, identification of the serotypes of the causative virus strains is important for vaccine selection and tracing the source of outbreaks. In this study, real-time reverse transcription polymerase chain reaction (rRT-PCR) assays using primer/probe sets designed from the VP1 coding region of the virus genomes were developed for the specific detection of serotype O, A and Asia-1 FMD viruses (FMDVs) circulating in the Middle East. These assays were evaluated using representative field samples of serotype O strains belonging exclusively to the PanAsia-2 lineage, serotype A strains of the Iran-05 lineage and serotype Asia-1 viruses from three relevant sub-groups. When RNA extracted from archival and contemporary field strains was tested using one- or two-step rRT-PCR assays, all three primer/probe sets detected the RNA from homotypic viruses and no cross-reactivity was observed with heterotypic viruses. Similar results were obtained using both single- and multiplex assay formats. Using plasmid standards, the minimum detection level of these tests was found to be lower than two copies. The results illustrate the potential of tailored rRT-PCR tools for the detection and categorization of viruses circulating in the Middle East belonging to distinct subgroups of serotypes O, A and Asia-1. These assays can also overcome the problem of serotyping samples which are found positive by the generic rRT-PCR diagnostic assays but negative by virus isolation and antigen-detection ELISA which would otherwise have to be serotyped by nucleotide sequencing. A similar approach could be used to develop serotyping assays for FMDV strains circulating in other regions of the world.
Thomson, K G; Dietzgen, R G; Gibbs, A J; Tang, Y C; Liesack, W; Teakle, D S; Stackebrandt, E
A reverse transcription-polymerase chain reaction (RT-PCR) method was used to identify Zucchini yellow mosaic virus (ZYMV) in leaves of infected cucurbits. Oligonucleotide primers which annealed to regions in the nuclear inclusion body (NIb) and the coat protein (CP) genes, generated a 300-bp product from ZYMV and also from the closely related watermelon mosaic virus type 2 (WMV-2). However, no product was obtained from papaya ringspot potyvirus which also infects cucurbits. ZYMV and WMV-2 were differentiated using a third primer which was complementary to a sequence in the 3'-untranslated region; a 1186-bp amplified product was obtained for ZYMV only. Nucleotide sequence analysis of the 300-bp fragments of Australian ZYMV and WMV-2 strains revealed 93.7-100% sequence identity between ZYMV strains. Multiple sequence alignments indicated that the nucleotide sequence which codes for the N-terminus of the CP was 74-100% identical for different isolates of ZYMV. The Australian isolate of WMV-2 was 43-46% identical to all isolates of ZYMV and was 84.6% identical to a Florida isolate of WMV-2.
Background Normalizing through reference genes, or housekeeping genes, can make more accurate and reliable results from reverse transcription real-time quantitative polymerase chain reaction (qPCR). Recent studies have shown that no single housekeeping gene is universal for all experiments. Thus, suitable reference genes should be the first step of any qPCR analysis. Only a few studies on the identification of housekeeping gene have been carried on plants. Therefore qPCR studies on important crops such as cotton has been hampered by the lack of suitable reference genes. Results By the use of two distinct algorithms, implemented by geNorm and NormFinder, we have assessed the gene expression of nine candidate reference genes in cotton: GhACT4, GhEF1α5, GhFBX6, GhPP2A1, GhMZA, GhPTB, GhGAPC2, GhβTUB3 and GhUBQ14. The candidate reference genes were evaluated in 23 experimental samples consisting of six distinct plant organs, eight stages of flower development, four stages of fruit development and in flower verticils. The expression of GhPP2A1 and GhUBQ14 genes were the most stable across all samples and also when distinct plants organs are examined. GhACT4 and GhUBQ14 present more stable expression during flower development, GhACT4 and GhFBX6 in the floral verticils and GhMZA and GhPTB during fruit development. Our analysis provided the most suitable combination of reference genes for each experimental set tested as internal control for reliable qPCR data normalization. In addition, to illustrate the use of cotton reference genes we checked the expression of two cotton MADS-box genes in distinct plant and floral organs and also during flower development. Conclusion We have tested the expression stabilities of nine candidate genes in a set of 23 tissue samples from cotton plants divided into five different experimental sets. As a result of this evaluation, we recommend the use of GhUBQ14 and GhPP2A1 housekeeping genes as superior references for normalization of gene
Tewari, Deepanker; Kim, Hyun; Feria, Willard; Russo, Brigite; Acland, Helen
West Nile virus (WNV) RNA was quantified in WNV infected crows and horses with the help of a real-time reverse transcriptase-PCR assay. A 5' nuclease assay, based on NS5 gene detection with a fluorescent probe was used for quantifying WNV RNA using formalin fixed paraffin embedded tissue specimens. Quantitative detection of WNV RNA showed the presence of a higher amount of the viral RNA in crow tissues compared to equine tissues and these results correlated well with the detection of WNV antigen by immunostaining. In crows, the highest amount of virus was seen in the intestine and in horses in the brain.
Andergassen, Ulrich; Kölbl, Alexandra C; Mahner, Sven; Jeschke, Udo
Cells, which detach from a primary epithelial tumour and migrate through lymphatic vessels and blood stream are called 'circulating tumour cells'. These cells are considered to be the main root of remote metastasis and are correlated to a worse prognosis concerning progression-free and overall survival of the patients. Therefore, the detection of the minimal residual disease is of great importance regarding therapeutic decisions. Many different detection strategies are already available, but only one method, the CellSearch® system, reached FDA approval. The present review focusses on the detection of circulating tumour cells by means of real-time PCR, a highly sensitive method based on differences in gene expression between normal and malignant cells. Strategies for an enrichment of tumour cells are mentioned, as well as a large panel of potential marker genes. Drawbacks and advantages of the technique are elucidated, whereas, the greatest advantage might be, that by selection of appropriate marker genes, also tumour cells, which have already undergone epithelial to mesenchymal transition can be detected. Finally, the application of real-time PCR in different gynaecological malignancies is described, with breast cancer being the most studied cancer entity.
Ma, Yue-jiao; Sun, Xiao-hong; Xu, Xiao-yan; Zhao, Yong; Pan, Ying-jie; Hwang, Cheng-An; Wu, Vivian C. H.
Vibrio parahaemolyticus is a significant human pathogen capable of causing foodborne gastroenteritis associated with the consumption of contaminated raw or undercooked seafood. Quantitative RT-PCR (qRT-PCR) is a useful tool for studying gene expression in V. parahaemolyticus to characterize its virulence factors and understand the effect of environmental conditions on its pathogenicity. However, there is not a stable gene in V. parahaemolyticus that has been identified for use as a reference gene for qRT-PCR. This study evaluated the stability of 6 reference genes (16S rRNA, recA, rpoS, pvsA, pvuA, and gapdh) in 5 V. parahaemolyticus strains (O3:K6-clinical strain-tdh+, ATCC33846-tdh+, ATCC33847-tdh+, ATCC17802-trh+, and F13-environmental strain-tdh+) cultured at 4 different temperatures (15, 25, 37 and 42°C). Stability values were calculated using GeNorm, NormFinder, BestKeeper, and Delta CT algorithms. The results indicated that recA was the most stably expressed gene in the V. parahaemolyticus strains cultured at different temperatures. This study examined multiple V. parahaemolyticus strains and growth temperatures, hence the finding provided stronger evidence that recA can be used as a reference gene for gene expression studies in V. parahaemolyticus. PMID:26659406
Fondevila, Norberto; Compaired, Diego; Maradei, Eduardo; Duffy, Sergio
A specific real time reverse transcription polymerase chain reaction (RT-PCRrt) for the detection of foot-and-mouth disease virus was validated using the LightCycler thermocycler 2.0 and its reagents as recommended by the World Organization for Animal Health and was assessed for the detection of the virus in acute infection of cattle experimentally vaccinated and challenged with virus A Argentina/2001 or A24 Cruzeiro. The technique proved to be robust, showing coefficients of variation lower than 4% for different ARN extractions, days or repetitions and was able to detect up to 0,4 TCID 50%, and/or up to 100 RNA molecules. In probang samples, diagnostic sensitivity was 93.1 (95% CI 86.5-96.6) and diagnostic specificity 100 (95% CI 96.3-100). The results of the challenge in vaccinated or multivaccinated bovines showed that although there were high levels of clinical protection in the vaccinated group, FMDV could be detected in all challenged groups. However, detection was 100 times lower in immunized animals.
Sánchez-Matamoros, A; Kukielka, D; De las Heras, A I; Sánchez-Vizcaíno, J M
Cytokine secretion is one of the main mechanisms by which the immune system is regulated in response to pathogens. Therefore, the measurement of cytokine expression is fundamental to characterizing the immune response to infections. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) is widely used to measure cytokine mRNA levels, but assay conditions should be properly evaluated before analyzing important equine infections through relative quantification of gene expression. The aim of this study was to develop and evaluate a set of RT-qPCR assays for a panel of the most common cytokines in horses involved in innate and adaptive immune responses. Eight cytokines (interleukin (IL)-1β, IL-2, IL-4, IL-10, IL-12, TNFα, IFNβ and IFNγ) and a housekeeping gene (β-actin) were detected and amplified with the same annealing temperature in a SYBR Green RT-qPCR assay of samples of mitogen-stimulated peripheral blood mononuclear cells from a healthy horse and whole blood from a horse infected with African horse sickness virus. The method gave good efficiency for all genes tested, allowing quantification of relative expression levels. These SYBR Green RT-qPCR assays may be useful for examining cytokine gene expression in horses in response to exposure to economically important pathogens.
Gu, Chun-Sun; Liu, Liang-qin; Xu, Chen; Zhao, Yan-hai; Zhu, Xu-dong; Huang, Su-Zhen
Quantitative real time PCR (RT-qPCR) has emerged as an accurate and sensitive method to measure the gene expression. However, obtaining reliable result depends on the selection of reference genes which normalize differences among samples. In this study, we assessed the expression stability of seven reference genes, namely, ubiquitin-protein ligase UBC9 (UBC), tubulin alpha-5 (TUBLIN), eukaryotic translation initiation factor (EIF-5A), translation elongation factor EF1A (EF1 α ), translation elongation factor EF1B (EF1b), actin11 (ACTIN), and histone H3 (HIS), in Iris. lactea var. chinensis (I. lactea var. chinensis) root when the plants were subjected to cadmium (Cd), lead (Pb), and salt stress conditions. All seven reference genes showed a relatively wide range of threshold cycles (C t ) values in different samples. GeNorm and NormFinder algorithms were used to assess the suitable reference genes. The results from the two software units showed that EIF-5A and UBC were the most stable reference genes across all of the tested samples, while TUBLIN was unsuitable as internal controls. I. lactea var. chinensis is tolerant to Cd, Pb, and salt. Our results will benefit future research on gene expression in response to the three abiotic stresses.
Abreu, Paolla M V; Piccin, João G; Rodrigues, Silas P; Buss, David S; Ventura, José A; Fernandes, Patricia M B
Papaya meleira virus (PMeV) is the causal agent of papaya sticky disease. This study describes two methods for molecular diagnosis of PMeV using conventional and real-time PCR. These methods were shown to be more efficient than current methods of viral detection using extraction of PMeV dsRNA and observation of symptoms in the field. The methods described here were used to evaluate the effect of inoculation of papaya plants with purified PMeV dsRNA on the progress of PMeV infection. A single inoculation with PMeV dsRNA was observed to delay the progress of the virus infection by several weeks. The possibility of vertical transmission of PMeV was also investigated. No evidence was found for PMeV transmission through seeds collected from diseased fruit. The implications of these results for the epidemiology of PMeV and the management of papaya sticky disease are discussed.
Vina-Rodriguez, Ariel; Eiden, Martin; Keller, Markus; Hinrichs, Winfried; Groschup, Martin H
Venezuelan equine encephalitis virus (VEEV) is an Alphavirus from the family Togaviridae that causes epizootic outbreaks in equids and humans in Central and South America. So far, most studies use conventional reverse transcriptase PCR assays for the detection of the different VEEV subtypes. Here we describe the development of a TaqMan quantitative real-time reverse transcriptase PCR assay for the specific detection and quantitation of all VEEV subtypes which uses in parallel a universal equine encephalitis virus control RNA carrying target sequences of the three equine encephalitis viruses. The control RNA was used to generate standard curves for the calculation of copy numbers of viral genome of Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), and VEEV. The new assay provides a reliable high-throughput method for the detection and quantitation of VEEV RNA in clinical and field samples and allows a rapid differentiation from potentially cocirculating EEEV and WEEV strains. The capability to detect all known VEEV variants was experimentally demonstrated and makes this assay suitable especially for the surveillance of VEEV.
Vina-Rodriguez, Ariel; Eiden, Martin; Keller, Markus; Hinrichs, Winfried
Venezuelan equine encephalitis virus (VEEV) is an Alphavirus from the family Togaviridae that causes epizootic outbreaks in equids and humans in Central and South America. So far, most studies use conventional reverse transcriptase PCR assays for the detection of the different VEEV subtypes. Here we describe the development of a TaqMan quantitative real-time reverse transcriptase PCR assay for the specific detection and quantitation of all VEEV subtypes which uses in parallel a universal equine encephalitis virus control RNA carrying target sequences of the three equine encephalitis viruses. The control RNA was used to generate standard curves for the calculation of copy numbers of viral genome of Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), and VEEV. The new assay provides a reliable high-throughput method for the detection and quantitation of VEEV RNA in clinical and field samples and allows a rapid differentiation from potentially cocirculating EEEV and WEEV strains. The capability to detect all known VEEV variants was experimentally demonstrated and makes this assay suitable especially for the surveillance of VEEV. PMID:28042576
Slomka, M J; Irvine, R M; Pavlidis, T; Banks, J; Brown, I H
Diagnosis and management of avian influenza outbreaks now include the use of validated real-time reverse transcription PCR (RRT-PCR) methods in many countries, including all member states of the European Union. Two outbreaks in poultry of notifiable avian influenza (H5 and H7 subtypes) that occurred in Great Britain during 2007 will serve as examples in which RRT-PCR demonstrated its value in 1) rapid diagnosis and confirmation of disease by sensitive and specific laboratory testing of samples derived from the index cases and 2) high-volume, rapid testing of surveillance samples. The two poultry outbreaks followed the incursion of a H7N2 low-pathogenicity notifiable avian influenza (LPNAI) virus (May-June 2007) and a Eurasian lineage H5N1 highly pathogenic notifiable avian influenza (HPNAI) virus (November 2007). Coupled with the use of high-throughput, robotic RNA extraction methods, a total of approximately 9300 and 20,300 field samples were tested by appropriate, validated RRT-PCR assays during the 4- and 5-wk duration of the H7N2 LPNAI and H5N1 HPNAI outbreaks, respectively. Fundamental features of the validated RRT-PCR assays used included their high degree of sensitivity, specificity, and rapidity, attributes that were invaluable in providing timely and accurate information for notifiable AI outbreak management.
Velloso, Alvaro Jorge; da Silva Junior, Haroldo Cid; Santos, Eneida Almeida; Baroni, Eliane Barbosa; de Moraes, Marcia Terezinha Baroni
Cells from various tissues and species are able to bind to Theiler's virus strain DA and allow it to replicate to some extent. Meanwhile, permissiveness in vitro to BeAn strains has not been well investigated. In this paper, the BeAn 8386 virus was subjected to five passages in BHK-21 cells and showed a persistent profile. In order to follow the in vitro infection, real-time RT-PCR to detect the IRES, L* and 3A3B regions of the Theiler's virus genome was carried out in the first and last passages. In addition, the expression of L* protein was detected. These findings confirm the persistence of the virus in vitro, even in the absence of cytopathic effect (CPE).
Salez, N; Ninove, L; Thirion, L; Gazin, C; Zandotti, C; de Lamballerie, X; Charrel, R N
Rapid documentation of respiratory specimens can have an impact on the management of patients and their relatives in terms of preventive and curative measures. We compared the results of the Xpert(®) Flu assay (Cepheid) with three real-time RT-PCR assays using 127 nasopharyngeal samples, of which 75 were positive for influenza A (with 52 identified as A/H1N1-2009) and 52 were positive for influenza B. The Xpert(®) Flu assay presented a quasi-absence of non-interpretable tests, and showed sensitivity and specificity of 100% and 100% for Flu A, 98.4% and 100% for A/H1N1-2009, and 80.7% and 100% for Flu B.
Gangisetty, Omkaram; Reddy, Doodipala Samba
The GABA-A receptor plays a critical role in inhibitory neurotransmission in the brain. Quantitation of GABA-A receptor subunits in various brain regions is essential to understand their role in plasticity and brain disorders. However, conventional RNA assays are tedious and less sensitive for use in studies of subunit plasticity. Here we describe optimization of a sensitive assay of GABA-A receptor subunit gene expression by TaqMan real-time PCR. For each subunit gene, a set of primers and TaqMan fluorogenic probe were designed to specifically amplify the target template. The TaqMan methodology was optimized for quantification of mouse GABA-A receptor subunits (α1–6, β1–3, γ2, and δ) and GAPDH. The TaqMan reaction detected very low levels of gene expression (~100 template copies of cDNA). A standard curve for GAPDH and one of the target genes, constructed using the cDNA, revealed slopes around −3.4 (r2=0.990), reflecting similar optimum PCR efficiencies. The methodology was utilized for quantification of the GABA-A receptor α4 subunit, which is known to upregulate following withdrawal from chronic progesterone or neurosteroids. Our results show that the α4-subunit expression increased threefold in the hippocampus following neurosteroid withdrawal in mice. The TaqMan PCR assay allows sensitive, high-throughput transcriptional profiling of complete GABA-A receptor subunit family, and thus provides specific tool for studies of GABA-A receptor subunit plasticity in neurological and psychiatric animal models. PMID:19406150
Reference Gene Selection for Quantitative Real-Time RT-PCR Normalization in the Half-Smooth Tongue Sole (Cynoglossus semilaevis) at Different Developmental Stages, in Various Tissue Types and on Exposure to Chemicals
Liu, Conghui; Xin, Nian; Zhai, Yi; Jiang, Liming; Zhai, Jieming; Zhang, Quanqi; Qi, Jie
Quantitative real time RT-PCR has been described as the most sensitive method for the detection of low abundance mRNA. To date, no reference genes have been screened in the half-smooth tongue sole (Cynoglossus semilaevis). The aim of this study was to select the most stable genes for quantitative real-time RT-PCR. Eight housekeeping genes (18S, TUBA, B2M, ACTB, EF1A, GAPDH, RPL17 and UBCE) were tested at different developmental stages, in different tissues, and following exposure to the drug SB-431542. Using geNorm, BestKeeper and NormFinder software, GAPDH/B2M, GAPDH/18S and UBCE/GAPDH were identified as the most suitable genes from samples taken of different developmental stages while 18S/RPL17 were consistently ranked as the best reference genes for different tissue types. Furthermore, TUBA/B2M, TUBA/UBCE and B2M/TUBA were found to be the most suitable genes in samples treated with the drug, SB-431542 by geNorm, BestKeeper and NormFinder respectively. Across both different developmental stages and tissue types, the combination of 18S and GAPDH was the most stable reference gene analyzed by Ref-Finder. To test and verify the screened reference genes, the expression profiles of LEFTY-normalized to the combination of GAPDH/18S and ACTB were presented. These results will be useful for future gene-expression studies in the half-smooth tongue sole. PMID:24667563
Taylor-Harris, Pamela M; Felkin, Leanne E; Birks, Emma J; Franklin, Rodney C G; Yacoub, Magdi H; Baines, Anthony J; Barton, Paul J R; Pinder, Jennifer C
The proteins, spectrin and 4.1 confer support and resilience to animal cell membranes, and promote assembly of multimeric, membrane-bound signalling complexes. Protein 4.1 also plays important roles in tumour suppression and the regulation of cell proliferation. To assess relative tissue expression of the four genes encoding human protein 4.1, we measured mRNA levels using quantitative real-time polymerase chain reaction. We compared 4.1 expression with that of a major splice variant of spectrin, betaIISigma2 that has a shortened C-terminus lacking a pleckstrin homology domain. mRNA for 4.1R is four-fold higher in bone marrow than in tissues with the next highest prevalence: cerebellum, lung, testis and thymus. 4.1G mRNA is highly expressed in brain, spinal cord and testis; 4.1N in brain, spinal cord and adrenal gland; 4.1B in testis, brain, spinal cord, and kidney. Thus, 4.1N, 4.1B and 4.1G all show high accumulation in nervous tissues. mRNA for betaIISigma2-spectrin is ubiquitous, but most abundant in cardiac and nervous tissues. Comparative transcript abundance was analysed in heart and brain. betaIISigma2-spectrin was the most abundant transcript in heart with levels 5 fold greater than 4.1G or 4.1N and at least 9 fold greater than 4.1B. In brain, 4.1N was the most abundant transcript, with levels 2.4 fold greater than 4.1B and at least 4 fold greater than 4.1G or betaIISigma2-spectrin. 4.1R abundance was very low in both tissues. Whilst we expected that 4.1 mRNAs would feature highly in muscle and nerve, we note their high abundance in testis, indicating previously unsuspected functions in reproduction.
Kong, Qiusheng; Yuan, Jingxian; Gao, Lingyun; Zhao, Shuang; Jiang, Wei; Huang, Yuan; Bie, Zhilong
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.
Ma, Kang-Sheng; Li, Fen; Liang, Ping-Zhuo; Chen, Xue-Wei; Liu, Ying; Gao, Xi-Wu
To obtain accurate and reliable results from quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis, it is necessary to select suitable reference genes as standards for normalizing target gene expression data. QRT-PCR is a popular analytical methodology for studying gene expression and it has been used widely in studies of Aphis gossypii Glover in recent years. However, there is absence of study on the stability of the expression of reference genes in A. gossypii. In this study, eight commonly used candidate reference genes, including 18S, 28S, β-ACT, GAPDH, EF1α, RPL7, α-TUB, and TBP, were evaluated under various experimental conditions to assess their suitability for use in the normalization of qRT-PCR data. The optimal number of reference genes was determined using the geNorm program, and the suitability of particular reference genes was empirically validated by performing normalizations of expression data for the HSP70 gene. The results showed the most suitable combinations of reference genes for the different experimental conditions. For experiments based on divergent developmental stages, EF1α, β-ACT, and RPL7 are the optimal reference gene combination, both EF1α and β-ACT are the optimal combination used in the experiments of different geographical populations, whereas for experiments of the temperature changes, the combination of GAPDH and RPL7 is optimal, both 18S and β-ACT are an optimal combination for feeding assay experiments. These research results should be useful for the selection of the suitable reference genes to obtain reliable qRT-PCR data in the gene expression study of A. gossypii.
Uccella, S; Cromi, A; Vigetti, D; Cimetti, L; Deleonibus, S; Casarin, J; Passi, A; Riva, C; Ghezzi, F
We investigated whether endometrial cancer (EC) cells can express fibrinogen. Consecutive patients treated for EC were enrolled (cases). A control group of women who had hysterectomy for benign conditions was identified in a case:control ratio of 4:1. Immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) were performed to identify the presence of fibrinogen and the mRNA of its three chains (α, β, γ) in the tissue specimens from both cases and controls. Sixteen EC cases and 4 benign controls were included. Immunohistochemistry failed in one case of EC. In 12/15 (80%) cases versus 0 controls, a moderate-to-intense positivity for fibrinogen was observed (p = 0.09; OR: 32.1; 95%CI: 1.4-752.9). Six (37.5%) women among the cases versus 0 controls expressed RNA for at least one chain of fibrinogen (p = 0.25). All the cases (6/6, 100%) with positive RT-PCR had moderate-to-intense positive immunohistochemistry. Molecular and immunohistochemistry show that some cases of EC have the capability to express fibrinogen and the mRNA of at least one of its chains.
Fernández-Aparicio, M; Huang, K; Wafula, E K; Honaas, L A; Wickett, N J; Timko, M P; Depamphilis, C W; Yoder, J I; Westwood, J H
Striga is a root parasitic weed that attacks many of the staple crops in Africa, India and Southeast Asia, inflicting tremendous losses in yield and for which there are few effective control measures. Studies of parasitic plant virulence and host resistance will be greatly facilitated by the recent emergence of genomic resources that include extensive transcriptome sequence datasets spanning all life stages of S. hermonthica. Functional characterization of Striga genes will require detailed analyses of gene expression patterns. Quantitative real-time PCR is a powerful tool for quantifying gene expression, but correct normalization of expression levels requires identification of control genes that have stable expression across tissues and life stages. Since no S. hermonthica housekeeping genes have been established for this purpose, we evaluated the suitability of six candidate housekeeping genes across key life stages of S. hermonthica from seed conditioning to flower initiation using qRT-PCR and high-throughput cDNA sequencing. Based on gene expression analysis by qRT-PCR and RNA-Seq across heterogeneous Striga life stages, we determined that using the combination of three genes, UBQ1, PP2A and TUB1 provides the best normalization for gene expression throughout the parasitic life cycle. The housekeeping genes characterized here provide robust standards that will facilitate powerful descriptions of parasite gene expression patterns.
Ciafaloni, E.; Silva, H.A.R. de; Roses, A.D.
Duchenne and Becker muscular dystrophies (DMD, BMD) are X-linked recessive disorders caused by mutations in the dystrophin (dys) gene. The majority of these mutations are intragenic deletions of duplications routinely detected by Southern biots and multiplex PCR. The remainder are very likely, smaller mutations, mostly point-mutations. Detection of these mutations is very difficult due to the size and complexity of the dys gene. We applied RT-PCR to analyse the entire dys mRNA of three DMD patients with no detectable genomic defect. In two unrelated patients, a duplication of the 62 bp exon 2 was identified. This causes a frameshift sufficient to explain the DMD phenotype. In the third patient, who had congenital DMD and severe mental retardation, a complex pattern of aberrant splicing at the 3-prime exons 67-79 was observed. Sural nerve biopsy in this patient showed the complete absence of Dp116. PCR-SSCP studies are presently in progress to identify the mutations responsible for the aberrant splicing patterns.
The analysis component of the Enrichment Diagnostic System (EDS) developed for the Atomic Vapor Laser Isotope Separation Program (AVLIS) at Lawrence Livermore National Laboratory (LLNL) is described. Four different types of analysis are performed on data acquired through EDS: (1) absorption spectroscopy on laser-generated spectral lines, (2) mass spectrometer analysis, (3) general purpose waveform analysis, and (4) separation performance calculations. The information produced from this data includes: measures of particle density and velocity, partial pressures of residual gases, and overall measures of isotope enrichment. The analysis component supports a variety of real-time modeling tasks, a means for broadcasting data to other nodes, and a great degree of flexibility for tailoring computations to the exact needs of the process. A particular data base structure and program flow is common to all types of analysis. Key elements of the analysis component are: (1) a fast access data base which can configure all types of analysis, (2) a selected set of analysis routines, (3) a general purpose data manipulation and graphics package for the results of real time analysis.
This paper describes the analysis component of the Enrichment Diagnostic System (EDS) developed for the Atomic Vapor Laser Isotope Separation Program (AVLIS) at Lawrence Livermore National Laboratory (LLNL). Four different types of analysis are performed on data acquired through EDS: (1) absorption spectroscopy on laser-generated spectral lines, (2) mass spectrometer analysis, (3) general purpose waveform analysis, and (4) separation performance calculations. The information produced from this data includes: measures of particle density and velocity, partial pressures of residual gases, and overall measures of isotope enrichment. The analysis component supports a variety of real-time modeling tasks, a means for broadcasting data to other nodes, and a great degree of flexibility for tailoring computations to the exact needs of the process. A particular data base structure and program flow is common to all types of analysis. Key elements of the analysis component are: (1) a fast access data base which can configure all types of analysis, (2) a selected set of analysis routines, (3) a general purpose data manipulation and graphics package for the results of real time analysis. Each of these components are described with an emphasis upon how each contributes to overall system capability. 3 figs.
Bachofen, Claudia; Willoughby, Kim; Zadoks, Ruth; Burr, Paul; Mellor, Dominic; Russell, George C
Studies of the molecular epidemiology of viral diseases are dependent on the analysis of large numbers of samples from infected individuals, and the assembly of relevant sequence databases are a prerequisite to investigate chains of infection. As part of research in support of the Scottish BVDV eradication campaign, we have established a direct RT-PCR method for the high throughput amplification and analysis of the informative 5'-untranslated region of the BVDV genome. Heat-treatment followed by a one-step RT-PCR, performed in 96-well plates, produced sufficient material for sequence analysis from 0.5 μl of serum or plasma. Of 93 samples assayed, only five failed to give full sequence data for the region amplified and these were subsequently successfully analysed in single tube format reactions. This approach improved the speed of analysis, reduced costs, operator time and the potential for contamination, and may allow analysis of samples for which volumes are too low for conventional RNA isolation. It also has the potential for wider application in both human and animal disease research in which high throughput and low cost would increase the size of datasets that can be obtained.
Petrov, Anja; Beer, Martin; Blome, Sandra
Dysregulation of cytokine responses plays a major role in the pathogenesis of severe and life-threatening infectious diseases like septicemia or viral hemorrhagic fevers. In pigs, diseases like African and classical swine fever are known to show exaggerated cytokine releases. To study these responses and their impact on disease severity and outcome in detail, reliable, highly specific and sensitive methods are needed. For cytokine research on the molecular level, real-time RT-PCRs have been proven to be suitable. Yet, the currently available and most commonly used SYBR Green I assays or heterogeneous gel-based RT-PCRs for swine show a significant lack of specificity and sensitivity. The latter is however absolutely essential for an accurate quantification of rare cytokine transcripts as well as for detection of small changes in gene expressions. For this reason, a harmonized TaqMan-based triplex real-time RT-PCR protocol for the quantitative detection of normalized gene expression profiles of seven porcine cytokines was designed and validated within the presented study. Cytokines were chosen to represent different immunological pathways and targets known to be involved in the pathogenesis of the above mentioned porcine diseases, namely interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, tumor necrosis factor (TNF)-α and interferon (IFN)-α. Beta-Actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as reference genes for normalization. For absolute quantification a synthetic standard plasmid was constructed comprising all target cytokines and reference genes within a single molecule allowing the generation of positive control RNA. The standard as well as positive RNAs from samples, and additionally more than 400 clinical samples, which were collected from animal trials, were included in the validation process to assess analytical sensitivity and applicability under routine conditions. The resulting assay allows the reliable assessment of gene expression
Petrov, Anja; Beer, Martin; Blome, Sandra
Dysregulation of cytokine responses plays a major role in the pathogenesis of severe and life-threatening infectious diseases like septicemia or viral hemorrhagic fevers. In pigs, diseases like African and classical swine fever are known to show exaggerated cytokine releases. To study these responses and their impact on disease severity and outcome in detail, reliable, highly specific and sensitive methods are needed. For cytokine research on the molecular level, real-time RT-PCRs have been proven to be suitable. Yet, the currently available and most commonly used SYBR Green I assays or heterogeneous gel-based RT-PCRs for swine show a significant lack of specificity and sensitivity. The latter is however absolutely essential for an accurate quantification of rare cytokine transcripts as well as for detection of small changes in gene expressions. For this reason, a harmonized TaqMan-based triplex real-time RT-PCR protocol for the quantitative detection of normalized gene expression profiles of seven porcine cytokines was designed and validated within the presented study. Cytokines were chosen to represent different immunological pathways and targets known to be involved in the pathogenesis of the above mentioned porcine diseases, namely interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, tumor necrosis factor (TNF)-α and interferon (IFN)-α. Beta-Actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as reference genes for normalization. For absolute quantification a synthetic standard plasmid was constructed comprising all target cytokines and reference genes within a single molecule allowing the generation of positive control RNA. The standard as well as positive RNAs from samples, and additionally more than 400 clinical samples, which were collected from animal trials, were included in the validation process to assess analytical sensitivity and applicability under routine conditions. The resulting assay allows the reliable assessment of gene expression
Huili, Wang; Xiaokai, Zhao; Meili, Lin; Dahlgren, Randy A.; Wei, Chen; Jaiopeng, Zhou; Chengyang, Xu; Chunlei, Jin; Yi, Xu; Xuedong, Wang; Li, Ding; Qiyu, Bao
Arthrospira (Spirulina) platensis (ASP) is a representative filamentous, non-N2-fixing cyanobacterium that has great potential to enhance the food supply and possesses several valuable physiological features. ASP tolerates high and low temperatures along with highly alkaline and salty environments, and can strongly resist oxidation and irradiation. Based on genomic sequencing of ASP, we compared the protein expression profiles of this organism under different temperature conditions (15°C, 35°Cand 45°C) using 2-DE and peptide mass fingerprinting techniques. A total of 122 proteins having a significant differential expression response to temperature were retrieved. Of the positively expressed proteins, the homologies of 116 ASP proteins were found in Arthrospira (81 proteins in Arthrospira platensis str. Paraca and 35 in Arthrospira maxima CS-328). The other 6 proteins have high homology with other microorganisms. We classified the 122 differentially expressed positive proteins into 14 functions using the COG database, and characterized their respective KEGG metabolism pathways. The results demonstrated that these differentially expressed proteins are mainly involved in post-translational modification (protein turnover, chaperones), energy metabolism (photosynthesis, respiratory electron transport), translation (ribosomal structure and biogenesis) and carbohydrate transport and metabolism. Others proteins were related to amino acid transport and metabolism, cell envelope biogenesis, coenzyme metabolism and signal transduction mechanisms. Results implied that these proteins can perform predictable roles in rendering ASP resistance against low and high temperatures. Subsequently, we determined the transcription level of 38 genes in vivo in response to temperature and identified them by qRT-PCR. We found that the 26 differentially expressed proteins, representing 68.4% of the total target genes, maintained consistency between transcription and translation levels. The
Adachi, Iwao P. (Inventor); Adachi, Yoshifumi (Inventor); Frazer, Robert E. (Inventor)
A computer assisted keratometer in which a fiducial line pattern reticle illuminated by CW or pulsed laser light is projected on a corneal surface through lenses, a prismoidal beamsplitter quarterwave plate, and objective optics. The reticle surface is curved as a conjugate of an ideal corneal curvature. The fiducial image reflected from the cornea undergoes a polarization shift through the quarterwave plate and beamsplitter whereby the projected and reflected beams are separated and directed orthogonally. The reflected beam fiducial pattern forms a moire pattern with a replica of the first recticle. This moire pattern contains transverse aberration due to differences in curvature between the cornea and the ideal corneal curvature. The moire pattern is analyzed in real time by computer which displays either the CW moire pattern or a pulsed mode analysis of the transverse aberration of the cornea under observation, in real time. With the eye focused on a plurality of fixation points in succession, a survey of the entire corneal topography is made and a contour map or three dimensional plot of the cornea can be made as a computer readout in addition to corneal radius and refractive power analysis.
Blanchard, Philippe; Ribière, Magali; Celle, Olivier; Lallemand, Perrine; Schurr, Frank; Olivier, Violaine; Iscache, Anne Laure; Faucon, Jean Paul
A two-step real-time RT-PCR assay, based on TaqMan technology using a fluorescent probe (FAM-TAMRA) was developed to quantify Chronic bee paralysis virus (CBPV) genome in bee samples. Standard curves obtained from a CBPV control RNA and from a plasmid containing a partial sequence of CBPV showed that this assay provided linear detection over a 7-log range (R(2)>0.99) with a limit of detection of 100 copies, and reliable inter-assay and intra-assay reproducibility. Standardisation including RNA purification and cDNAs synthesis was also validated. The CBPV TaqMan methodology was first evaluated by quantifying the CBPV genomic load in bee samples from an experimental infection obtained by topical application. Up to 1.9 x 10(10) CBPV copies per segment of insect body (head, thorax and abdomen) were revealed whereas a lower CBPV genomic load was detected in dissected organs such as mandibular and hypopharyngeal glands, brain and alimentary canal (up to 7.2 x 10(6) CBPV copies). The CBPV genomic loads in different categories of bees from a hive presenting the trembling symptoms typical of Chronic paralysis were then quantified. Significantly higher CBPV loads were found in guard, symptomatic and dead bees (up to 1.9 x 10(13) CBPV copies) than in forager, drones and house bees (up to 3.4 x 10(6) CBPV copies). The results obtained for symptomatic or dead bees support the correlation between high CBPV genomic load and pathology expression. Moreover, the high CBPV genomic load revealed in guard bees highlights the possible pivotal role played by this category of bees in CBPV infection.
Agüero, Montserrat; Sánchez, Azucena; San Miguel, Elena; Gómez-Tejedor, Concepción; Jiménez-Clavero, Miguel Angel
This work describes the development of a real-time RT-PCR (RRT-PCR) procedure for detection of the N1 gene from avian influenza virus (AIV), based on the use of specific primers and a TaqMan-MGB (minor groove binder) probe. Nucleotide sequences of the neuraminidase type 1 gene from a collection of H5N1 Eurasian strains of AIV were aligned using ClustalW software. Conserved regions were located and used to design specific primers and a TaqMan-MGB probe using Primer Express software. A one-step RRT-PCR method was optimized using RNA from the Turkey 2005 H5N1 strain of AIV and can be completed in about 2 hr once the RNA is extracted from the sample. The specificity of the assay was assessed with non-N1 AIV strains, another related avian virus, and different avian tissue samples from healthy animals. Sensitivity was determined using 10-fold serial dilutions of the H5N1 Turkey 2005 strain and was compared with the generic RRT-PCR detection method, targeted at the matrix protein gene of AIV, commonly used at the Spanish AIV National Reference Laboratory. The N1 detection method proved to be even more sensitive than the generic (matrix-based) method, allowing a very quick confirmation (or discarding) of any Eurasian N1 strain when a positive result was obtained with the matrix RRT-PCR assay. Combined with RRT-PCR tests for general detection of AIV and H5 typing in use at the NRL, the procedure here described allows characterizing of any H5N1 Eurasian AIV strain in a field sample within a working day.
Petit, F; Craquelin, S; Guespin-Michel, J; Buffet-Janvresse, C
We describe an extraction protocol for genomic DNA and RNA of both viruses and bacteria from polluted estuary water. This procedure was adapted to the molecular study of microflora of estuarine water where bacteria and viruses are found free, forming low-density biofilms, or intimately associated with organo-mineral particles. The sensitivity of the method was determined with seeded samples for RT-PCR and PCR analysis of viruses (10 virions/mL), and bacteria (1 colony-forming unit mL). We report an example of molecular detection of both poliovirus and Salmonella in the Seine estuary (France) and an approach to studying their association with organo-mineral particles.
Gibert, Elisa; Martín-Valls, Gerard; Mateu, Enric
The detection of porcine reproductive and respiratory syndrome virus (PRRSV) in oral fluids (OF) by quantitative real-time polymerase chain reaction (qRT-PCR) is gaining increasing popularity. However, the different steps leading to a result have not been extensively evaluated. The aim of the present study was to examine the effect on the performance of qRT-PCR with different sampling materials, conditions of storage of the OF, the need for centrifuging OF, as well as to compare RNA extraction methods and PCR mixes. For the assays, pen-based oral fluids were used, which were pooled and spiked in a serial dilution (up to genotype 10(0) TCID50/mL) of type 1 PRRSV isolate 3267. Centrifugation at 15,000g for 15min resulted in an increase in sensitivity (1-2 PCR cycles) that was significant (P<0.05) at the lowest dilution tested. The TRIzol and MagMAX RNA extraction methods gave the maximum sensitivity, lowest threshold cycle (Ct), at equivalent virus concentrations. The AgPath-ID One-Step RT-PCR Kit PCR mix reagents were more sensitive for the detection of PRRSV using a purified plasmid as standard, but LSI VetMAX PRRSV EU/NA PRRSV reagents resulted in a slightly better sensitivity with OF (p<0.05). The present results may be useful to standardize protocols for optimizing detection of type 1 PRRSV in OF by qRT-PCR.
Aad, Pauline Y; Voge, Justin L; Santiago, Consuelo A; Malayer, Jerry R; Spicer, Leon J
Ovarian follicular growth and dominance are controlled by a series of hormonal and intraovarian events including a decrease in intrafollicular IGF-binding proteins -2, -4 and -5 levels. Proteolytic enzymes such as pregnancy-associated plasma protein-A (PAPP-A) degrade IGFBPs and increase bioavailability of IGF-I and -II during follicular development. The objective of this study was to determine the effect of IGF-I, IGF-II, insulin (INS), LH, FSH, estradiol (E2), leptin or cortisol on ovarian PAPP-A mRNA levels. Granulosa (GC) from small (SM) (1-5 mm) and large (LG) (8-22 mm) follicles as well as theca cells (TC) from LG follicles were collected from bovine ovaries and cultured for 48 h in medium containing 10% FCS and then treated with various hormones in serum-free medium for an additional 24 h. Cells were treated with various concentrations (3-500 ng/ml) and combinations of IGF-I, IGF-II, FSH, LH, E2, INS, leptin and (or) cortisol for 24 h (Experiments 1-10). PAPP-A mRNA levels were measured using quantitative real-time RT-PCR. In SM-GC and LG-GC, none of the treatments significantly affected (P>0.10) PAPP-A mRNA abundance. In LG-TC, IGF-I, LH or cortisol did not affect (P>0.10) PAPP-A mRNA levels, whereas INS with or without LH decreased (P<0.05) PAPP-A mRNA. E2 alone decreased PAPP-A mRNA levels in LG-TC, and E2 amplified the insulin-induced inhibition of PAPP-A mRNA abundance in LG-TC. We conclude that control of PAPP-A mRNA abundance in granulosa and theca cells differs, and that E2 may be part of an intraovarian negative feedback system which may reduce the bioavailable IGFs in the theca layer during growth and selection of follicles.
Wright, C.; Haddad, F.; Qin, A. X.; Baldwin, K. M.
An assay was developed for rapid and sensitive analysis of myosin heavy chain (MHC) mRNA expression in rodent skeletal muscle. Only 2 microg of total RNA were necessary for the simultaneous analysis of relative mRNA expression of six different MHC genes. We designed synthetic DNA fragments as internal standards, which contained the relevant primer sequences for the adult MHC mRNAs type I, IIa, IIx, IIb as well as the embryonic and neonatal MHC mRNAs. A known amount of the synthetic fragment was added to each polymerase chain reaction (PCR) and yielded a product of different size than the amplified MHC mRNA fragment. The ratio of amplified MHC fragment to synthetic fragment allowed us to calculate percentages of the gene expression of the different MHC genes in a given muscle sample. Comparison with the traditional Northern blot analysis demonstrated that our reverse transcriptase-PCR-based assay was reliable, fast, and quantitative over a wide range of relative MHC mRNA expression in a spectrum of adult and neonatal rat skeletal muscles. Furthermore, the high sensitivity of the assay made it very useful when only small quantities of tissue were available. Statistical analysis of the signals for each MHC isoform across the analyzed samples showed a highly significant correlation between the PCR and the Northern signals as Pearson correlation coefficients ranged between 0.77 and 0.96 (P < 0.005). This assay has potential use in analyzing small muscle samples such as biopsies and samples from pre- and/or neonatal stages of development.
Ukaji, Takao; Kai, Osamu
We designed primer sets specific to the immunoglobulin (Ig) heavy-chain constant region (IGHC) genes in Mongolian gerbil (Meriones unguiculatus) to amplify five gerbil IGHC cDNA sequences, Cμ, Cγ1, Cγ2, Cε, and Cα. Five gerbil-mouse heterohybridomas B11D2(C2), B11E2(D5).M, B5-3, D5, and C11 respectively expressed Cγ1, Cμ, Cγ2, Cγ2, and Cγ1. In contrast, a commercial isotyping kit for mouse Igs identified Cγ1, Cμ, Cγ3, Cγ3, and Cγ1, respectively, misidentifying gerbil IgG2 as IgG3 by cross-reactivity with anti-mouse IgG3 polyclonal antibody. These primer sets will allow the accurate estimation of gerbil Ig classes and IgG subclasses. These results from three gerbil strains indicate that the primer sets can be used for isotype analysis of gerbil mAbs and for evaluation of humoral immunity.
Silberberg, George G.
By the early 1970s, classical photo-optical range instrumentation technology (as a means of gathering weapons' system performance data) had become a costly and inefficient process. Film costs were increasing due to soaring silver prices. Time required to process, read, and produce optical data was becoming unacceptable as a means of supporting weapon system development programs. NWC investigated the feasibility of utilizing Closed Circuit Television (CCTV) technology as an alternative solution for providing optical data. In 1978 a program entitled Metric Video (measurements from video images) was formulated at the Naval Weapons Center, China Lake, California. The purpose of this program was to provide timely data, to reduce the number of operating personnel, and to lower data acquisition costs. Some of the task elements for this program included a near real-time vector miss-distance system, a weapons scoring system, a velocity measuring system, a time-space position system, and a system to replace film cameras for gathering real-time engineering sequential data. These task elements and the development of special hardware and techniques to achieve real-time data will be discussed briefly in this paper.
Zhao, Junfeng; Zhang, Chong; Lu, Jing; Lu, Zhaoxin
Genome shuffling is an efficient approach for the rapid engineering of microbial strains with desirable industrial phenotypes. In this study, we used genome shuffling in an attempt to improve fengycin production of the wild-type strain Bacillus amyloliquefaciens ES-2-4. After 2 rounds of genome shuffling, a high-yield recombinant F2-72 (FMB72) strain that exhibited 8.30-fold increases in fengycin production was obtained. Comparative analysis of synthetase gene (fenA) expression was conducted between the initial and shuffled strains using fluorescent quantitation RT-PCR. Delta CT (threshold cycle) relative quantitation analysis revealed that fengycin synthetase gene (fenA) expression at the transcriptional level in the FMB72 strain was 12.77-fold greater than in the ES-2-4 wild type. The shuffled strain has a potential application in food and pharmaceutical industries. At the same time, the analysis of improved phenotypes will provide more valuable data for inverse metabolic engineering.
Walker, R.; Gupta, N.
The important algorithm issues necessary to achieve a real time flutter monitoring system; namely, the guidelines for choosing appropriate model forms, reduction of the parameter convergence transient, handling multiple modes, the effect of over parameterization, and estimate accuracy predictions, both online and for experiment design are addressed. An approach for efficiently computing continuous-time flutter parameter Cramer-Rao estimate error bounds were developed. This enables a convincing comparison of theoretical and simulation results, as well as offline studies in preparation for a flight test. Theoretical predictions, simulation and flight test results from the NASA Drones for Aerodynamic and Structural Test (DAST) Program are compared.
Romanutti, Carina; Gallo Calderón, Marina; Keller, Leticia; Mattion, Nora; La Torre, José
During 2007-2014, 84 out of 236 (35.6%) samples from domestic dogs submitted to our laboratory for diagnostic purposes were positive for Canine Distemper Virus (CDV), as analyzed by RT-PCR amplification of a fragment of the nucleoprotein gene. Fifty-nine of them (70.2%) were from dogs that had been vaccinated against CDV. The full-length gene encoding the Fusion (F) protein of fifteen isolates was sequenced and compared with that of those of other CDVs, including wild-type and vaccine strains. Phylogenetic analysis using the F gene full-length sequences grouped all the Argentinean CDV strains in the SA2 clade. Sequence identity with the Onderstepoort vaccine strain was 89.0-90.6%, and the highest divergence was found in the 135 amino acids corresponding to the F protein signal-peptide, Fsp (64.4-66.7% identity). In contrast, this region was highly conserved among the local strains (94.1-100% identity). One extra putative N-glycosylation site was identified in the F gene of CDV Argentinean strains with respect to the vaccine strain. The present report is the first to analyze full-length F protein sequences of CDV strains circulating in Argentina, and contributes to the knowledge of molecular epidemiology of CDV, which may help in understanding future disease outbreaks.
Guichet, Emilande; Aghokeng, Avelin; Eymard-Duvernay, Sabrina; Vidal, Nicole; Ayouba, Ahidjo; Mpoudi Ngole, Eitel; Delaporte, Eric; Ciaffi, Laura; Peeters, Martine
With the increasing demand of HIV viral load (VL) tests in resource-limited countries (RLCs) there is a need for assays at affordable cost and able to quantify all known HIV-1 variants. VLs obtained with a recently developed open and polyvalent universal HIV-1/SIVcpz/SIVgor RT-qPCR were compared to Abbott RealTime HIV-1 assay in Cameroon. On 474 plasma samples, characterized by a wide range of VLs and a broad HIV-1 group M genetic diversity, 97.5% concordance was observed when using the lower detection limit of each assay. When using the threshold of 3.00 log10 copies/mL, according to WHO guidelines to define virological failure (VF) in RLCs, the concordance was 94.7%, 360/474 versus 339/474 patients were identified with VF with the new assay and Abbott RealTime HIV-1, respectively. Higher VLs were measured with the new assay, +0.47 log10 copies/mL (95% CI; 0.42-0.52) as shown with Bland-Altman analysis. Eleven samples from patients on VF with drug resistance were not detected by Abbott RealTime HIV-1 versus two only with the new assay. Overall, our study showed that the new assay can be easily implemented in a laboratory in RLCs with VL experience and showed good performance on a wide diversity of HIV-1 group M variants.
Balasuriya, Udeni B R; Lee, Pei-Yu Alison; Tiwari, Ashish; Skillman, Ashley; Nam, Bora; Chambers, Thomas M; Tsai, Yun-Long; Ma, Li-Juan; Yang, Pai-Chun; Chang, Hsiao-Fen Grace; Wang, Hwa-Tang Thomas
Equine influenza (EI) is an acute, highly contagious viral respiratory disease of equids. Currently, equine influenza virus (EIV) subtype H3N8 continues to be the most important respiratory pathogen of horses in many countries around the world. The need to achieve a rapid diagnosis and to implement effective quarantine and movement restrictions is critical in controlling the spread of EIV. In this study, a novel, inexpensive and user-friendly assay based on an insulated isothermal RT-PCR (iiRT-PCR) method on the POCKIT™, a field-deployable device, was described and validated for point-of-need detection of EIV-H3N8 in clinical samples. The newly established iiRT-PCR assay targeting the EIV HA3 gene was evaluated for its sensitivity using in vitro transcribed (IVT) RNA, as well as ten-fold serial dilutions of RNA extracted from the prototype H3N8 strain A/equine/Miami/1/63. Inclusivity and exclusivity panels were tested for specificity evaluation. Published real-time RT-PCR (rRT-PCR) assays targeting the NP and HA3 genes were used as the reference standards for comparison of RNA extracted from field strains and from nasal swab samples collected from experimentally infected horses, respectively. Limit of detection with a 95% probability (LoD95%) was estimated to be 11copies of IVT RNA. Clinical sensitivity analysis using RNA prepared from serial dilutions of a prototype EIV (Miami 1/63/H3N8) showed that the iiRT-PCR assay was about 100-fold more sensitive than the rRT-PCR assay targeting the NP gene of EIV subtype H3N8. The iiRT-PCR assay identified accurately fifteen EIV H3N8 strains and two canine influenza virus (CIV) H3N8 strains, and did not cross-react with H6N2, H7N7, H1N1 subtypes or any other equine respiratory viral pathogens. Finally, 100% agreement was found between the iiRT-PCR assay and the universal influenza virus type A rRT-PCR assay in detecting the EIV A/equine/Kentucky/7/07 strain in 56 nasal swab samples collected from experimentally inoculated
Orencio-Trejo, Montserrat; Flores, Noemí; Escalante, Adelfo; Hernández-Chávez, Georgina; Bolívar, Francisco; Gosset, Guillermo; Martinez, Alfredo
Background A metabolic regulation study was performed, based upon measurements of enzymatic activities, fermentation performance, and RT-PCR analysis of pathways related to central carbon metabolism, in an ethanologenic Escherichia coli strain (CCE14) derived from lineage C. In comparison with previous engineered strains, this E coli derivative has a higher ethanol production rate in mineral medium, as a result of the elevated heterologous expression of the chromosomally integrated genes encoding PDCZm and ADHZm (pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis). It is suggested that this behavior might be due to lineage differences between E. coli W and C. Results This study demonstrated that the glycolytic flux is controlled, in this case, by reactions outside glycolysis, i.e., the fermentative pathways. Changes in ethanol production rate in this ethanologenic strain result in low organic acid production rates, and high glycolytic and ethanologenic fluxes, that correlate with enhanced transcription and enzymatic activity levels of PDCZm and ADHZm. Furthermore, a higher ethanol yield (90% of the theoretical) in glucose-mineral media was obtained with CCE14 in comparison with previous engineered E. coli strains, such as KO11, that produces a 70% yield under the same conditions. Conclusion Results suggest that a higher ethanol formation rate, caused by ahigher PDCZm and ADHZm activities induces a metabolic state that cells compensate through enhanced glucose transport, ATP synthesis, and NAD-NADH+H turnover rates. These results show that glycolytic enzymatic activities, present in E. coli W and C under fermentative conditions, are sufficient to contend with increases in glucose consumption and product formation rates. PMID:18471274
Rocha-Martins, Maurício; Njaine, Brian; Silveira, Mariana S.
Background Housekeeping genes have been commonly used as reference to normalize gene expression and protein content data because of its presumed constitutive expression. In this paper, we challenge the consensual idea that housekeeping genes are reliable controls for expression studies in the retina through the investigation of a panel of reference genes potentially suitable for analysis of different stages of retinal development. Methodology/Principal Findings We applied statistical tools on combinations of retinal developmental stages to assess the most stable internal controls for quantitative RT-PCR (qRT-PCR). The stability of expression of seven putative reference genes (Actb, B2m, Gapdh, Hprt1, Mapk1, Ppia and Rn18s) was analyzed using geNorm, BestKeeper and Normfinder software. In addition, several housekeeping genes were tested as loading controls for Western blot in the same sample panel, using Image J. Overall, for qRT-PCR the combination of Gapdh and Mapk1 showed the highest stability for most experimental sets. Actb was downregulated in more mature stages, while Rn18s and Hprt1 showed the highest variability. We normalized the expression of cyclin D1 using various reference genes and demonstrated that spurious results may result from blind selection of internal controls. For Western blot significant variation could be seen among four putative internal controls (β-actin, cyclophilin b, α-tubulin and lamin A/C), while MAPK1 was stably expressed. Conclusion Putative housekeeping genes exhibit significant variation in both mRNA and protein content during retinal development. Our results showed that distinct combinations of internal controls fit for each experimental set in the case of qRT-PCR and that MAPK1 is a reliable loading control for Western blot. The results indicate that biased study outcomes may follow the use of reference genes without prior validation for qRT-PCR and Western blot. PMID:22916200
Burns, Cara C; Kilpatrick, David R; Iber, Jane C; Chen, Qi; Kew, Olen M
Virologic surveillance is essential to the success of the World Health Organization initiative to eradicate poliomyelitis. Molecular methods have been used to detect polioviruses in tissue culture isolates derived from stool samples obtained through surveillance for acute flaccid paralysis. This chapter describes the use of realtime PCR assays to identify and serotype polioviruses. In particular, a degenerate, inosine-containing, panpoliovirus (panPV) PCR primer set is used to distinguish polioviruses from NPEVs. The high degree of nucleotide sequence diversity among polioviruses presents a challenge to the systematic design of nucleic acid-based reagents. To accommodate the wide variability and rapid evolution of poliovirus genomes, degenerate codon positions on the template were matched to mixed-base or deoxyinosine residues on both the primers and the TaqMan™ probes. Additional assays distinguish between Sabin vaccine strains and non-Sabin strains. This chapter also describes the use of generic poliovirus specific primers, along with degenerate and inosine-containing primers, for routine VP1 sequencing of poliovirus isolates. These primers, along with nondegenerate serotype-specific Sabin primers, can also be used to sequence individual polioviruses in mixtures.
Viroids are small, infectious, single-stranded RNA molecules that cause several important citrus diseases. Viroids are transmitted primarily in budwood, however, spread can also occur mechanically on pruning equipment, budding knives, hedging and topping equipment. Exocortis and cachexia are two we...
Arun, Alok; Baumlé, Véronique; Amelot, Gaël; Nieberding, Caroline M.
Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera) have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae), two developmental stages (pupal and adult) and two sexes (male and female), all of which were subjected to two food treatments (food stress and control feeding ad libitum). The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm. Both methods identified RpS8 as the best suitable reference gene for expression data normalization. We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana. Finally, we tested the effect of choosing reference genes with different stability on the normalization of the transcript abundance of a candidate gene involved in olfactory communication in B. anynana, the Fatty Acyl Reductase 2, and we confirmed that using an unstable reference gene can drastically alter the expression
Arun, Alok; Baumlé, Véronique; Amelot, Gaël; Nieberding, Caroline M
Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera) have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae), two developmental stages (pupal and adult) and two sexes (male and female), all of which were subjected to two food treatments (food stress and control feeding ad libitum). The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm. Both methods identified RpS8 as the best suitable reference gene for expression data normalization. We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana. Finally, we tested the effect of choosing reference genes with different stability on the normalization of the transcript abundance of a candidate gene involved in olfactory communication in B. anynana, the Fatty Acyl Reductase 2, and we confirmed that using an unstable reference gene can drastically alter the expression
In recent years, a few structures have been instrumented to provide continuous vibration data in real time, recording not only large-amplitude motions generated by extreme loads, but also small-amplitude motions generated by ambient loads. The main objective in continuous recording is to track any changes in structural characteristics, and to detect damage after an extreme event, such as an earthquake or explosion. The Fourier-based spectral analysis methods have been the primary tool to analyze vibration data from structures. In general, such methods do not work well for real-time data, because real-time data are mainly composed of ambient vibrations with very low amplitudes and signal-to-noise ratios. The long duration, linearity, and the stationarity of ambient data, however, allow us to utilize statistical signal processing tools, which can compensate for the adverse effects of low amplitudes and high noise. The analysis of real-time data requires tools and techniques that can be applied in real-time; i.e., data are processed and analyzed while being acquired. This paper presents some of the basic tools and techniques for processing and analyzing real-time vibration data. The topics discussed include utilization of running time windows, tracking mean and mean-square values, filtering, system identification, and damage detection.
Kao, Simon A.; Laffey, Thomas J.; Schmidt, James L.; Read, Jackson Y.; Dunham, Larry L.
This paper descibes a knowledge-based system for performing real-time monitoring and analysis of telemetry data from the NASA Hubble Space Telescope (HST). In order to handle asynchronous inputs and perform in real time the system consists of three or more separate processes, which run concurrently and communicate via a message passing scheme. The data management process gathers, compresses, and scales the incoming telemetry data befoe sending it to the other tasks. The inferencing process uses the incoming data to perform a real-time analysis of the state and health of the Space Telescope. The I/O process receives telemetry monitors from the data management process, updates its graphical displays in real time, and acts as the interface to the console operator. The three processes may run on the same or different computers. This system is currently under development and is being used to monitor testcases produced by the Bass Telemetry System in the Hardware/Software Integration Facility at Lockheed Missile and Space Co. in Sunnyvale, California.
Dedkov, V G; Magassouba, N' F; Safonova, M V; Deviatkin, A A; Dolgova, A S; Pyankov, O V; Sergeev, A A; Utkin, D V; Odinokov, G N; Safronov, V A; Agafonov, A P; Maleev, V V; Shipulin, G A
In early February 2014, an outbreak of the Ebola virus disease caused by Zaire ebolavirus (EBOV) occurred in Guinea; cases were also recorded in other West African countries with a combined population of approximately 25 million. A rapid, sensitive and inexpensive method for detecting EBOV is needed to effectively control such outbreak. Here, we report a real-time reverse-transcription PCR assay for Z. ebolavirus detection used by the Specialized Anti-epidemic Team of the Russian Federation during the Ebola virus disease prevention mission in the Republic of Guinea. The analytical sensitivity of the assay is 5 × 10(2) viral particles per ml, and high specificity is demonstrated using representative sampling of viral, bacterial and human nucleic acids. This assay can be applied successfully for detecting the West African strains of Z. ebolavirus as well as on strains isolated in the Democratic Republic of the Congo in 2014.
Ebadzad, Ghazal; Cravador, Alfredo
cDNA-AFLP methodology was used to gain insight into gene fragments differentially present in the mRNA profiles of Quercus suber roots infected with zoospores of Phytophthora cinnamomi at different post challenge time points. Fifty-three transcript-derived fragments (TDFs) were identified and sequenced. Six candidate genes were selected based on their expression patterns and homology to genes known to play a role in defence. They encode a cinnamyl alcohol dehydrogenase2 (QsCAD2), a protein disulphide isomerase (QsPDI), a CC-NBS-LRR resistance protein (QsRPc), a thaumatin-like protein (QsTLP), a chitinase (QsCHI) and a 1,3-β-glucanase (QsGlu). Evaluation of the expression of these genes by quantitative polymerase chain reaction (qPCR) revealed that transcript levels of QsRPc, QsCHI, QsCAD2 and QsPDI increased during the first 24 h post-inoculation, while those of thaumatin-like protein decreased. No differential expression was observed for 1,3-β-glucanase (QsGlu). Four candidate reference genes, polymerase II (QsRPII), eukaryotic translation initiation factor 5A (QsEIF-5A), β-tubulin (QsTUB) and a medium subunit family protein of clathrin adaptor complexes (QsCACs) were assessed to determine the most stable internal references for qRT-PCR normalization in the Phytophthora-Q. suber pathosystem in root tissues. Those found to be more stable, QsRPII and QsCACs, were used as internal reference in the present work. Knowledge on the Quercus defence mechanisms against biotic stress is scarce. This study provides an insight into the gene profiling of a few important genes of Q. suber in response to P. cinnamomi infection contributing to the knowledge of the molecular interactions involving Quercus and root pathogens that can be useful in the future to understand the mechanisms underlying oak resistance to soil-borne oomycetes.
Savabi, Omid; Kazemi, Mohammad; Kamali, Sara; Salehi, Ahmad Reza; Eslami, Gilda; Tahmourespour, Arezoo; Salehi, Rasoul
Background: The Streptococci are the pioneer strains in plaque formation and Streptococcus mutans are the main etiological agent of dental plaque and caries. In general, biofilm formation is a step-wise process, which begins by adhesion of planktonic cells to the surfaces. Evidences show that expression of glucosyltransferase B and C (gtfB and gtfC) and fructosyltransferase (ftf) genes play critical role in initial adhesion of S. mutans to the tooth surface which results in formation of dental plaques and consequently caries and other periodontal disease. Materials and Methods: The aim of this study was to determine the effect of biosurfactants produced by a probiotic strain; Lactobacillus casei (ATCC39392) on gene expression profile of gftB/C and tft of S. mutans (ATCC35668) using quantitative real-time PCR. Results: The application of the prepared biosurfactant caused dramatic down regulation of all the three genes under study. The reduction in gene expression was statistically highly significant (for gtfB, P > 0.0002; for gtfC, P > 0.0063, and for ftf, P > 0.0057). Conclusion: Considerable downregulation of all three genes in the presence of the prepared biosurfactant comparing to untreated controls is indicative of successful inhibition of influential genes in bacterial adhesion phenomena. In view of the importance of glucosyltransferase gene products for S.mutans attachment to the tooth surface which is the initial important step in biofilm production and dental caries, further research in this field may lead to an applicable alternative for successful with least adverse side effects in dental caries prevention. PMID:25538917
Efforts continue to develop a low cost real time interactive analysis system for the reception of satellite data. A multi-purpose ingest hardware software frame formatter was demonstrated for GOES and TIROS data and work is proceeding on extending the capability to receive GMS data. A similar system was proposed as an archival and analysis system for use with INSAT data and studies are underway to modify the system to receive the planned SeaWiFS (ocean color) data. This system was proposed as the core of a number of international programs in support of U.S. AID activities. Systems delivered or nearing final testing are listed.
Morten, Brianna C.; Scott, Rodney J.; Avery-Kiejda, Kelly A.
p53 is expressed as multiple smaller isoforms whose functions in cancer are not well understood. The p53 isoforms demonstrate abnormal expression in different cancers, suggesting they are important in modulating the function of full-length p53 (FLp53). The quantification of relative mRNA expression has routinely been performed using real-time PCR (qPCR). However, there are serious limitations when detecting p53 isoforms using this method, particularly for formalin-fixed paraffin-embedded (FFPE) tissues. The use of FFPE tumours would be advantageous to correlate expression of p53 isoforms with important clinical features of cancer. One alternative method of RNA detection is the hybridization-based QuantiGene 2.0 Assay, which has been shown to be advantageous for the detection of RNA from FFPE tissues. In this pilot study, we compared the QuantiGene 2.0 Assay to qPCR for the detection of FLp53 and its isoform Δ40p53 in matched fresh frozen (FF) and FFPE breast tumours. FLp53 mRNA expression was detected using qPCR in FF and FFPE tissues, but Δ40p53 mRNA was only detectable in FF tissues. Similar results were obtained for the QuantiGene 2.0 Assay. FLp53 relative mRNA expression was shown to be strongly correlated between the two methods (R2 = 0.9927, p = 0.0031) in FF tissues, however Δ40p53 was not (R2 = 0.4429, p = 0.3345). When comparing the different methods for the detection of FLp53 mRNA from FFPE and FF samples, no correlation (R2 = 0.0002, p = 0.9863) was shown using the QuantiGene 2.0 Assay, and in contrast, the level of expression was highly correlated between the two tissues using qPCR (R2 = 0.8753, p = 0.0644). These results suggest that both the QuantiGene 2.0 Assay and qPCR methods are inadequate for the quantification of Δ40p53 mRNA in FFPE tissues. Therefore, alternative methods of RNA detection and quantification are required to study the relative expression of Δ40p53 in FFPE samples. PMID:27832134
Jamal, Syed M; Belsham, Graham J
Rapid and accurate diagnosis of foot-and-mouth disease (FMD) and virus serotyping are of paramount importance for control of this disease in endemic areas where vaccination is practiced. Ideally this virus characterization should be achieved without the need for virus amplification in cell culture. Due to the heterogeneity of FMD viruses (FMDVs) in different parts of the world, region specific diagnostic tests are required. In this study, hydrolysable probe-based real time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays were developed for specific detection and serotyping of the FMDVs currently circulating in West Eurasia. These assays were evaluated, in parallel with pan-FMDV diagnostic assays and earlier serotype-specific assays, using field samples originating from Pakistan and Afghanistan containing FMD viruses belonging to different sublineages of O-PanAsia, A-Iran05 and Asia-1 (Group-II and Group-VII (Sindh-08)). In addition, field samples from Iran and Bulgaria, containing FMDVs belonging to the O-PanAsiaANT-10 sublineage were also tested. Each of the three primer/probe sets was designed to be specific for just one of the serotypes O, A and Asia-1 of FMDV and detected the RNA from the target viruses with cycle threshold (CT) values comparable with those obtained with the serotype-independent pan-FMDV diagnostic assays. No cross-reactivity was observed in these assays between the heterotypic viruses circulating in the region. The assays reported here have higher diagnostic sensitivity (100% each for serotypes O and Asia-1, and 92% [95% CI = 81.4-100%] for serotype A positive samples) and specificity (100% each for serotypes O, A and Asia-1 positive samples) for the viruses currently circulating in West Eurasia compared to the serotyping assays reported earlier. Comparisons of the sequences of the primers and probes used in these assays and the corresponding regions of the circulating viruses provided explanations for the poor
Parra, Macarena; Jung, Jimmy; Almeida, Eduardo; Boone, Travis; Tran, Luan; Schonfeld, Julie
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
Large-scale analysis of gene expression using cDNA microarrays promises the
rapid detection of the mode of toxicity for drugs and other chemicals. cDNA
microarrays were used to examine chemically-induced alterations of gene
expression in HepG2 cells exposed to oxidative ...
Herget, William F.; Tromp, Marianne L.; Anderson, Charles R.
A Fourier transform infrared (FT-IR) - based system has been developed and is undergoing evaluation for near real time multicomponent quantitative analysis of undiluted gaseous automotive exhaust emissions. The total system includes: (1) a gas conditioning system (GCS) for tracer gas injection, gas mixing, and temperature stabilization; and (2) an exhaust gas analyzer (EGA) consisting of a sample cell, an FT-IR system, and a computerized data processing system. Tests have shown that the system can monitor about 20 individual species (concentrations down to the 1-20 ppm range) with a time resolution of one second. Tests have been conducted on a chassis dynamometer system utilizing different autos, different fuels, and different driving cycles. Results were compared with those obtained using a standard constant volume sampling (CVS) system.
Xu, Jiang; Xu, ZhiChao; Zhu, YingJie; Luo, HongMei; Qian, Jun; Ji, AiJia; Hu, YuanLei; Sun, Wei; Wang, Bo; Song, JingYuan; Sun, Chao; Chen, ShiLin
Quantitative real-time reverse transcription PCR (qRT-PCR) is a rapid, sensitive, and reliable technique for gene expression studies. The accuracy and reliability of qRT-PCR results depend on the stability of the reference genes used for gene normalization. Therefore, a systematic process of reference gene evaluation is needed. Ganoderma lucidum is a famous medicinal mushroom in East Asia. In the current study, 10 potential reference genes were selected from the G. lucidum genomic data. The sequences of these genes were manually curated, and primers were designed following strict criteria. The experiment was conducted using qRT-PCR, and the stability of each candidate gene was assessed using four commonly used statistical programs-geNorm, NormFinder, BestKeeper, and RefFinder. According to our results, PP2A was expressed at the most stable levels under different fermentation conditions, and RPL4 was the most stably expressed gene in different tissues. RPL4, PP2A, and β-tubulin are the most commonly recommended reference genes for normalizing gene expression in the entire sample set. The current study provides a foundation for the further use of qRT-PCR in G. lucidum gene analysis.
Viral isolation is the conventional method for influenza virus diagnosis but it is less useful for immediate patient management. RT-PCR is the sensitive and rapid assay for the detection of respiratory viruses. Single step and multiplex RT-PCR is able to detect several viruses simultaneously in a single reaction. Real time PCR(TaqMan method) is able to detect the amplicon directly by release of a fluorescent reporter of the probe during the amplification reactions. This procedure can save time since it eliminates post-PCR processing steps. These RT-PCR methods should be useful for the accurate and rapid diagnosis of influenza virus infection, especially severe cases such as pneumonia and encephalopathy.
Duong, Vu; Duong, Tuan
A recently written computer program implements dominant-element-based gradient descent and dynamic initial learning rate (DOGEDYN), which was described in Method of Real-Time Principal-Component Analysis (NPO-40034) NASA Tech Briefs, Vol. 29, No. 1 (January 2005), page 59. To recapitulate: DOGEDYN is a method of sequential principal-component analysis (PCA) suitable for such applications as data compression and extraction of features from sets of data. In DOGEDYN, input data are represented as a sequence of vectors acquired at sampling times. The learning algorithm in DOGEDYN involves sequential extraction of principal vectors by means of a gradient descent in which only the dominant element is used at each iteration. Each iteration includes updating of elements of a weight matrix by amounts proportional to a dynamic initial learning rate chosen to increase the rate of convergence by compensating for the energy lost through the previous extraction of principal components. In comparison with a prior method of gradient-descent-based sequential PCA, DOGEDYN involves less computation and offers a greater rate of learning convergence. The sequential DOGEDYN computations require less memory than would parallel computations for the same purpose. The DOGEDYN software can be executed on a personal computer.
In a real-time environment, the results of changes or failures in a complex, interconnected system need evaluation quickly. Tabulations showing the effects of changes and/or failures of a given item in the system are generally only useful for a single input, and only with regard to that item. Subsequent changes become harder to evaluate as combinations of failures produce a cascade effect. When confronted by multiple indicated failures in the system, it becomes necessary to determine a single cause. In this case, failure tables are not very helpful. CRANS, the Configurable Real-time ANalysis System, can interpret a logic tree, constructed by the user, describing a complex system and determine the effects of changes and failures in it. Items in the tree are related to each other by Boolean operators. The user is then able to change the state of these items (ON/OFF FAILED/UNFAILED). The program then evaluates the logic tree based on these changes and determines any resultant changes to other items in the tree. CRANS can also search for a common cause for multiple item failures, and allow the user to explore the logic tree from within the program. A "help" mode and a reference check provide the user with a means of exploring an item's underlying logic from within the program. A commonality check determines single point failures for an item or group of items. Output is in the form of a user-defined matrix or matrices of colored boxes, each box representing an item or set of items from the logic tree. Input is via mouse selection of the matrix boxes, using the mouse buttons to toggle the state of the item. CRANS is written in C-language and requires the MIT X Window System, Version 11 Revision 4 or Revision 5. It requires 78K of RAM for execution and a three button mouse. It has been successfully implemented on Sun4 workstations running SunOS, HP9000 workstations running HP-UX, and DECstations running ULTRIX. No executable is provided on the distribution medium; however
Zhao, Junfeng; Li, Yuanhong; Zhang, Chong; Yao, Zhengying; Zhang, Li; Bie, Xiaomei; Lu, Fengxia; Lu, Zhaoxin
Genome shuffling is an efficient approach for the rapid improvement of the yield of secondary metabolites. This study was undertaken to enhance the yield of surfactin produced by Bacillus amyloliquefaciens ES-2-4 using genome shuffling and to examine changes in SrfA expression of the improved phenotype at the transcriptional level. Six strains with subtle improvements in lipopeptide yield were obtained from populations generated by ultraviolet irradiation, nitrosoguanidine, and ion beam mutagenesis. These strains were then subjected to recursive protoplast fusion. A strain library that was likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both ultraviolet irradiation and heat treatments. After two rounds of genome shuffling, a high-yield recombinant F2-38 strain that exhibited 3.5- and 10.3-fold increases in surfactin production in shake flask and fermenter respectively, was obtained. Comparative analysis of synthetase gene expression was conducted between the initial and shuffled strains using FQ (fluorescent quantitation) RT-PCR. Delta CT (threshold cycle) relative quantitation analysis revealed that surfactin synthetase gene (srfA) expression at the transcriptional level in the F2-38 strain was 15.7-fold greater than in the ES-2-4 wild-type. The shuffled strain has a potential application in food and pharmaceutical industries. At the same time, the analysis of improved phenotypes will provide more valuable data for inverse metabolic engineering.
Salihah, Nur Thaqifah; Hossain, Mohammad Mosharraf; Lubis, Hamadah; Ahmed, Minhaz Uddin
Analyses to ensure food safety and quality are more relevant now because of rapid changes in the quantity, diversity and mobility of food. Food-contamination must be determined to maintain health and up-hold laws, as well as for ethical and cultural concerns. Real-time polymerase chain reaction (RT-PCR), a rapid and inexpensive quantitative method to detect the presence of targeted DNA-segments in samples, helps in determining both accidental and intentional adulterations of foods by biological contaminants. This review presents recent developments in theory, techniques, and applications of RT-PCR in food analyses, RT-PCR addresses the limitations of traditional food analyses in terms of sensitivity, range of analytes, multiplexing ability, cost, time, and point-of-care applications. A range of targets, including species of plants or animals which are used as food ingredients, food-borne bacteria or viruses, genetically modified organisms, and allergens, even in highly processed foods can be identified by RT-PCR, even at very low concentrations. Microfluidic RT-PCR eliminates the separate sample-processing step to create opportunities for point-of-care analyses. We also cover the challenges related to using RT-PCR for food analyses, such as the need to further improve sample handling.
Chamizo, C; Rubio, J M; Moreno, J; Alvar, J
Cytokines play an important role in the regulation of the immune system, but low circulating levels in plasma make routine measurement a difficult task. A new methodology based on single tube RT-PCR has been developed to determine the expression of multiple canine cytokines (TNF-alpha, IL-2, IFN-gamma, IL-18, IL-4, IL-6 and IL-10) using primers and protocols designed allow specific amplification of the mRNAs. The technique is performed in one tube in two consecutive steps, a specific transcription of the mRNA of a given cytokine and amplification of the corresponding gene by PCR. The technique was used to analyse the mRNA cytokine profile of peripheral blood mononuclear cells (PBMCs) from healthy dogs using two approaches: (i) analysis of PBMC isolated ex vivo; (ii) analysis of PBMC after in vitro cultures with or without the mitogen ConA. The samples were separated in agarose gels and the intensity of ethidium bromide signals quantified using standard video imaging equipment. Results were interpreted as the ratio of cytokine to GAPDH expression. The results obtained show that the method is easy to use and reproducible. Therefore, this method of monitoring the mRNA cytokine expression might be an useful tool for understanding the immune response in dogs.
Shi, Caihua; Yang, Fengshan; Zhu, Xun; Du, Erxia; Yang, Yuting; Wang, Shaoli; Wu, Qingjun; Zhang, Youjun
The soil insect Bradysia odoriphaga (Diptera: Sciaridae) causes substantial damage to Chinese chive. Suitable reference genes in B. odoriphaga (Bradysia odoriphaga) have yet to be identified for normalizing target gene expression among samples by quantitative real-time PCR (qRT-PCR). This study was focused on identifying the expression stability of 12 candidate housekeeping genes in B. odoriphaga under various experiment conditions. The final stability ranking of 12 housekeeping genes was obtained with RefFinder, and the most suitable number of reference genes was analyzed by GeNorm. The results revealed that the most appropriate sets of internal controls were RPS15, RPL18, and RPS18 across developmental phases; RPS15, RPL28, and GAPDH across temperatures; RPS15 and RPL18 across pesticide treatments; RSP5, RPS18, and SDHA across photoperiods; ACTb, RPS18, and RPS15 across diets; RPS13 and RPL28 across populations; and RPS15, ACTb, and RPS18 across all samples. The use of the most suitable reference genes versus an arbitrarily selected reference gene resulted in significant differences in the analysis of a target gene expression. HSP23 in B. odoriphaga was found to be up-regulated under low temperatures. These results will contribute to the standardization of qRT-PCR and will also be valuable for further research on gene function in B. odoriphaga. PMID:27399679
Lü, Zhi-Chuang; Liu, Wan-Xue; Wan, Fang-Hao
The Bemisia tabaci Mediterranean (MED) cryptic species has been rapidly invading to most parts of the world owing to its strong ecological adaptability, which is considered as a model insect for stress tolerance studies under rapidly changing environments. Selection of a suitable reference gene for quantitative stress-responsive gene expression analysis based on qRT-PCR is critical for elaborating the molecular mechanisms of thermotolerance. To obtain accurate and reliable normalization data in MED, eight candidate reference genes (β-act, GAPDH, β-tub, EF1-α, GST, 18S, RPL13A and α-tub) were examined under various thermal stresses for varied time periods by using geNorm, NormFinder and BestKeeper algorithms, respectively. Our results revealed that β-tub and EF1-α were the best reference genes across all sample sets. On the other hand, 18S and GADPH showed the least stability for all the samples studied. β-act was proved to be highly stable only in case of short-term thermal stresses. To our knowledge this was the first comprehensive report on validation of reference genes under varying temperature stresses in MED. The study could expedite particular discovery of thermotolerance genes in MED. Further, the present results can form the basis of further research on suitable reference genes in this invasive insect and will facilitate transcript profiling in other invasive insects. PMID:28323834
Dai, Tian-Mei; Lü, Zhi-Chuang; Liu, Wan-Xue; Wan, Fang-Hao
The Bemisia tabaci Mediterranean (MED) cryptic species has been rapidly invading to most parts of the world owing to its strong ecological adaptability, which is considered as a model insect for stress tolerance studies under rapidly changing environments. Selection of a suitable reference gene for quantitative stress-responsive gene expression analysis based on qRT-PCR is critical for elaborating the molecular mechanisms of thermotolerance. To obtain accurate and reliable normalization data in MED, eight candidate reference genes (β-act, GAPDH, β-tub, EF1-α, GST, 18S, RPL13A and α-tub) were examined under various thermal stresses for varied time periods by using geNorm, NormFinder and BestKeeper algorithms, respectively. Our results revealed that β-tub and EF1-α were the best reference genes across all sample sets. On the other hand, 18S and GADPH showed the least stability for all the samples studied. β-act was proved to be highly stable only in case of short-term thermal stresses. To our knowledge this was the first comprehensive report on validation of reference genes under varying temperature stresses in MED. The study could expedite particular discovery of thermotolerance genes in MED. Further, the present results can form the basis of further research on suitable reference genes in this invasive insect and will facilitate transcript profiling in other invasive insects.
sequences at the 3′- termini of cleaved mRNA fragments in human cells under physiological conditions. Our results demonstrated the great potential...siRNA action in mul- ticellular organisms under physiological conditions. In this study, we used a novel stem-loop array RT-PCR (SLA-RT-PCR) assay to... physiological condition in human cells. We used the synthetic siRNAs with an ectopically expressed mRNA target templates as testing models, and the wild-type
Wenzel, F.; Daniell, J.; Khazai, B.; Mühr, B.; Kunz-Plapp, T.; Markus, M.; Vervaeck, A.
The Center for Disaster Management and Risk Reduction Technology (CEDIM, www.cedim.de) - an interdisciplinary research center founded by the German Research Centre for Geoscience (GFZ) and Karlsruhe Institute of Technology (KIT) - has embarked on a new style of disaster research known as Forensic Disaster Analysis. The notion has been coined by the Integrated Research on Disaster Risk initiative (IRDR, www.irdrinternational.org) launched by ICSU in 2010. It has been defined as an approach to studying natural disasters that aims at uncovering the root causes of disasters through in-depth investigations that go beyond the reconnaissance reports and case studies typically conducted after disasters. In adopting this comprehensive understanding of disasters CEDIM adds a real-time component to the assessment and evaluation process. By comprehensive we mean that most if not all relevant aspects of disasters are considered and jointly analysed. This includes the impact (human, economy, and infrastructure), comparisons with recent historic events, social vulnerability, reconstruction and long-term impacts on livelihood issues. The forensic disaster analysis research mode is thus best characterized as "event-based research" through systematic investigation of critical issues arising after a disaster across various inter-related areas. The forensic approach requires (a) availability of global data bases regarding previous earthquake losses, socio-economic parameters, building stock information, etc.; (b) leveraging platforms such as the EERI clearing house, relief-web, and the many sources of local and international sources where information is organized; and (c) rapid access to critical information (e.g., crowd sourcing techniques) to improve our understanding of the complex dynamics of disasters. The main scientific questions being addressed are: What are critical factors that control loss of life, of infrastructure, and for economy? What are the critical interactions
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...
This research used reverse transcription polymerase chain reaction (RT-PCR) method to help detect active bacteria in a single-tank deammonification reactor combining partial nitritation and anammox. The single-tank aerobic deammonification reactor effectively removed the ammonia in anaerobically di...
Gutierrez, Laurent; Mauriat, Mélanie; Guénin, Stéphanie; Pelloux, Jérôme; Lefebvre, Jean-François; Louvet, Romain; Rusterucci, Christine; Moritz, Thomas; Guerineau, François; Bellini, Catherine; Van Wuytswinkel, Olivier
Reverse transcription-polymerase chain reaction (RT-PCR) approaches have been used in a large proportion of transcriptome analyses published to date. The accuracy of the results obtained by this method strongly depends on accurate transcript normalization using stably expressed genes, known as references. Statistical algorithms have been developed recently to help validate reference genes, and most studies of gene expression in mammals, yeast and bacteria now include such validation. Surprisingly, this important approach is under-utilized in plant studies, where putative housekeeping genes tend to be used as references without any appropriate validation. Using quantitative RT-PCR, the expression stability of several genes commonly used as references was tested in various tissues of Arabidopsis thaliana and hybrid aspen (Populus tremula x Populus tremuloides). It was found that the expression of most of these genes was unstable, indicating that their use as references is inappropriate. The major impact of the use of such inappropriate references on the results obtained by RT-PCR is demonstrated in this study. Using aspen as a model, evidence is presented indicating that no gene can act as a universal reference, implying the need for a systematic validation of reference genes. For the first time, the extent to which the lack of a systematic validation of reference genes is a stumbling block to the reliability of results obtained by RT-PCR in plants is clearly shown.
Recently developed universal external RNA controls allow comparison of expression data generated from microarray and real time qRT-PCR, including SYBR Green and TaqMan-probe based chemistry. It provides reliable controls for data normalization and analysis. In this study, we further developed stra...
Svingen, Terje; Letting, Heidi; Hadrup, Niels; Hass, Ulla; Vinggaard, Anne Marie
In biological research the analysis of gene expression levels in cells and tissues can be a powerful tool to gain insights into biological processes. For this, quantitative RT-PCR (RT-qPCR) is a popular method that often involve the use of constitutively expressed endogenous reference (or 'housekeeping') gene for normalization of data. Thus, it is essential to use reference genes that have been verified to be stably expressed within the specific experimental setting. Here, we have analysed the expression stability of 12 commonly used reference genes (Actb, B2m, Gapdh, Hprt, Pgk1, Rn18s, Rpl13a, Rps18, Rps29, Sdha, Tbp and Ubc) across several juvenile and adult rat tissues (liver, adrenal, prostate, fat pad, testis and ovaries), both under normal conditions and following exposure to various chemicals during development. Employing NormFinder and BestKeeper softwares, we found Hprt and Sdha to be amongst the most stable genes across normal and manipulated tissues, with several others also being suitable for most tissues. Tbp and B2m displayed highest variability in transcript levels between tissues and developmental stages. It was also observed that the reference genes were most unstable in liver and testis following toxicological exposure. For future studies, we propose the use of more than one verified reference gene and the continuous monitoring of their suitability under various experimental conditions, including toxicological studies, based on changes in threshold (Ct) values from cDNA samples having been reverse-transcribed from a constant input concentration of RNA.
Forrester, N L; Boag, B; Moss, S R; Turner, S L; Trout, R C; White, P J; Hudson, P J; Gould, E A
Because Rabbit haemorrhagic disease virus (RHDV) is highly pathogenic for rabbits, farmers illegally introduced it as a bio-control agent onto New Zealand farms in 1997. The virus was dispersed rapidly, initially causing high fatality rates in rabbits. Nevertheless, many survived and these surviving rabbits have been investigated for evidence of infection by RHDV. Livers from healthy rabbits contained RHDV-specific RNA, as shown by nested RT-PCR sequencing. The sequences of the viral capsids were related closely to the released Czech strain of RHDV, although the sequence from one rabbit was related most closely to a Spanish strain of RHDV. Phylogenetic analysis of the capsid sequences of 38 samples implied that there have been at least two introductions of the Czech virus into New Zealand, probably corresponding firstly to the original illegal introduction by farmers and secondly to the introduction of the same virus under governmental control. Genomic length sequence of two samples was obtained, suggesting that they may have retained the potential to be infectious, although this has not yet been demonstrated. The detection of genomic-length RNA in the liver of healthy rabbits suggests that even though a highly virulent virus was introduced into New Zealand, it rapidly established persistent or latent infections in a proportion of rabbits. This might account for their ability to survive in the face of virulent released virus. Moreover, the co-circulation of other strains of RHDV in the same rabbit population, such as the Spanish strain, might also impact on their susceptibility to the bio-control agent.
Kucharova, Veronika; Strand, Trine Aakvik; Almaas, Eivind; Naas, Adrian E.; Brautaset, Trygve; Valla, Svein
mRNA stability is one among many parameters that can potentially affect the level of recombinant gene expression in bacteria. Blocking of the entire prokaryotic transcription machinery by addition of rifampicin is commonly used in protocols for analysis of mRNA stability. Here we show that such treatment can be effectively replaced by a simple, non-invasive method based on removal of the relevant transcriptional inducers and that the mRNA decay can then be followed by qRT-PCR. To establish the methodology we first used the m-toluate-inducible XylS/Pm expression cassette as a model system and analyzed several examples of DNA modifications causing gene expression stimulation in Escherichia coli. The new method allowed us to clearly discriminate whether an improvement in mRNA stability contributes to observed increases in transcript amounts for each individual case. To support the experimental data a simple mathematical fitting model was developed to calculate relative decay rates. We extended the relevance of the method by demonstrating its application also for an IPTG-inducible expression cassette (LacI/Ptac) and by analyzing features of the bacteriophage T7-based expression system. The results suggest that the methodology is useful in elucidating factors controlling mRNA stability as well as other specific features of inducible expression systems. Moreover, as expression systems based on diffusible inducers are almost universally available, the concept can be most likely used to measure mRNA decay for any gene in any cell type that is heavily used in molecular biology research. PMID:23840466
Development of a rapid automated influenza A, influenza B, and respiratory syncytial virus A/B multiplex real-time RT-PCR assay and its use during the 2009 H1N1 swine-origin influenza virus epidemic in Milwaukee, Wisconsin.
Beck, Eric T; Jurgens, Lisa A; Kehl, Sue C; Bose, Michael E; Patitucci, Teresa; LaGue, Elizabeth; Darga, Patrick; Wilkinson, Kimberly; Witt, Lorraine M; Fan, Jiang; He, Jie; Kumar, Swati; Henrickson, Kelly J
Rapid, semiautomated, and fully automated multiplex real-time RT-PCR assays were developed and validated for the detection of influenza (Flu) A, Flu B, and respiratory syncytial virus (RSV) from nasopharyngeal specimens. The assays can detect human H1N1, H3N2, and swine-origin (S-OIV) H1N1 Flu A viruses and were effectively used to distinguish Flu A infections (of all subtypes) from Flu B and RSV infections during the current S-OIV outbreak in Milwaukee, WI. The analytical limits of detection were 10(-2) to 10(1) TCID(50)/ml depending on the platform and analyte and showed only one minor cross-reaction among 23 common respiratory pathogens (intermittent cross-reaction to adenovirus at >10(7) TCID(50)/ml). A total of 100 clinical samples were tested by tissue culture, both automated assays, and the US Food and Drug Administration-approved ProFlu+ assay. Both the semiautomated and fully automated assays exhibited greater overall (Flu A, Flu B, and RSV combined) clinical sensitivities (93 and 96%, respectively) and individual Flu A sensitivities (100%) than the Food and Drug Administration-approved test (89% overall sensitivity and 93% Flu A sensitivity). All assays were 99% specific. During the S-OIV outbreak in Milwaukee, WI, the fully automated assay was used to test 1232 samples in 2 weeks. Flu A was detected in 134 clinical samples (126 H1N1 S-OIV, 5 H1N1 [human], and 1 untyped) with 100% positive agreement compared with other "in-house" validated molecular assays, with only 2 false-positive results. Such accurate testing using automated high-throughput molecule systems should allow clinicians and public health officials to react quickly and effectively during viral outbreaks.
Wei, Libin; Miao, Hongmei; Zhao, Ruihong; Han, Xiuhua; Zhang, Tide; Zhang, Haiyang
Sesame (Sesamum indicum L.) is an ancient and important oilseed crop. However, few sesame reference genes have been selected for quantitative real-time PCR until now. Screening and validating reference genes is a requisite for gene expression normalization in sesame functional genomics research. In this study, ten candidate reference genes, i.e., SiACT, SiUBQ6, SiTUB, Si18S rRNA, SiEF1α, SiCYP, SiHistone, SiDNAJ, SiAPT and SiGAPDH, were chosen and examined systematically in 32 sesame samples. Three qRT-PCR analysis methods, i.e., geNorm, NormFinder and BestKeeper, were evaluated systematically. Results indicated that all ten candidate reference genes could be used as reference genes in sesame. SiUBQ6 and SiAPT were the optimal reference genes for sesame plant development; SiTUB was suitable for sesame vegetative tissue development, SiDNAJ for pathogen treatment, SiHistone for abiotic stress, SiUBQ6 for bud development and SiACT for seed germination. As for hormone treatment and seed development, SiHistone, SiCYP, SiDNAJ or SiUBQ6, as well as SiACT, SiDNAJ, SiTUB or SiAPT, could be used as reference gene, respectively. To illustrate the suitability of these reference genes, we analyzed the expression variation of three functional sesame genes of SiSS, SiLEA and SiGH in different organs using the optimal qRT-PCR system for the first time. The stability levels of optimal and worst reference genes screened for seed development, anther sterility and plant development were validated in the qRT-PCR normalization. Our results provided a reference gene application guideline for sesame gene expression characterization using qRT-PCR system.
Rubio-Guerri, Consuelo; Melero, Mar; Rivera-Arroyo, Belén; Bellière, Edwige Nina; Crespo, Jose Luis; García-Párraga, Daniel; Esperón, Fernando; Sánchez-Vizcaíno, Jose Manuel
A highly sensitive and specific real-time (rt) RT-PCR assay has been developed for rapid, simultaneous detection of three strains of cetacean morbillivirus (CeMV). In this assay, two PCR primers and a hydrolysis probe from a commercially available Universal Probe Library (UPL) are used to amplify a highly conserved region within the fusion protein gene. RT-PCR is carried out on the same sample using two primer sets in parallel: one set detects the more virulent strains, dolphin morbillivirus (DMV) and porpoise morbillivirus (PMV), and the other set detects the least virulent and least common strain, pilot whale morbillivirus (PWMV). Sensitivity analysis using dilute samples containing purified DMV, PMV and PWMV showed that viral RNA detection limits in this UPL RT-PCR assay were lower than in a conventional RT-PCR assay. Our method gave no amplification signal with field samples positive for viruses related and unrelated to CeMV, such as phocine distemper virus (PDV). The reliability and robustness of the UPL RT-PCR assay were verified using tissue samples previously analyzed by conventional methods, as well as a panel of clinical samples suspected of containing CeMV. Using the UPL RT-PCR assay, we were able to associate DMV with a mass stranding of striped dolphins in the Spanish Mediterranean in 2011 with greater reliability than was possible with a conventional RT-PCR method. These results suggest that this UPL RT-PCR method is more sensitive and specific than the conventional approach, and that it may be an affordable and rapid test for routine diagnosis of three CeMV strains.
Kang, Zhi-Wei; Liu, Fang-Hua; Tian, Hong-Gang; Zhang, Meng; Guo, Shan-Shan; Liu, Tong-Xian
The green peach aphid, Myzus persicae Sulzer (Hemiptera, Aphididae), is an important cosmopolitan pest. Real time qRT-PCR has been used for target gene expression analysis on M. persicae. Using real time qRT-PCR, the expression levels are normalized on the basis of the reliable reference genes. However, to date, the stability of available reference genes has been insufficient. In this study, we evaluated nine candidate reference genes from M. persicae under diverse experimental conditions. The tested candidate genes were comprehensively ranked based on five alternative methods (RefFinder, geNorm, Normfinder, BestKeeper and the comparative ΔCt method). 18s, Actin and ribosomal protein L27 (L27) were recommended as the most stable reference genes for M. persicae, whereas ribosomal protein L27 (L27) was found to be the least stable reference genes for abiotic studies (photoperiod, temperature and insecticide susceptibility). Our finding not only sheds light on establishing an accurate and reliable normalization of real time qRT-PCR data in M. persicae but also lays a solid foundation for further studies of M. persicae involving RNA interference and functional gene research.
The RNA genome of Hop stunt viroid (HSVd) contains five to six nucleotides in a variable (V) domain, called the cachexia expression motif, which is associated with pathogenic and non-pathogenic variants in citrus. Current methods to differentiate HSVd variants rely on lengthy greenhouse biological i...
Consolidated storage of swine manure is associated with the production of a variety of odors and emissions which result from anaerobic digestion of materials present in the manure. Methanogenic archaea are a diverse group of anaerobic microorganisms responsible for the production of methane. In th...
Sun, Meng; Lu, Ming-Xing; Tang, Xiao-Tian; Du, Yu-Zhou
The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR) is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study, seven candidate reference genes, 18S ribosomal RNA (18S rRNA), elongation factor 1 (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S13 (RPS13), ribosomal protein S20 (RPS20), tubulin (TUB), and β-actin (ACTB) were evaluated for their suitability in normalizing gene expression under different experimental conditions. The results indicated that three genes (RPS13, RPS20, and EF1) were optimal for normalizing gene expression in different insect tissues (head, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes, and salivary glands). 18S rRNA, EF1, and GAPDH were best for normalizing expression with respect to developmental stages and sex (egg masses; first, second, third, fourth, fifth, and sixth instar larvae; male and female pupae; and one-day-old male and female adults). 18S rRNA, RPS20, and TUB were optimal for fifth instars exposed to different temperatures (−8, −6, −4, −2, 0, and 27°C). To validate this recommendation, the expression profile of a target gene heat shock protein 83 gene (hsp83) was investigated, and results showed the selection was necessary and effective. In conclusion, this study describes reference gene sets that can be used to accurately measure gene expression in S. inferens. PMID:25585250
Sun, Meng; Lu, Ming-Xing; Tang, Xiao-Tian; Du, Yu-Zhou
The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR) is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study, seven candidate reference genes, 18S ribosomal RNA (18S rRNA), elongation factor 1 (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S13 (RPS13), ribosomal protein S20 (RPS20), tubulin (TUB), and β-actin (ACTB) were evaluated for their suitability in normalizing gene expression under different experimental conditions. The results indicated that three genes (RPS13, RPS20, and EF1) were optimal for normalizing gene expression in different insect tissues (head, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes, and salivary glands). 18S rRNA, EF1, and GAPDH were best for normalizing expression with respect to developmental stages and sex (egg masses; first, second, third, fourth, fifth, and sixth instar larvae; male and female pupae; and one-day-old male and female adults). 18S rRNA, RPS20, and TUB were optimal for fifth instars exposed to different temperatures (-8, -6, -4, -2, 0, and 27°C). To validate this recommendation, the expression profile of a target gene heat shock protein 83 gene (hsp83) was investigated, and results showed the selection was necessary and effective. In conclusion, this study describes reference gene sets that can be used to accurately measure gene expression in S. inferens.
A SYBR® Green-based real-time quantitative reverse transcription PCR (qRT-PCR) assay in combination with melt curve analysis (MCA) was developed for the detection of nine grapevine viruses. The detection limits for singleplex qRT-PCR for all nine grapevine viruses were determined to be in the range ...
Mérens, Audrey; Guérin, Philippe Jean; Guthmann, Jean-Paul; Nicand, Elisabeth
Biological samples collected in refugee camps during an outbreak of hepatitis E were used to compare the accuracy of hepatitis E virus RNA amplification by real-time reverse transcription-PCR (RT-PCR) for sera and dried blood spots (concordance of 90.6%). Biological profiles (RT-PCR and serology) of asymptomatic individuals were also analyzed.
Kuo, F. Y.
The Space Shuttle Main Engine (SSME) is a reusable, high performance, liquid rocket engine with variable thrust. The engine control system continuously monitors the engine parameters and issues propellant valve control signals in accordance with the thrust and mixture ratio commands. A real time engine simulation lab was installed at MSFC to verify flight software and to perform engine dynamic analysis. A real time engine model was developed on the AD100 computer system. This model provides sufficient fidelity on the dynamics of major engine components and yet simplified enough to be executed in real time. The hardware-in-the-loop type simulation and analysis becomes necessary as NASA is continuously improving the SSME technology, some with significant changes in the dynamics of the engine. The many issues of interfaces between new components and the engine can be better understood and be resolved prior to the firing of the engine. In this paper, the SSME real time simulation Lab at the MSFC, the SSME real time model, SSME engine and control system stability analysis, both in real time and non-real time is presented.
Parra, Macarena; Almeida, Eduardo; Boone, Travis; Jung, Jimmy; Schonfeld, Julie
The objective of NASA Ames Research Centers WetLab-2 Project is to place on the ISS a research platform capable of conducting gene expression analysis via quantitative real-time PCR (qRT-PCR) of biological specimens sampled or cultured on orbit. The project has selected a Commercial-Off-The-Shelf (COTS) qRT-PCR system, the Cepheid SmartCycler and will fly it in its COTS configuration. The SmartCycler has a number of advantages including modular design (16 independent PCR modules), low power consumption, rapid ramp times and the ability to detect up to four separate fluorescent channels at one time enabling multiplex assays that can be used for normalization and to study multiple genes of interest in each module. The team is currently working with Cepheid to enable the downlink of data from the ISS to the ground and provide uplink capabilities for programming, commanding, monitoring, and instrument maintenance. The project has adapted commercial technology to design a module that can lyse cells and extract RNA of sufficient quality and quantity for use in qRT-PCR reactions while using a housekeeping gene to normalize RNA concentration and integrity. The WetLab-2 system is capable of processing multiple sample types ranging from microbial cultures to animal tissues dissected on-orbit. The ability to conduct qRT-PCR on-orbit eliminates 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. The system can be used to validate terrestrial analyses of samples returned from ISS by providing on-orbit gene expression benchmarking prior to sample return. The ability to get on orbit data will provide investigators with the opportunity to adjust experiment parameters for subsequent trials based on the real-time data analysis without need for sample return and re-flight. Researchers will also be able to sample multigenerational changes in organisms. Finally, the system can be
Shoaf, S.; APS Engineering Support Division
A real-time image analysis system was developed for beam imaging diagnostics. An Apple Power Mac G5 with an Active Silicon LFG frame grabber was used to capture video images that were processed and analyzed. Software routines were created to utilize vector-processing hardware to reduce the time to process images as compared to conventional methods. These improvements allow for more advanced image processing diagnostics to be performed in real time.
Zhang, Yanjun; Mao, Haiyan; Yan, Juying; Wang, Xinying; Zhang, Lei; Guus, Koch; Li, Hui; Li, Zhen; Chen, Yin; Gong, Liming; Chen, Zhiping; Xia, Shichang
Recently, human deaths have resulted from infection with low-pathogenicity avian influenza virus H7N9 strains that have emerged recently in China. To strengthen H7N9 surveillance and outbreak control, rapid and reliable diagnostic methods are needed. To develop a sensitive quantitative real-time RT-PCR assay for rapid detection of H7N9 viral RNA, primers and AllGlo probes were designed to target the HA and NA genes of H7N9. Conserved sequences in the HA and NA genes were identified by phylogenic analysis and used as targets for H7N9 virus detection. The similarities of the targeted HA and NA gene sequences from different H7 and N9 influenza virus strains were 93.2-99.9 % and 96.0-99.6 %, respectively The specificity and sensitivity of the new multiplex real-time qRT-PCR was established. The test was used for the detection of viral RNA in human pharyngeal swabs and environmental samples. The detection limit of the multiplex qRT-PCR was estimated to be about 10(-1) TCID50/reaction. Finally, the diagnostic sensitivities of the multiplex qRT-PCR, virus isolation and TaqMan qRT-PCR were compared using pharyngeal swabs and environmental samples. These analyses yielded positive results in 46.7 %, 43.3 % and 20.0 % of the samples, respectively. The novel multiplex AllGlo qRT-PCR is a rapid and sensitive method to identify H7N9 virus in clinical and environmental samples and can be used to facilitate studies on the epidemiology of H7N9 virus.
Faulkner, C.; Shaw, A.; Wragg, C.; Greenslade, M.; Haynes, H.; Williams, H.; Lowis, S.; Williams, M.; Kurian, K.M.
INTRODUCTION: Pilocytic astrocytomas can show a wide morphological spectrum making definitive histological diagnosis challenging. The FISH test for KIAA1549-BRAF fusions is most commonly used, but this is difficult to interpret. We aimed to develop a real-time PCR (RT-PCR) test as a first-line screen for the three most common KIAA1549-BRAF fusion variants. METHOD: A RT-PCR method for detecting KIAA1549-BRAF fusions from formalin-fixed paraffin-embedded (FFPE) brain tumour tissues (pilocytic astrocytoma). The three most common fusion variants are detected using this assay: exon 16 of KIAA1549 fused to exon 9 of BRAF, exon 15 of KIAA1549 fused to exon 9 of BRAF and exon 16 of KIAA1549 fused to exon 11 of BRAF fusion. GAPDH expression was used as a control. RESULTS: The RT-PCR assay was initially validated on 12 samples previously tested by FISH or RT-PCR in a different laboratory. The RT-PCR assay had a sensitivity of 89% (8/9 - one sample tested positive by FISH but negative on RT-PCR) and a specificity of 100% (2/2). The failure rate was 8.3% (1/12). Sensitivity experiments showed that the fusion can be detected when present at a least 5% of the total cDNA content. 51 Neuropathology diagnostic FFPE samples from 42 pilocytic astrocytoma patients were then tested using the BRAF fusion RT-PCR assay. The overall pick-up rate was 54% (20/37 patients) Of the positive patients (20), 55% (11/20) had the 16-9 fusion and 45% (9/20) had the 15-9 fusion. Two patients had multiple fusions (2/20 positive patients, 10%) showing the 16-9 fusion and a low-level 16-11 fusion. No patients exclusively had the 16-11 fusion. CONCLUSION: We propose RTPCR first line for fusion analysis followed by FISH, for pilocytic astrocytoma.
Lossi, Laura; Gambino, Graziana; Salio, Chiara; Merighi, Adalberto
In situ polymerase chain reaction (PCR) is a histological technique that exploits the advantages of PCR for detection of mRNA directly in tissue sections. It somehow conjugates together PCR and in situ hybridization that is more traditionally employed for mRNA localization in cell organelles, intact cells, or tissue sections. This chapter describes the application of in situ PCR for neuropeptide mRNA localization. We provide here a detailed protocol for direct in situ reverse transcription (RT) PCR (RT-PCR) with nonradioactive probes after fixation and paraffin embedding or cryosectioning. Digoxigenin-labeled nucleotides (digoxigenin-11-dUTP) are incorporated in the PCR product after RT and subsequently detected with an anti-digoxigenin antibody conjugated with alkaline phosphatase. The procedure can be modified for use with fluorescent probes and employed in combination with enzyme/fluorescence immunocytochemical labeling.
Cassol, Daniela; Cruz, Fernanda P; Espindola, Kauê; Mangeon, Amanda; Müller, Caroline; Loureiro, Marcelo Ehlers; Corrêa, Régis L; Sachetto-Martins, Gilberto
Quantitative real-time PCR (RT-qPCR) is one of the most powerful and sensitive techniques to the study of gene expression. Several factors influence RT-qPCR performance though, including the stability of the reference genes used for data normalization. While the selection of appropriate reference genes is crucial for accurate and reliable gene expression analysis, no suitable reference genes have been previously identified in castor bean under drought stress. In this study, the expression stability of eleven mRNAs, thirteen microRNAs (miRNAs) and one small nuclear RNA were analyzed in roots and leaves across different levels of water deficit. Three different algorithms were employed to analyze the RT-qPCR data, and the resulting outputs were merged using a non-weighted unsupervised rank aggregation method. Our analysis indicated that the Elongation factor 1-beta (EF1B), Protein phosphatase 2A (PP2A) and ADP-ribosylation factor (ADP) ranked as the best candidates across diverse samples submitted to different levels of drought conditions. EF1B and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and EF1B and SKP1/ASK-interacting protein 16 (SKIP16) were found as the most suitable reference genes for expression analysis in roots and leaves, respectively. In addition, miRNAs miR168, miR160 and miR397 were selected as optimal reference genes across all tissues and treatments. miR168 and miR156 were recommended as reference for roots, while miR168 and miR160 were recommended for leaves. Together, our results constitute the first attempt to identify and validate the most suitable reference genes for accurate normalization of gene expression in castor bean under drought stress.
Liu, Juanjuan; Tan, Yang; Yang, Xiaohong; Chen, Xiaohua; Li, Fuli
Clostridium ljungdahlii DSM 13528 is a promising platform organism for biofuel production from syngas. Gene expression analysis permits a better understanding of the important molecular biological characteristics of this organism, such as carbon fixation and solvent adaptation. Normalization is a prerequisite for accurate gene expression analysis, but until now, no valid reference genes have been proposed for quantitative real-time polymerase chain reaction (qRT-PCR) analysis of C. ljungdahlii DSM 13528. In this study, seven candidate reference genes (gyrA, rho, fotl, rpoA, gukl, recA, 16S rRNA) were selected for qRT-PCR quantification of their expression levels in various culture conditions that corresponded to different carbon sources and stresses. Two analytical programs, geNorm and NormFinder, were used to evaluate reference gene stability. The results showed that gyrA, rho and fotl exhibited the most stable expression levels across all tested samples and can be confidently used as reference genes to normalize the transcriptional data of target genes in qRT-PCR analyses of C. ljungdahlii DSM 13528. This study presents the first attempt to explore the validity of candidate reference genes and provide a set of valid reference genes for normalizing C. ljungdahlii DSM 13528 target gene expression and transcriptome analysis.
Arthrospira (Spirulina) platensis as a representative species of cyanobacteria has been recognized and used worldwide as a source of protein in the food, which possesses some unusual and valuable physiological characteristics, such as alkali and salt tolerance. Based on complete genome sequencing of Arthrospira (Spirulina) plantensis-YZ, we compared the protein expression profiles of this organism under different salt-stress conditions (i.e. 0.02 M, 0.5 M and 1.0 M NaCl, respectively), using 2-D electrophoresis and peptide mass fingerprinting, and retrieved 141 proteins showing significantly differential expression in response to salt-stress. Of the 141 proteins, 114 Arthrospira (Spirulina) plantensis-YZ proteins were found with significant homology to those found in Arthrospira (76 proteins in Arthrospira platensis str. Paraca and 38 in Arthrospira maxima CS-328). The remaining 27 proteins belong to other bacteria. Subsequently, we determined the transcriptional level of 29 genes in vivo in response to NaCl treatments and verified them by qRT-PCR. We found that 12 genes keep consistency at both transcription and protein levels, and transcription of all of them but one were up-regulated. We classified the 141 differentially expressed proteins into 18 types of function categories using COG database, and linked them to their respective KEGG metabolism pathways. These proteins are involved in 31 metabolism pathways, such as photosynthesis, glucose metabolism, cysteine and methionine metabolism, lysine synthesis, fatty acid metabolism, glutathione metabolism. Additionally, the SRPs, heat shock protein and ABC transporter proteins were identified, which probably render Arthrospira (Spirulina) plantensis’s resistance against high salt stress. PMID:23363438
Tham, Nguyen thi; Hang, Vu thi Ty; Khanh, Trong Huu; Viet, Do Chau; Hien, Tran Tinh; Farrar, Jeremy; van Vinh Chau, Nguyen; van Doorn, H. Rogier
Background Real-time PCR can be considered the gold standard for detection of influenza viruses due to its high sensitivity and specificity. Roche has developed the RealTime ready Influenza A/H1N1 Detection Set, consisting of a generic influenza virus A PCR targeting the M2 gene (M2 PCR) and a specific PCR targeting the HA of A/H1N1-pdm09 (HA PCR, 2009 H1N1), with the intention to make a reliable, rapid, and simple test to detect and quantify 2009 H1N1 in clinical samples. Methods We evaluated this kit against the USCDC/WHO real-time PCR for influenza virus using 419 nose and throat swabs from 210 patients collected in 3 large hospitals in Ho Chi Minh city, Vietnam. Results In the per patient analysis, when compared to CDC PCR, the sensitivity and specificity of the M2 PCR were 85.8 and 97.6%, respectively; the sensitivity and specificity of HA PCR were 88.2 and 100%, respectively. In the per sample analysis, the sensitivity and specificity in nose swabs were higher than in throat swabs for both M2 and HA PCRs. The viral loads as determined with the M2 and HA PCRs correlated well with the Ct values of the CDC PCR. Conclusion Compared with the CDC PCR, the kit has a reasonable sensitivity and very good specificity for the detection and quantification of Influenza A virus and A/H1N1-pdm09. However, given the current status of 2009 H1N1, a kit that can detect all circulating seasonal influenza viruses would be preferable. PMID:22785431
Tekin, Nilgun; Conget, Paulette; Bruna, Flavia; Timar, Botond; Gagyi, Eva; Basak, Ranjan; Naik, Omkar; Auewarakul, Chirayu; Sritana, Narongrit; Levy, Debora; Cerci, Juliano Julio; Bydlowski, Sergio Paulo; Pereira, Juliana; Dimamay, Mark Pierre; Natividad, Filipinas; Chung, June-Key; Belder, Nevin; Kuzu, Isinsu; Paez, Diana; Dondi, Maurizio; Carr, Robert
As a part of an international study on the molecular analysis of Diffuse Large B-cell Lymphoma (DLBCL), a robust protocol for gene expression analysis from RNA extraction to qRT-PCR using Formalin Fixed Paraffin Embedded tissues was developed. Here a study was conducted to define a strategy to validate the previously reported 6-gene (LMO2, BCL6, FN1, CCND2, SCYA3 and BCL2) model as predictor of prognosis in DLBCL. To avoid variation, all samples were tested in a single centre and single platform. This study comprised 8 countries (Brazil, Chile, Hungary, India, Philippines, S. Korea, Thailand and Turkey). Using the Kaplan-Meier and log rank test on patients (n=162) and two mortality risk groups (with those above and below the mean representing high and low risk groups) confirmed that the 6-gene predictor score correlates significantly with overall survival (OS, p<0.01) but not with event free survival (EFS, p=0.18). Adding the International Prognostic Index (IPI) shows that the 6-gene predictor score correlates significantly with high IPI scores for OS (p<0.05), whereas those with low IPI scores show a trend not reaching significance (p=0.08). This study defined an effective and economical qRT-PCR strategy and validated the 6-gene score as a predictor of OS in an international setting. PMID:27825111
Tekin, Nilgun; Omidvar, Nader; Morris, Tim Peter; Conget, Paulette; Bruna, Flavia; Timar, Botond; Gagyi, Eva; Basak, Ranjan; Naik, Omkar; Auewarakul, Chirayu; Sritana, Narongrit; Levy, Debora; Cerci, Juliano Julio; Bydlowski, Sergio Paulo; Pereira, Juliana; Dimamay, Mark Pierre; Natividad, Filipinas; Chung, June-Key; Belder, Nevin; Kuzu, Isinsu; Paez, Diana; Dondi, Maurizio; Carr, Robert; Ozdag, Hilal; Padua, Rose Ann
As a part of an international study on the molecular analysis of Diffuse Large B-cell Lymphoma (DLBCL), a robust protocol for gene expression analysis from RNA extraction to qRT-PCR using Formalin Fixed Paraffin Embedded tissues was developed. Here a study was conducted to define a strategy to validate the previously reported 6-gene (LMO2, BCL6, FN1, CCND2, SCYA3 and BCL2) model as predictor of prognosis in DLBCL. To avoid variation, all samples were tested in a single centre and single platform. This study comprised 8 countries (Brazil, Chile, Hungary, India, Philippines, S. Korea, Thailand and Turkey). Using the Kaplan-Meier and log rank test on patients (n=162) and two mortality risk groups (with those above and below the mean representing high and low risk groups) confirmed that the 6-gene predictor score correlates significantly with overall survival (OS, p<0.01) but not with event free survival (EFS, p=0.18). Adding the International Prognostic Index (IPI) shows that the 6-gene predictor score correlates significantly with high IPI scores for OS (p<0.05), whereas those with low IPI scores show a trend not reaching significance (p=0.08). This study defined an effective and economical qRT-PCR strategy and validated the 6-gene score as a predictor of OS in an international setting.
Prompt detection of influenza A and B viruses using the BD Veritor™ System Flu A+B, Quidel® Sofia® Influenza A+B FIA, and Alere BinaxNOW® Influenza A&B compared to real-time reverse transcription-polymerase chain reaction (RT-PCR).
Dunn, Jim; Obuekwe, Joy; Baun, Traci; Rogers, Justin; Patel, Twinkle; Snow, Linda
The performance characteristics of rapid influenza diagnostic tests vary widely. This study evaluated the BD Veritor™ System Flu A+B (Veritor; BD Diagnostics, Sparks, MD, USA), Quidel® Sofia® Influenza A+B FIA (Sofia; Quidel Corp., San Diego, CA, USA), and Alere BinaxNOW® Influenza A&B (Binax; Alere Scarborough, Inc., Scarborough, ME, USA) compared to reverse transcription-polymerase chain reaction (RT-PCR) for detection of influenza viruses in nasal wash specimens from 240 pediatric patients. Positive percent agreements for influenza A and B virus detection were 93.8% and 94.2%, 95.8% and 98.1%, and 79.2% and 80.8% for Veritor, Sofia, and Binax, respectively. The Veritor and Binax tests demonstrated negative percent agreements >97.9% for detection of both influenza viruses, but the negative percent agreement of the Sofia test was 91.1% for influenza A and 70.7% for influenza B virus. Overall, the Veritor and Sofia tests were nearly as sensitive as RT-PCR and considerably more sensitive than Binax for detection of influenza viruses. However, the accuracy of the Sofia test was significantly lower than either Veritor or Binax.
Optical detection of bacteria has been approached in recent years as a bacteria detection method that can counter time restraints of traditional plating or the high reoccurring cost of real-time polymerase chain reaction (RT-PCR). The goal of optical detection is to identify bacteria with spectral s...
Spinsanti, Giacomo; Panti, Cristina; Lazzeri, Elisa; Marsili, Letizia; Casini, Silvia; Frati, Francesco; Fossi, Cristina Maria
Background Odontocete cetaceans occupy the top position of the marine food-web and are particularly sensitive to the bioaccumulation of lipophilic contaminants. The effects of environmental pollution on these species are highly debated and various ecotoxicological studies have addressed the impact of xenobiotic compounds on marine mammals, raising conservational concerns. Despite its sensitivity, quantitative real-time PCR (qRT-PCR) has never been used to quantify gene induction caused by exposure of cetaceans to contaminants. A limitation for the application of qRT-PCR is the need for appropriate reference genes which allow the correct quantification of gene expression. A systematic evaluation of potential reference genes in cetacean skin biopsies is presented, in order to validate future qRT-PCR studies aiming at using the expression of selected genes as non-lethal biomarkers. Results Ten commonly used housekeeping genes (HKGs) were partially sequenced in the striped dolphin (Stenella coeruleoalba) and, for each gene, PCR primer pairs were specifically designed and tested in qRT-PCR assays. The expression of these potential control genes was examined in 30 striped dolphin skin biopsy samples, obtained from specimens sampled in the north-western Mediterranean Sea. The stability of selected control genes was determined using three different specific VBA applets (geNorm, NormFinder and BestKeeper) which produce highly comparable results. Glyceraldehyde-3P-dehydrogenase (GAPDH) and tyrosine 3-monooxygenase (YWHAZ) always rank as the two most stably expressed HKGs according to the analysis with geNorm and Normfinder, and are defined as optimal control genes by BestKepeer. Ribosomal protein L4 (RPL4) and S18 (RPS18) also exhibit a remarkable stability of their expression levels. On the other hand, transferrin receptor (TFRC), phosphoglycerate kinase 1 (PGK1), hypoxanthine ribosyltransferase (HPRT1) and β-2-microglobin (B2M) show variable expression among the studied
Zhao, Xiting; Zhang, Xiaoli; Guo, Xiaobo; Li, Shujie; Han, Linlin; Song, Zhihui; Wang, Yunying; Li, Junhua; Li, Mingjun
Quantitative real-time polymerase chain reaction (qRT-PCR) is one of the most common methods for gene expression studies. Data normalization based on reference genes is essential for obtaining reliable results for qRT-PCR assays. This study evaluated potential reference genes of Chinese yam (Dioscorea opposita Thunb.), which is an important tuber crop and medicinal plant in East Asia. The expression of ten candidate reference genes across 20 samples from different organs and development stages was assessed. We identified the most stable genes for qRT-PCR studies using combined samples from different organs. Our results also suggest that different suitable reference genes or combinations of reference genes for normalization should be applied according to different organs and developmental stages. To validate the suitability of the reference genes, we evaluated the relative expression of PE2.1 and PE53, which are two genes that may be associated with microtuber formation. Our results provide the foundation for reference gene(s) selection in D. opposita and will contribute toward more accurate gene analysis studies of the genus Dioscorea. PMID:27314014
Zhao, Xiting; Zhang, Xiaoli; Guo, Xiaobo; Li, Shujie; Han, Linlin; Song, Zhihui; Wang, Yunying; Li, Junhua; Li, Mingjun
Quantitative real-time polymerase chain reaction (qRT-PCR) is one of the most common methods for gene expression studies. Data normalization based on reference genes is essential for obtaining reliable results for qRT-PCR assays. This study evaluated potential reference genes of Chinese yam (Dioscorea opposita Thunb.), which is an important tuber crop and medicinal plant in East Asia. The expression of ten candidate reference genes across 20 samples from different organs and development stages was assessed. We identified the most stable genes for qRT-PCR studies using combined samples from different organs. Our results also suggest that different suitable reference genes or combinations of reference genes for normalization should be applied according to different organs and developmental stages. To validate the suitability of the reference genes, we evaluated the relative expression of PE2.1 and PE53, which are two genes that may be associated with microtuber formation. Our results provide the foundation for reference gene(s) selection in D. opposita and will contribute toward more accurate gene analysis studies of the genus Dioscorea.
Wijnands, T.; Parlange, F.; Couturier, B.; Moulin, D.
Four different techniques for the fast analysis of multichannel data in plasma physics are discussed. All four of these techniques are general and sufficiently fast to be used in real time applications. Function parametrization, canonical correlation analysis and a neural network of the multilayer perceptron (MLP) type are compared with a unique linear mapping based on a singular value decomposition, which is used as a reference. Applications deal with the identification of the plasma boundary and some global plasma parameters in the DIII-D and the Tore Supra tokamaks by using magnetic measurements. The results of an MLP-1 neural network, employed for the real time plasma position determination in Tore Supra, are presented
Yang, Mo; Reilly, P.T.A.; Gieray, R.A.; Whitten, W.B.; Ramsey, J.M.
Real-time mass spectrometry of individual aerosol particles using an ion trap mass spectrometer is described. The microparticles are sampled directly from the air by a particle inlet system into the vacuum chamber. An incoming particle is detected as it passes through two CW laser beams and a pulsed laser is triggered to intercept the particle for laser ablation ionization at the center of the ion trap. The produced ions are analyzed by the ion trap mass spectrometer. Ions of interest are selected and dissociated through collision with buffer gas atoms for further fragmentation analysis. Real-time chemical analyses of inorganic, organic, and bacterial aerosol articles have been demonstrated. It has been confirmed that the velocity and the size of the incoming particles highly correlate to each other. The performance of the inlet system, particle detection, and preliminary results are discussed.
Carranza-Fulmer, T. L.; Gannon, J. L.; Love, J. J.
The Dst is commonly used to specify geomagnetic disturbance periods and characterize the resulting ring current enhancements from ground-based horizontal magnetic field intensity measurements. Real-time versions of the Dst index are produced for operational purposes, and are of interest to many users, including the US military, airline industry, and power companies. USGS Real time Dst, Kyoto Quicklook Dst, and Space Environment Corporation RDst use preliminary data and use a variety of contributing observatories and processing methods. Both USGS and RDst use a combined time-and-frequency domain method and Kyoto uses a time domain only method in creating the Dst index. We perform an analysis of these three real time Dst indices for the time period of October 1, 2009 to May 31, 2010. The USGS 3, using three observatories instead of the standard four, and the Kyoto Sym-H index, are introduced in the analysis for comparison of observatory location with the three main Dst indices. We present a statistical study of the differences due to algorithm, output time resolution, and location of contributing observatories. Higher time resolution shows higher frequency fluctuations during disturbances and more defined storm features. There were small differences in mid- to low-latitude observatories during quiet to moderate storm time periods. The average impact on the index due to the different algorithms used was approximately 9 nT, and greater for individual storms.
Kunz, Michael; Zschau, Jochen; Wenzel, Friedemann; Khazai, Bijan; Kunz-Plapp, Tina; Trieselmann, Werner
The impacts of extreme hydro-meteorological and geophysical events are controlled by various factors including severity of the event (intensity, duration, spatial extent), amplification with other phenomena (multihazard or cascading effects), interdependencies of technical systems and infrastructure, preparedness and resilience of the society. The Center for Disaster Management and Risk Reduction Technology (CEDIM) has adopted the comprehensive understanding of disasters and develops methodologies of near real-time FDA as a complementing component of the FORIN program of IRDR. The new research strategy 'Near Real-Time Forensic Disaster Analysis (FDA)' aims at scrutinizing disasters closely with a multi-disciplinary approach in order to assess the various aspects of disasters and to identify mechanisms most relevant for an extreme event to become a disaster (e.g., causal loss analysis). Recent technology developments - which have opened unprecedented opportunities for real-time hazard, vulnerability and loss assessment - are used for analyzing disasters and their impacts in combination with databases of historical events. The former covers modern empirical and analytical methods available in engineering and remote sensing for rapid impact assessments, rapid information extraction from crowd sourcing as well as rapid assessments of socio-economic impacts and economic losses. The event-driven science-based assessments of CEDIM are compiled based on interdisciplinary expertise and include the critical evaluation, assessment, validation, and quantification of an event. An important component of CEDIM's FDA is the near real-time approach which is expected to significantly speed up our understanding of natural disasters and be used to provide timely, relevant and valuable information to various user groups within their respective contexts. Currently, CEDIM has developed models and methodologies to assess different types of hazard. These approaches were applied to several
Chan, Wai-Fong; Maharjan, Ram P; Reeves, Peter R; Sintchenko, Vitali; Gilbert, Gwendolyn L; Lan, Ruiting
Real Time-PCR (RT-PCR) and high resolution melt (HRM) analyses were used for rapid typing of genes encoding components of the pertussis acellular vaccine, namely prn, ptxA, fhaB, fim2 and fim3. The length polymorphisms in prn were detected by RT-PCR followed by HRM; single nucleotide polymorphisms in prn and other genes were detected by hairpin primer RT-PCR. These rapid methods are suitable for large-scale studies of vaccine-driven evolution of Bordetella pertussis.
Hamlet, Stephen M
The development of analytical methods enabling the accurate identification and enumeration of bacterial species colonizing the oral cavity has led to the identification of a small number of bacterial pathogens that are major factors in the etiology of periodontal disease. Further, these methods also underpin more recent epidemiological analyses of the impact of periodontal disease on general health. Given the complex milieu of over 700 species of microorganisms known to exist within the complex biofilms found in the oral cavity, the identification and enumeration of oral periodontopathogens has not been an easy task. In recent years however, some of the intrinsic limitations of the more traditional microbiological analyses previously used have been overcome with the advent of immunological and molecular analytical methods. Of the plethora of methodologies reported in the literature, the enzyme-linked immunosorbent assay (ELISA), which combines the specificity of antibody with the sensitivity of simple enzyme assays and the polymerase chain reaction (PCR), has been widely utilized in both laboratory and clinical applications. Although conventional PCR does not allow quantitation of the target organism, real-time PCR (rtPCR) has the ability to detect amplicons as they accumulate in "real time" allowing subsequent quantitation. These methods enable the accurate quantitation of as few as 10(2) (using rtPCR) to 10(4) (using ELISA) periodontopathogens in dental plaque samples.
Nagaraj, Sowmya; Ramlal, Shylaja; Venkataswamachari, Bhavani Peddayelachagiri; Paul, Soumya; Kingston, Joseph; Batra, Harsh Vardhan
In spite of their involvement in foodborne illness, the epidemiological relevance of staphylococcal enterotoxin C (SEC) subtypes is poorly documented may be due to high sequence similarity. Among subtypes, SEC1, SEC2, and SEC3 exhibit more than 97 % homology because of which specific detection tools are seldom available to identify and differentiate them. In this study, a SYBR Green-based RT-PCR followed by melt curve analysis was developed for differentiation of entC1 from entC2/entC3 using a single primer pair. Nucleotide sequences of all three subtypes were analyzed using Clustal Omega program and the region with significant sequence variation/heterogeneity (where utmost SNPs were closely located and accessible for RT-PCR) was selected for amplification by designing a single primer pair that could amplify all three subtypes. In spite of same amplicon size, entC1 showed distinct melt peak at 76 °C. However, due to high similarity between entC2 and entC3, the developed format was deficient to discriminate between them and both showed melt peak at 82 °C. Reliability of developed RT-PCR was evaluated using various naturally contaminated samples and 91 food and clinical Staphylococcus aureus isolates where satisfactory results were obtained in comparison with commercial immunoassay kit and conventional PCRs using validated primers. To the best of our knowledge, this is the first method being reported to differentiate entC1 from entC2/entC3 using single primer pair which is unachievable by conventional PCR due to same amplicon size. As benefits, the method is sensitive, rapid, and inexpensive with no requirement of fluorescent probes, multiple primers, and post-PCR procedures. Thus, the assay might find its utility as a detection tool in epidemiological survey of foodborne outbreaks for simultaneous identification and differentiation of entC1 from entC2/entC3.
The intelligent system for telemetry analysis in real-time (ISTAR) is an advanced vehicle monitoring environment incorporating expert systems, analysis tools, and on-line hypermedia documentation. The system was developed for the Air Force Space and Missile Systems Center (SMC) in Los Angeles, California, in support of the inertial upper stage (IUS) booster vehicle. Over a five year period the system progressed from rapid prototype to operational system. ISTAR has been used to support five IUS missions and countless mission simulations. There were a significant number of lessons learned with respect to integrating an expert system capability into an existing ground system.
Glasser, Alexander; Kolemen, Egemen; Glasser, Alan
ITER is intended to operate at plasma parameters approaching the frontier of achievable stability limits. And yet, plasma disruptions at ITER must be kept to a bare minimum to avoid damage to its plasma-facing structures. These competing goals necessitate real-time plasma stability analysis and feedback control at ITER. This work aims to develop a mechanism for real-time analysis of a large and virulent class of disruptions driven by the rapid growth of ideal MHD unstable modes in tokamak equilibria. Such modes will be identified by a parallelized, low-latency implementation of A.H. Glasser's well-tested DCON (Direct Criterion of Newcomb) code, which measures the energetics of modes in the bulk plasma fluid, as well as M.S. Chance's VACUUM code, which measures the same in the vacuum between the plasma and tokamak chamber wall. Parallelization of these codes is intended to achieve a time-savings of 40x, thereby reducing latency to a timescale of order 100ms and making the codes viable for ideal MHD stability control at ITER. The hardware used to achieve this parallelization will be an Intel Xeon Phi server with 77 cores (308 threads). Supported by the US DOE under DE-AC02-09CH11466.
Heider, Susanne; Metzner, Christoph
Providing information about single virus particles has for a long time been mainly the domain of electron microscopy. More recently, technologies have been developed—or adapted from other fields, such as nanotechnology—to allow for the real-time quantification of physical virion particles, while supplying additional information such as particle diameter concomitantly. These technologies have progressed to the stage of commercialization increasing the speed of viral titer measurements from hours to minutes, thus providing a significant advantage for many aspects of virology research and biotechnology applications. Additional advantages lie in the broad spectrum of virus species that may be measured and the possibility to determine the ratio of infectious to total particles. A series of disadvantages remain associated with these technologies, such as a low specificity for viral particles. In this review we will discuss these technologies by comparing four systems for real-time single virus particle analysis and quantification. - Highlights: • We introduce four methods for virus particle-based quantification of viruses. • They allow for quantification of a wide range of samples in under an hour time. • The additional measurement of size and zeta potential is possible for some.
Marhavý, Peter; Benková, Eva
Plants maintain capacity to form new organs such as leaves, flowers, lateral shoots and roots throughout their postembryonic lifetime. Lateral roots (LRs) originate from a few pericycle cells that acquire attributes of founder cells (FCs), undergo series of anticlinal divisions, and give rise to a few short initial cells. After initiation, coordinated cell division and differentiation occur, giving rise to lateral root primordia (LRP). Primordia continue to grow, emerge through the cortex and epidermal layers of the primary root, and finally a new apical meristem is established taking over the responsibility for growth of mature lateral roots [for detailed description of the individual stages of lateral root organogenesis see Malamy and Benfey (1997)]. To examine this highly dynamic developmental process and to investigate a role of various hormonal, genetic and environmental factors in the regulation of lateral root organogenesis, the real time imaging based analyses represent extremely powerful tools (Laskowski et al., 2008; De Smet et al., 2012; Marhavý et al., 2013 and 2014). Herein, we describe a protocol for real time lateral root primordia (LRP) analysis, which enables the monitoring of an onset of the specific gene expression and subcellular protein localization during primordia organogenesis, as well as the evaluation of the impact of genetic and environmental perturbations on LRP organogenesis. PMID:27331080
Muratore, John F.
During early manned spacecraft operations, the primary role of ground telemetry systems was data display to flight controllers. As manned spaceflights have increased in complexity, greater demands have been placed on flight controllers to simultaneously monitor systems and replan systems operations. This has led to interest in automated telemetry monitoring systems to decrease the workload on flight controllers. The Mission Operations Directorate at the Lyndon B. Johnson Space Center has developed a five layer model to integrate various monitoring and analysis technologies such as digital filtering, fault detection algorithms, and expert systems. The paper describes the five layer model and explains how it has been used to guide prototyping efforts at Mission Control. Results from some initial expert systems are presented. The paper also describes the integrated prototype currently under development which implements a real time expert system to assist flight controllers in the Mission Control Center in monitoring Space Shuttle communications systems.
Duong, Tuan; Duong, Vu
Dominant-element-based gradient descent and dynamic initial learning rate (DOGEDYN) is a method of sequential principal-component analysis (PCA) that is well suited for such applications as data compression and extraction of features from sets of data. In comparison with a prior method of gradient-descent-based sequential PCA, this method offers a greater rate of learning convergence. Like the prior method, DOGEDYN can be implemented in software. However, the main advantage of DOGEDYN over the prior method lies in the facts that it requires less computation and can be implemented in simpler hardware. It should be possible to implement DOGEDYN in compact, low-power, very-large-scale integrated (VLSI) circuitry that could process data in real time.
Karbon, Maria; Soja, Benedikt; Nilson, Tobias; Heinkelmann, Robert; Liu, Li; Lu, Ciuxian; Xu, Minghui; Raposo-Pulido, Virginia; Mora-Diaz, Julian; Schuh, Harald
Geodetic Very Long Baseline Interferometry (VLBI) is one of the primary space geodetic techniques. It provides the full set of Earth Orientation Parameter (EOP) and is unique for observing long term Universal Time (UT1) and precession/nutation. Currently the VLBI products are delivered with a delay of about two weeks from the moment of the observation. However, the need for near-real time estimates of the parameters is increasing, e.g. for satellite based navigation and positioning or for enabling precise tracking of interplanetary spacecraft. The goal is thus to reduce the time span between observation and the final result to less than one day. This can be archived by replacing the classical least squares method with an adaptive Kalman filter. We have developed a Kalman filter for VLBI data analysis. This method has the advantage that it is simultaneously possible to estimate stationary parameters, e.g. station positions, and to model the highly variable stochastic behavior of non-stationary parameters like clocks or atmospheric parameters. The filter is able to perform without any human interaction, making it a completely autonomous tool. In this work we describe the filter and discuss its application for EOP determination and prediction. We discuss the implementation of the stochastic models to statistically account for unpredictable changes in EOP. Furthermore, additional data like results from other techniques can be included to improve the performance. For example, atmospheric angular momentum calculated from numerical weather models can be introduced to supplement the short-term prediction of UT1 and polar motion. This Kalman filter will be extended and embedded in the newly developed Vienna VLBI Software (VieVS) as a completely autonomous tool enabling the VLBI analysis in near real-time and providing all the parameters of interest with the highest possible accuracy.
Lin, Pengfei; Lan, Xiangli; Chen, Fenglei; Yang, Yanzhou; Jin, Yaping; Wang, Aihua
The study of uterine gene expression patterns is valuable for understanding the biological and molecular mechanisms that occur during embryo implantation. Real-time quantitative RT-PCR (qRT-PCR) is an extremely sensitive technique that allows for the precise quantification of mRNA abundance; however, selecting stable reference genes suitable for the normalization of qRT-PCR data is required to avoid the misinterpretation of experimental results and erroneous analyses. This study employs several mouse models, including an early pregnancy, a pseudopregnancy, a delayed implantation and activation, an artificial decidualization and a hormonal treatment model; ten candidate reference genes (PPIA, RPLP0, HPRT1, GAPDH, ACTB, TBP, B2M, 18S, UBC and TUBA) that are found in uterine tissues were assessed for their suitability as internal controls for relative qRT-PCR quantification. GeNorm(PLUS), NormFinder, and BestKeeper were used to evaluate these candidate reference genes, and all of these methods identified RPLP0 and GAPDH as the most stable candidates and B2M and 18S as the least stable candidates. However, when the different models were analyzed separately, the reference genes exhibited some variation in their expression levels.
Xiong, Fusheng; Frasch, Wayne D.
Padlock probe-mediated quantitative real time PCR (PLP-qRT-PCR) was adapted to quantify the abundance of sequential 10mer DNA sequences for use in DNA computing to identify optimal answers of traveling salesman problems. The protocol involves: (i) hybridization of a linear PLP with a target DNA sequence; (ii) PLP circularization through enzymatic ligation; and (iii) qRT-PCR amplification of the circularized PLP after removal of non-circularized templates. The linear PLP was designed to consist of two 10-mer sequence-detection arms at the 5′ and 3′ ends separated by a core sequence composed of universal PCR primers, and a qRT-PCR reporter binding site. Circularization of each PLP molecule is dependent upon hybridization with target sequence and high-fidelity ligation. Thus, the number of PLP circularized is determined by the abundance of target in solution. The amplification efficiency of the PLP was 98.7% within a 0.2 pg–20 ng linear detection range between thermal cycle threshold (Ct value) and target content. The Ct values derived from multiplex qRT-PCR upon three targets did not differ significantly from those obtained with singleplex assays. The protocol provides a highly sensitive and efficient means for the simultaneous quantification of multiple short nucleic acid sequences that has a wide range of applications in biotechnology. PMID:21691417
Smith, Peter R.; Eustaquio-Martin, Almudena; Thomason, Harry; Bennett, M.; Thurston, H.
The physiology and pathology of dissected blood vessels are studied by perfusion myography combined with video microscopy. Images of the vessels are formed under diffuse white light illumination and contrast is achieved by differential absorption with respect to the vessel wall. To obtain the vessel dimensional information in quasi real time an edge-tracking algorithm is used, allowing the edges to be found by applying common image processing tools to a very small number of pixels rather than the whole image. Employing a low order optical model of the light transmission properties of vessels with circular cross section, a relationship between the positions of edges found by a typical image processing algorithm and actual dimensions is derived. The dimensional analysis is demonstrated on rat mesenteric resistance arteries (internal diameter less than 300 micrometer) mounted in a perfusion arteriograph. Segments of vessels are secured on two glass cannulae using single strands of a nylon braided suture. The artery is perfused with physiological salt solution and the perfusion pressure maintained at 60 mmHg before starting the experiment. Changes in vascular diameter to the vasoconstrictor noradrenaline and the endothelium-dependent vasodilator acetylcholine were then observed.
Green, Richard D.; Guan, Ling; Burne, J. A.
This paper describes a video analysis system, free of markers and set-up procedures, that quantitatively identified gait abnormalities in real-time from standard video images. A novel color 3D body model was sized and texture mapped to the exact characteristics of a person from video images. The kinematics of the body model was represented by a transformation tree to track the position and orientation of a person relative to the camera. Joint angles were used to track the location and orientation of each body part, with the range of joint angles being constrained by associating degrees of freedom with each joint. To stabilize tracking, the joint angles were estimated for the next frame. The calculation of joint angles, for the next frame, was cast as an estimation problem which was solved using an iterated extended Kalman filter. Patients with dopa-responsive parkinsonism, and age matched normals, were video taped during several gait cycles with walking movements successfully tracked and classified. The results suggested that this approach has the potential to guide clinicians on the relative sensitivity of specific postural/gait features in diagnosis.
Background Yersiniosis is a zoonotic disease reported worldwide. Culture and PCR based protocols are the most common used methods for detection of pathogenic Yersinia species in animal samples. PCR sensitivity could be increased by an initial enrichment step. This step is particularly useful in surveillance programs, where PCR is applied to samples from asymptomatic animals. The aim of this study was to evaluate the improvement in pathogenic Yersinia species detection using a suitable enrichment method prior to the real time PCR (rtPCR). Nine different enrichment protocols were evaluated including six different broth mediums (CASO, ITC, PSB, PBS, PBSMSB and PBSSSB). Results The analysis of variance showed significant differences in Yersinia detection by rtPCR according to the enrichment protocol used. These differences were higher for Y. pseudotuberculosis than for Y. enterocolitica. In general, samples incubated at lower temperatures yielded the highest detection rates. The best results were obtained with PBSMSB and PBS2. Application of PBSMSB protocol to free-ranging wild board samples improved the detection of Y. enterocolitica by 21.2% when compared with direct rtPCR. Y. pseudotuberculosis detection was improved by 10.6% when results obtained by direct rtPCR and by PBSMSB enrichment before rtPCR were analyzed in combination. Conclusions The data obtained in the present study indicate a difference in Yersinia detection by rtPCR related to the enrichment protocol used, being PBSMSB enrichment during 15 days at 4°C and PBS during 7 days at 4°C the most efficient. The use of direct rtPCR in combination with PBSMSB enrichment prior to rtPCR resulted in an improvement in the detection rates of pathogenic Yersinia in wild boar and could be useful for application in other animal samples. PMID:25168886
Fornaser, A.; De Cecco, M.; Leuci, M.; Conci, N.; Daldoss, M.; Armanini, A.; Maule, L.; De Natale, F.; Da Lio, M.
Techniques for tracking the eyes took place since several decades for different applications that range from military, to education, entertainment and clinics. The existing systems are in general of two categories: precise but intrusive or comfortable but less accurate. The idea of this work is to calibrate an eye tracker of the second category. In particular we have estimated the uncertainty both in nominal and in case of variable operating conditions. We took into consideration different influencing factors such as: head movement and rotation, eyes detected, target position on the screen, illumination and objects in front of the eyes. Results proved that the 2D uncertainty can be modelled as a circular confidence interval as far as there is no stable principal directions in both the systematic and the repeatability effects. This confidence region was also modelled as a function of the current working conditions. In this way we can obtain a value of the uncertainty that is a function of the operating condition estimated in real time opening the field to new applications that reconfigure the human machine interface as a function of the operating conditions. Examples can range from option buttons reshape, local zoom dynamically adjusted, speed optimization to regulate interface responsiveness, the possibility to take into account the uncertainty associated to a particular interaction. Furthermore, in the analysis of visual scanning patterns, the resulting Point of Regard maps would be associated with proper confidence levels thus allowing to draw accurate conclusions. We conducted an experimental campaign to estimate and validate the overall modelling procedure obtaining valid results in 86% of the cases.
The report gives results of analyses using real-time dynamometer test emissions data from 13 passenger cars to examine variations in emissions during different speeds or modes of travel. The resulting data provided a way to separately identify idle, cruise, acceleration, and dece...
Theroux, James M.
In 2005, M. Hopkins and J. Theroux implemented the second example of an experimental case study, at 11 business schools in the United States and Canada. The new type of case study, named the "real-time case (RTC) study," uses the Internet to bring business reality to business courses and to facilitate communication among faculty,…
Quantitative real-time polymerase chain reaction (qRT-PCR) is a commonly used technique for measuring gene expression levels due to its simplicity, specificity, and sensitivity. Reliable reference selection for the accurate quantification of gene expression under various experimental conditions is a...
Recently a real-time PCR (RT-PCT) assay based on sequence from the gene for pertactin was proposed for identification of Bordetella pertussis. Here we report that the B. pertussis pertactin gene sequence for the region encompassing the RT-PCR probe and primers is nearly identical to that of many B....
Schibler, Manuel; Yerly, Sabine; Vieille, Gaël; Docquier, Mylène; Turin, Lara; Kaiser, Laurent; Tapparel, Caroline
Rhinoviruses are the most frequent cause of human respiratory infections, and quantitative rhinovirus diagnostic tools are needed for clinical investigations. Although results obtained by real-time reverse-transcription PCR (RT-PCR) assays are frequently converted to viral RNA loads, this presents several limitations regarding accurate virus RNA quantification, particularly given the need to reliably quantify all known rhinovirus genotypes with a single assay. Using an internal extraction control and serial dilutions of an in vitro-transcribed rhinovirus RNA reference standard, we validated a quantitative one-step real-time PCR assay. We then used chimeric rhinovirus genomes with 5'-untranslated regions (5'UTRs) originating from the three rhinovirus species and from one enterovirus to estimate the impact of the 5'UTR diversity. Respiratory specimens from infected patients were then also analyzed. The assay quantification ability ranged from 4.10 to 9.10 log RNA copies/ml, with an estimated error margin of ±10%. This variation was mainly linked to target variability and interassay variability. Taken together, our results indicate that our assay can reliably estimate rhinovirus RNA load, provided that the appropriate error margin is used. In contrast, due to the lack of a universal rhinovirus RNA standard and the variability related to sample collection procedures, accurate absolute rhinovirus RNA quantification in respiratory specimens is currently hardly feasible.
Mani, Seethambal S.; van Bloemen Waanders, Bart Gustaaf; Cooper, Scott Patrick; Jakaboski, Blake Elaine; Normann, Randy Allen; Jennings, Jim; Gilbert, Bob; Lake, Larry W.; Weiss, Chester Joseph; Lorenz, John Clay; Elbring, Gregory Jay; Wheeler, Mary Fanett; Thomas, Sunil G.; Rightley, Michael J.; Rodriguez, Adolfo; Klie, Hector; Banchs, Rafael; Nunez, Emilio J.; Jablonowski, Chris
The project objective was to detail better ways to assess and exploit intelligent oil and gas field information through improved modeling, sensor technology, and process control to increase ultimate recovery of domestic hydrocarbons. To meet this objective we investigated the use of permanent downhole sensors systems (Smart Wells) whose data is fed real-time into computational reservoir models that are integrated with optimized production control systems. The project utilized a three-pronged approach (1) a value of information analysis to address the economic advantages, (2) reservoir simulation modeling and control optimization to prove the capability, and (3) evaluation of new generation sensor packaging to survive the borehole environment for long periods of time. The Value of Information (VOI) decision tree method was developed and used to assess the economic advantage of using the proposed technology; the VOI demonstrated the increased subsurface resolution through additional sensor data. Our findings show that the VOI studies are a practical means of ascertaining the value associated with a technology, in this case application of sensors to production. The procedure acknowledges the uncertainty in predictions but nevertheless assigns monetary value to the predictions. The best aspect of the procedure is that it builds consensus within interdisciplinary teams The reservoir simulation and modeling aspect of the project was developed to show the capability of exploiting sensor information both for reservoir characterization and to optimize control of the production system. Our findings indicate history matching is improved as more information is added to the objective function, clearly indicating that sensor information can help in reducing the uncertainty associated with reservoir characterization. Additional findings and approaches used are described in detail within the report. The next generation sensors aspect of the project evaluated sensors and packaging
Lin, Meng; Jia, Renyong; Wang, Mingshu; Gao, Xinghong; Zhu, Dekang; Chen, Shun; Yin, Zhongqiong; Wang, Yin; Chen, Xiaoyue; Cheng, Anchun
Duck enteritis virus (DEV) UL49.5 encoding glycoprotein N was a conserved gene. The transcription dynamic process of UL49.5 homologous genes in herpesviruses was reported. However, the transcription dynamic process of DEV UL49.5 gene has not yet been established. In this study, a real-time quantitative reverse transcription PCR (real-time qRT-PCR) assay was established to test the transcription dynamic process of DEV UL49.5 gene, and the recombinant plasmid pUCm-T/UL49.5 was constructed as the standard DNA. The samples prepared from DEV-infected (at different time points) and uninfected cell were detected and calculated. The results demonstrated that the real-time qRT-PCR assay was successfully established. The transcription product of DEV UL49.5 gene was first detected at 0.5 h post infection (p.i.), increased at 8 h p.i. and reached a peak at 60 h p.i. Our results illustrated that DEV UL49.5 gene could be regarded as a late gene. The transcription dynamic process of DEV UL49.5 gene may provide a significant clue for further studies of DEV UL49.5 gene.
Hindson, B J; Reid, S M; Baker, B R; Ebert, K; Ferris, N P; Bentley Tammero, L F; Lenhoff, R J; Naraghi-Arani, P; Vitalis, E A; Slezak, T R; Hullinger, P J; King, D P
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-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 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.
Mazzatenta, Andrea; Pokorski, Mieczyslaw; Di Giulio, Camillo
Centenarians are a model to study human longevity and the physiological process of aging. A plethora of studies on this model show the complexity of the system. Laboratory studies fail to find a biomarker of senescence. The real time exhaled breath volatile organic compounds (VOCs) has been suggested as a new biomarker to detect and monitor physiological processes in the respiratory system. VOCs exhaled by centenarians have not been studied in the general population and across-age-groups. In the present study we investigated, in real time, the breath properties and VOC exhaled content in healthy centenarians as compared with non-centenarian seniors and young healthy subjects. We found distinctly different breath pattern and distribution profiles of VOCs in the centenarians. Thus, the VOCs measurement allowed to discriminate the differences between the age-groups. We propose a VOCs fingerprint as a biomarker underlying the physiological mechanisms of aging and longevity. Longevity should be considered physiologically as a new phase of life, characteristic of the well adapted subject.
Lynch, Robert E. (Inventor); Chidester, Thomas R. (Inventor); Lawrence, Robert E. (Inventor)
Method and system for monitoring and comparing, in real time, performance of an aircraft during an approach to touchdown along a conventional approach path and along a contemplated modified approach path to touchdown. In a first procedure, a flight parameter value at a selected location is compared and displayed, for the planned path and for the modified path. In a second procedure, flight parameter values FP(t(sub m)) at a sequence (t(sub n)}n, of measurement times is compared and displayed, for the planned path and for a contemplated or presently-executed modified path. If the flight parameter for the planned path and for the modified path differ too much from each other, the pilot in command has an option of terminating the approach along the modified path.
Savolaine, C. G.
Inexpensive ways to organize and conduct system testing that were used on a real-time satellite network control system are outlined. This system contains roughly 50,000 lines of executable source code developed by a team of eight people. For a small investment of staff, the system was thoroughly tested, including automated regression testing, before field release. Detailed records were kept for fourteen months, during which several versions of the system were written. A separate testing group was not established, but testing itself was structured apart from the development process. The errors found during testing are examined by frequency per subsystem by size and complexity as well as by type. The code was released to the user in March, 1983. To date, only a few minor problems found with the system during its pre-service testing and user acceptance has been good.
Rocha-Silva, Fabiana; Gomes, Luciana Inácia; Góes, Alfredo Miranda; Graciele-Melo, Cidiane; Caligiorne, Rachel Basques
Paracoccidioidomycosis (PCM) is a systemic mycosis caused by dimorphic fungi Paracoccidioides brasiliensis and P. lutzii. It is prevalent in Latin American, mainly in Brazil. Therefore, PCM has fundamental impact in the Brazilian global economy, especially in public health system, since it is affecting economical active population in different country regions.
Price, Andy; Álvarez, Laura Acuña; Whitby, Corinne; Larsen, Jan
Quantitative reverse transcription PCR (qRT-PCR) is a variation of conventional quantitative or real-time PCR, whereby mRNA is first converted into the complementary DNA (cDNA) by reverse transcription, the cDNA is then subsequently quantified by qPCR. The use of mRNA as the initial template allows the quantification of gene transcripts, rather than gene copy numbers. mRNA is only produced by actively metabolising cells and is produced by its corresponding gene to provide a 'blueprint' in order for a cell to manufacture a specific protein. Conventional qPCR detects not only DNA present in actively metabolising cells but also inactive and dead cells. qRT-PCR has the advantage that only actively metabolising cells are detected, hence provides a more reliable measure of microbial activity in oilfield samples. When qRT-PCR is combined with primers and probes for specific genes, the activity of microbial processes important in the oilfield, such as sulphate reduction, methanogenesis and nitrate reduction can be monitored.
Incineration has long been, and continues to be, one of the most cost-effective technologies for disposing of the world's growing volume of municipal and hazardous waste. Yet anyone who has been involved in an attempt to site an incinerator in recent years knows the political nightmare this process has become. The public has become extremely suspicious of the health and environmental impact of incinerators, and not without reason. Incinerators have been known to release unacceptably high levels of toxic substances into the air, including dioxins, furans, and other pollutants. Worse, there are no monitoring devices that can continuously measure trace gases in incinerator emissions to allow operators to know exactly what substances are being released and allow for quick corrective action. To address the problems, several teams of university scientists are developing techniques for real-time emissions monitoring that may simultaneously allow industry to operate incinerators in the most efficient manner and assure the public that their health is being protected.
Further improvement and validation of MagMAX-96 AI/ND viral RNA isolation for efficient removal of RT-PCR inhibitors from cloacal swabs and tissues for rapid diagnosis of avian influenza virus by RT reverse transcription PCR
Real time RT-PCR (RRT-PCR) is a high throughput molecular diagnostic test used for rapid detection of avian influenza virus (AIV) in clinical samples. However the performance of RRT-PCR can be adversely affected by RT-PCR inhibitors present in the sample. The tested commercial RNA extraction kits ...
Sasmono, R Tedjo; Aryati, Aryati; Wardhani, Puspa; Yohan, Benediktus; Trimarsanto, Hidayat; Fahri, Sukmal; Setianingsih, Tri Y; Meutiawati, Febrina
Diagnostic tests based on detection of dengue virus (DENV) genome are available with varying sensitivities and specificities. The Simplexa Dengue assay (Focus Diagnostics) is a newly developed real-time RT-PCR method designed to detect and serotype DENV simultaneously. To assess the performance of the Simplexa Dengue assay, we performed comparison with conventional RT-PCR and SYBR Green real-time RT-PCR on patients sera isolated from eight cities across Indonesia, a dengue endemic country. A total of 184 sera that were confirmed using NS1 and/or IgM and IgG ELISA were examined. Using conventional and SYBR Green real-time RT-PCR, we detected DENV in 53 (28.8%) and 81 (44.0%) out of 184 sera, respectively. When the Simplexa Dengue assay was employed, the detection rate was increased to 76.6% (141 out of 184 samples). When tested in 40 sera that were confirmed by virus isolation as the gold standard, the conventional RT-PCR yielded 95% sensitivity while the sensitivity of SYBR Green real-time RT-PCR and Simplexa Dengue assay reached 97.5% and 100%, respectively. The specificities of all methods were 100% when tested in 43 non-dengue illness and 20 healthy human samples. Altogether, our data showed the higher detection rate of Simplexa Dengue compared to conventional and SYBR Green real-time RT-PCR in field/surveillance setting. In conclusion, Simplexa Dengue offers rapid and accurate detection and typing of dengue infection and is suitable for both routine diagnostic and surveillance.
Cascini, Fidelia; Passerotti, Stella; Boschi, Ilaria
In this paper we describe analyses performed by Real-Time Reverse-Transcriptase Polymerase Chain Reaction (real-time RT-PCR) on RNA of 12 samples, carried out for forensic purposes to investigate a correlation between tetrahydrocannabinol (THC) concentration in Cannabis and the tetrahydrocannabinol acid synthase (THCAS) gene expression. Samples were obtained from an experimental cultivation of declared potency Cannabis variety seeds and from seizures. The Rubisco gene and the 26S ribosomal RNA gene were used as internal control genes for their constant expression and stability. As results we found minor gene expression in samples from leaves of young plants. Further, grouping results for cannabis samples with similar characteristics, we have found an increased relative expression in samples with the highest percentage of THC coming from seized sample and adult plants.
Hindson, B J; Baker, B R; Bentley Tammero, L F; Lenhoff, R J; Naraghi-Arani, P; Vitalis, E A; Slezak, T R; Hullinger, P J; Reid, S M; Ebert, K; Ferris, N P; King, D P
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 suspect 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.
Deepak, Sa; Kottapalli, Kr; Rakwal, R; Oros, G; Rangappa, Ks; Iwahashi, H; Masuo, Y; Agrawal, Gk
Invention of polymerase chain reaction (PCR) technology by Kary Mullis in 1984 gave birth to real-time PCR. Real-time PCR - detection and expression analysis of gene(s) in real-time - has revolutionized the 21(st) century biological science due to its tremendous application in quantitative genotyping, genetic variation of inter and intra organisms, early diagnosis of disease, forensic, to name a few. We comprehensively review various aspects of real-time PCR, including technological refinement and application in all scientific fields ranging from medical to environmental issues, and to plant.
Deepak, SA; Kottapalli, KR; Rakwal, R; Oros, G; Rangappa, KS; Iwahashi, H; Masuo, Y; Agrawal, GK
Invention of polymerase chain reaction (PCR) technology by Kary Mullis in 1984 gave birth to real-time PCR. Real-time PCR — detection and expression analysis of gene(s) in real-time — has revolutionized the 21st century biological science due to its tremendous application in quantitative genotyping, genetic variation of inter and intra organisms, early diagnosis of disease, forensic, to name a few. We comprehensively review various aspects of real-time PCR, including technological refinement and application in all scientific fields ranging from medical to environmental issues, and to plant. PMID:18645596
Hassani-Mehraban, A; Westenberg, M; Verhoeven, J T J; van de Vossenberg, B T L H; Kormelink, R; Roenhorst, J W
A set of tests for generic detection and identification of tospoviruses has been developed. Based on a multiple sequence alignment of the nucleocapsid gene and its 5' upstream untranslated region sequence from 28 different species, primers were designed for RT-PCR detection of tospoviruses from all recognized clades, i.e. the American, Asian and Eurasian clades, and from the small group of distinct and floating species. Pilot experiments on isolates from twenty different species showed that the designed primer sets successfully detected all species by RT-PCR, as confirmed by nucleotide sequence analysis of the amplicons. In a final optimized design, the primers were applied in a setting of five RT-PCR tests. Seven different tospoviruses were successfully identified from diagnostic samples and in addition a non-described tospovirus species from alstroemeria plants. The results demonstrate that the newly developed generic RT-PCR tests provide a relevant tool for broad detection and identification of tospoviruses in plant quarantine and diagnostic laboratories.
Singh, Varinder; Kaul, Sunil C.; Wadhwa, Renu; Pati, Pratap Kumar
Quantitative real-time PCR (qRT-PCR) is now globally used for accurate analysis of transcripts levels in plants. For reliable quantification of transcripts, identification of the best reference genes is a prerequisite in qRT-PCR analysis. Recently, Withania somnifera has attracted lot of attention due to its immense therapeutic potential. At present, biotechnological intervention for the improvement of this plant is being seriously pursued. In this background, it is important to have comprehensive studies on finding suitable reference genes for this high valued medicinal plant. In the present study, 11 candidate genes were evaluated for their expression stability under biotic (fungal disease), abiotic (wounding, salt, drought, heat and cold) stresses, in different plant tissues and in response to various plant growth regulators (methyl jasmonate, salicylic acid, abscisic acid). The data as analyzed by various software packages (geNorm, NormFinder, Bestkeeper and ΔCt method) suggested that cyclophilin (CYP) is a most stable gene under wounding, heat, methyl jasmonate, different tissues and all stress conditions. T-SAND was found to be a best reference gene for salt and salicylic acid (SA) treated samples, while 26S ribosomal RNA (26S), ubiquitin (UBQ) and beta-tubulin (TUB) were the most stably expressed genes under drought, biotic and cold treatment respectively. For abscisic acid (ABA) treated samples 18S-rRNA was found to stably expressed gene. Finally, the relative expression level of the three genes involved in the withanolide biosynthetic pathway was detected to validate the selection of reliable reference genes. The present work will significantly contribute to gene analysis studies in W. somnifera and facilitate in improving the quality of gene expression data in this plant as well as and other related plant species. PMID:25769035
Teng, Qiaoyang; Shen, Weixia; Yan, Dawei; Yan, Liping; Li, Xuesong; Li, Guoxin; Yang, Jianmei; Li, Zejun
Previous studies demonstrated that the H3 avian influenza virus (AIV) in China is isolated most frequently from wild birds and live poultry markets. However, there is no subtype-specific real-time polymerase chain reaction (RT-PCR) available for the rapid and highly sensitive identification of H3 AIV. In this study, a TaqMan minor groove binder (MGB) probe and a pair of primers were designed based on a conserved region in the hemagglutinin gene of H3 AIV. These were used to generate an H3-MGB RT-PCR assay that recognizes only H3 AIV. The detection limit of the H3-MGB RT-PCR was 10 copies of DNA per reaction when 10-fold serial dilutions of T-H3HA plasmid were used as the template. This was 1000-times more sensitive than conventional RT-PCR. In experimental samples obtained from oropharyngeal swabs or cloacal swabs, the virus was detected in all ducks using H3-MGB RT-PCR, whereas only one duck tested positive for the virus in oropharyngeal swabs tested using conventional RT-PCR. The H3-MGB RT-PCR assay developed in this study is a sensitive and rapid tool for screening H3 AIV in China.
Varkonyi-Gasic, Erika; Wu, Rongmei; Wood, Marion; Walton, Eric F; Hellens, Roger P
MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 μl of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression. PMID:17931426
Alm, Erik; Lesko, Birgitta; Lindegren, Gunnel; Ahlm, Clas; Söderholm, Sandra; Falk, Kerstin I.; Lagerqvist, Nina
Background Dengue is a mosquito-borne viral disease that has become more prevalent in the last few decades. Most patients are viremic when they present with symptoms, and early diagnosis of dengue is important in preventing severe clinical complications associated with this disease and also represents a key factor in differential diagnosis. Here, we designed and validated a hydrolysis-probe-based one-step real-time RT-PCR assay that targets the genomes of dengue virus serotypes 1–4. Methodology/Principal Findings The primers and probe used in our RT-PCR assay were designed to target the 3′ untranslated region of all complete genome sequences of dengue virus available in GenBank (n = 3,305). Performance of the assay was evaluated using in vitro transcribed RNA, laboratory-adapted virus strains, external control panels, and clinical specimens. The linear dynamic range was found to be 104–1011 GCE/mL, and the detection limit was between 6.0×102 and 1.1×103 GCE/mL depending on target sequence. The assay did not cross-react with human RNA, nor did it produce false-positive results for other human pathogenic flaviviruses or clinically important etiological agents of febrile illnesses. We used clinical serum samples obtained from returning travelers with dengue-compatible symptomatology (n = 163) to evaluate the diagnostic relevance of our assay, and laboratory diagnosis performed by the RT-PCR assay had 100% positive agreement with diagnosis performed by NS1 antigen detection. In a retrospective evaluation including 60 archived serum samples collected from confirmed dengue cases 1–9 days after disease onset, the RT-PCR assay detected viral RNA up to 9 days after appearance of symptoms. Conclusions/Significance The validation of the RT-PCR assay presented here indicates that this technique can be a reliable diagnostic tool, and hence we suggest that it be introduced as the method of choice during the first 5 days of dengue symptoms. PMID:25522325
Delporte, Marianne; Legrand, Guillaume; Hilbert, Jean-Louis; Gagneul, David
Plant polyphenols represent a huge reservoir of bioactive compounds. Industrial chicory, an important crop from northwestern Europe, accumulates an original combination of such compounds, i.e., chlorogenic, isochlorogenic, caftaric, and chicoric acids arising from the phenylpropanoid pathway. For a complete understanding of these biochemical pathways, analyses of gene expression using quantitative real-time PCR (qRT-PCR) should be considered. Because cell cultures are a model of choice for specialized metabolism investigations, this study described for the first time the validation of reference genes for this system in chicory. Eighteen potential reference genes were obtained by mining expressed sequence tag databases of chicory for orthologs of Arabidopsis thaliana genes currently used as reference genes. Twelve genes passed the qRT-PCR standard requirements and their expression stability across different samples was tested using three distinct softwares: geNorm, NormFinder, and BestKeeper. In cell cultures grown under various conditions, TIP41 (TIP41 like protein) was shown to be the most stable gene. Further validation of the proposed reference genes was done by normalization of expression levels of a group of genes of interest. In order to assess the potentiality of the proposed list of candidate reference genes, theses genes were in parallel tested on another experimental design, i.e., chicory seedlings. In this case, the best reference gene identified was Clath (Clathrin adaptator complex subunit). The results highlight the importance of the use of properly validated reference genes to achieve relevant interpretation of qRT-PCR analyses. Here, we provide a list of reference genes suitable for future gene expression studies in chicory. PMID:26347767
Pierce, Lindsey R; Willey, James C; Crawford, Erin L; Palsule, Vrushalee V; Leaman, Douglas W; Faisal, Mohamed; Kim, Robert K; Shepherd, Brian S; Stanoszek, Lauren M; Stepien, Carol A
Viral Hemorrhagic Septicemia virus (VHSv) causes one of the world's most important finfish diseases, killing >80 species across Eurasia and North America. A new and especially virulent strain (IVb) emerged in the North American Great Lakes in 2003, threatening fisheries, baitfish, and aquaculture industries. Weeks-long and costly cell culture is the OIE and USDA-APHIS approved diagnostic. A new Standardized Reverse Transcriptase Polymerase Chain Reaction (StaRT-PCR) assay that uniquely incorporates internal standards to improve accuracy and prevent false negatives was developed and evaluated for its ability to detect and quantify VHSv. Results from StaRT-PCR, SYBR(®) green real time qRT-PCR, and cell culture were compared, as well as the effects of potential PCR inhibitors (EDTA and high RNA). Findings show that StaRT-PCR is sensitive, detecting a single molecule, with 100% accuracy at six molecules, and had no false negatives. In comparison, false negatives ranged from 14 to 47% in SYBR(®) green real time qRT-PCR tests, and 47-70% with cell culture. StaRT-PCR uniquely controlled for EDTA and RNA interference. Range of VHSv quantitation by StaRT-PCR was 1.0×10(0)-1.2×10(5) VHSv/10(6)actb1 molecules in wild caught fishes and 1.0×10(0)-8.4×10(5) molecules in laboratory challenged specimens. In the latter experiments, muskellunge with skin lesions had significantly more viral molecules (mean=1.9×10(4)) than those without (1.1×10(3)) (p<0.04). VHSv infection was detected earlier in injection than in immersion challenged yellow perch (two versus three days), with molecule numbers in both being comparable and relatively consistent over the remaining course of the experiment. Our results show that the StaRT-PCR test accurately and reliably detects and quantifies VHSv.
Howard, Ayanna; Padgett, Curtis; Brown, Kenneth
We present an algorithm for detecting a specified set of targets for an Automatic Target Recognition (ATR) application. ATR involves processing images for detecting, classifying, and tracking targets embedded in a background scene. We address the problem of discriminating between targets and nontarget objects in a scene by evaluating 40x40 image blocks belonging to an image. Each image block is first projected onto a set of templates specifically designed to separate images of targets embedded in a typical background scene from those background images without targets. These filters are found using directed principal component analysis which maximally separates the two groups. The projected images are then clustered into one of n classes based on a minimum distance to a set of n cluster prototypes. These cluster prototypes have previously been identified using a modified clustering algorithm based on prior sensed data. Each projected image pattern is then fed into the associated cluster's trained neural network for classification. A detailed description of our algorithm will be given in this paper. We outline our methodology for designing the templates, describe our modified clustering algorithm, and provide details on the neural network classifiers. Evaluation of the overall algorithm demonstrates that our detection rates approach 96% with a false positive rate of less than 0.03%.
Wen, Shuxiang; Chen, Xiaoling; Xu, Fuzhou; Sun, Huiling
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. PMID:27942007
Ma, Rui; Xu, Sheng; Zhao, Yucheng; Xia, Bing; Wang, Ren
Lycoris aurea (L' Hér.) Herb, a perennial grass species, produces a unique variety of pharmacologically active Amaryllidaceae alkaloids. However, the key enzymes and their expression pattern involved in the biosynthesis of Amaryllidaceae alkaloids (especially for galanthamine) are far from being fully understood. Quantitative real-time polymerase chain reaction (qRT-PCR), a commonly used method for quantifying gene expression, requires stable reference genes to normalize its data. In this study, to choose the appropriate reference genes under different experimental conditions, 14 genes including YLS8 (mitosis protein YLS8), CYP2 (Cyclophilin 2), CYP 1 (Cyclophilin 1), TIP41 (TIP41-like protein), EXP2 (Expressed protein 2), PTBP1 (Polypyrimidine tract-binding protein 1), EXP1 (Expressed protein 1), PP2A (Serine/threonine-protein phosphatase 2A), β-TUB (β-tubulin), α-TUB (α-tubulin), EF1-α (Elongation factor 1-α), UBC (Ubiquitin-conjugating enzyme), ACT (Actin) and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) were selected from the transcriptome datasets of L. aurea. And then, expressions of these genes were assessed by qRT-PCR in various tissues and the roots under different treatments. The expression stability of the 14 candidates was analyzed by three commonly used software programs (geNorm, NormFinder, and BestKeeper), and their results were further integrated into a comprehensive ranking based on the geometric mean. The results show the relatively stable genes for each subset as follows: (1) EXP1 and TIP41 for all samples; (2) UBC and EXP1 for NaCl stress; (3) PTBP1 and EXP1 for heat stress, polyethylene glycol (PEG) stress and ABA treatment; (4) UBC and CYP2 for cold stress; (5) PTBP1 and PP2A for sodium nitroprusside (SNP) treatment; (6) CYP1 and TIP41 for methyl jasmonate (MeJA) treatment; and (7) EXP1 and TIP41 for various tissues. The reliability of these results was further enhanced through comparison between part qRT-PCR result and RNA
Li, Wei; Matsuoka, Masanori; Kai, Masanori; Thapa, Pratibha; Khadge, Saraswoti; Hagge, Deanna A; Brennan, Patrick J; Vissa, Varalakshmi
Drug resistance surveillance and strain typing of Mycobacterium leprae are necessary to investigate ongoing transmission of leprosy in regions of endemicity. To enable wider implementation of these molecular analyses, novel real-time PCR-high-resolution melt (RT-PCR-HRM) assays without allele-specific primers or probes and post-PCR sample handling were developed. For the detection of mutations within drug resistance-determining regions (DRDRs) of folP1, rpoB, and gyrA, targets for dapsone, rifampin, and fluoroquinolones, real-time PCR-HRM assays were developed. Wild-type and drug-resistant mouse footpad-derived strains that included three folP1, two rpoB, and one gyrA mutation types in a reference panel were tested. RT-PCR-HRM correctly distinguished the wild type from the mutant strains. In addition, RT-PCR-HRM analyses aided in recognizing samples with mixed or minor alleles and also a mislabeled sample. When tested in 121 sequence-characterized clinical strains, HRM identified all the folP1 mutants representing two mutation types, including one not within the reference panel. The false positives (<5%) could be attributed to low DNA concentration or PCR inhibition. A second set of RT-PCR-HRM assays for identification of three previously reported single nucleotide polymorphisms (SNPs) that have been used for strain typing were developed and validated in 22 reference and 25 clinical strains. Real-time PCR-HRM is a sensitive, simple, rapid, and high-throughput tool for routine screening known DRDR mutants in new and relapsed cases, SNP typing, and detection of minor mutant alleles in the wild-type background at lower costs than current methods and with the potential for quality control in leprosy investigations.
Nine isolates of Grapevine Leafroll-associated Virus 7 (GLRaV-7) from California have been sequenced to design more sensitive molecular diagnostic tools. These sequences were from the coat protein (CP) and the homologous heat shock protein (hHSP70) genes. Sequence identity among these isolates rang...
Chen, Kuan-Fu; Rothman, Richard E; Ramachandran, Padmini; Blyn, Lawrence; Sampath, Rangarajan; Ecker, David J; Valsamakis, Alexandra; Gaydos, Charlotte A
Diagnosis of the etiologic agent of respiratory viral infection relies traditionally on culture or antigen detection. This pilot evaluation compared performance characteristics of the RT-PCR and electrospray ionization mass spectrometry (RT-PCR/ESI-MS) platform to conventional virologic methods for identifying multiple clinically relevant respiratory viruses in nasopharyngeal aspirates. The RT-PCR/ESI-MS respiratory virus surveillance kit was designed to detect respiratory syncytial virus, influenza A and B, parainfluenza types 1-4, adenoviridae types A-F, coronaviridae, human bocavirus, and human metapneumovirus. Patients (N=192) attending an emergency department during the 2007-2008 respiratory season consented, and "excess" frozen archived nasopharyngeal aspirates were analysed; 46 were positive by conventional virology and 69 by RT-PCR/ESI-MS, among which there were six samples with multiple viral pathogens detected. The sensitivity and specificity of the assay were 89.1% and 80.3%, respectively. Additional viruses that were not identified by conventional virology assays were detected (4 human bocaviruses and 7 coronaviruses). Samples in which the RT-PCR/ESI-MS results disagreed with conventional virology were sent for analysis by a third method using a commercial RT-PCR-based assay, which can identify viruses not detectable by conventional virologic procedures. Time to first result of RT-PCR/ESI-MS was 8h. RT-PCR/ESI-MS demonstrated capacity to detect respiratory viruses identifiable and unidentifiable by conventional methods rapidly.
Omar, Ahmad A; Dekkers, Marty G H; Graham, James H; Grosser, Jude W
Quantitative real-time PCR (qRT-PCR) was adapted to estimate transgene copy number of the rice Xa21 gene in transgenic citrus plants. This system used TaqMan qRT-PCR and the endogenous citrus gene encoding for lipid transfer protein (LTP). Transgenic "Hamlin" sweet orange plants were generated using two different protoplast-GFP transformation systems: cotransformation and single plasmid transformation. A dilution series of genomic DNA from one of the transgenic lines was used to generate a standard curve for the endogenous LTP and the transgene Xa21. This standard curve was used for relative quantification of the endogenous gene and the transgene. Copy numbers of the transgene Xa21 detected from qRT-PCR analysis correlated with that from Southern blot analysis (r = 0.834). Thus, qRT-PCR is an efficient means of estimating copy number in transgenic citrus plants. This analysis can be performed at much earlier stages of transgenic plant development than southern blot analysis, which expedites investigation of transgenes in slow-growing woody plants.
Carrasco, Adriano de Oliveira Torres; Rodrigues, Juliana Nogueira Martins; Seki, Meire Christina; de Moraes, Fabricio Edgar; Silva, Jaqueline Raymondi; Durigon, Edison Luis; Pinto, Aramis Augusto
The aim of this study was to evaluate a simple molecular method of reverse transcriptase polymerase chain reaction (RT-PCR) to differentiate Newcastle disease virus strains according to their pathogenicity, in order to use it in molecular screening of Newcastle disease virus in poultry and free-living bird populations. Specific primers were developed to differentiate LaSota--LS--(vaccine strain) and Sao Joao do Meriti--SJM--strain (highly pathogenic strain). Chickens and pigeons were experimentally vaccinated/infected for an in vivo study to determine virus shedding in feces. Validation of sensitivity and specificity of the primers (SJM and LS) by experimental models used in the present study and results obtained in the molecular analysis of the primers by BLAST made it possible to generalize results. The development of primers that differentiate the level of pathogenicity of NDV stains is very important, mainly in countries where real-time RT-PCR is still not used as a routine test. These primers were able to determine the presence of the agent and to differentiate it according to its pathogenicity.
Wang, Xueliang; Liu, Fen; Jiang, Lingli; Bao, Yun; Xiao, Yanqun; Wang, Hualiang
Real-time quantitative reverse transcriptase polymerase chain reaction (rRT-PCR) is now widely used to detect viral pathogens in various human specimens. The application of internal controls to validate the entire process of these assays is necessary to prevent false-negative results caused by unexpected inhibition or inefficient extraction. In the present study, we describe a strategy to produce a stable internal control for rRT-PCR by packaging foreign RNA into influenza virions using plasmid-based reverse genetics technology. The envelope structure of influenza virus can effectively protect RNA segments from RNase digestion, which provides an advantage for its routine use as an internal control. Utilizing this approach, we successfully generated a recombinant influenza virus (rPR8-HCV) containing the 5′ untranslated region (5′UTR) of the hepatitis C virus (HCV) RNA genome. After inactivation and purification, the rPR8-HCV particles were demonstrated to be RNase resistant and stable at 4 °C for at least 252 days in human plasma, with no degradation even after being frozen and thawed multiple times. These results were reproducible in the COBAS TaqMan HCV test for 164 days. Moreover, the chimeric influenza virus particles could be easily produced in embryonated eggs and were noninfectious after inactivation treatment. Additionally, this strategy could also be adapted for real-time clinical applications of other RNA targets, providing a universal approach with broad clinical applications in rRT-PCR assays.
Southard, Jonathan N
Instrumentation for real-time PCR is used primarily for amplification and quantitation of nucleic acids. The capability to measure fluorescence while controlling temperature in multiple samples can also be applied to the analysis of proteins. Conformational stability and changes in stability due to ligand binding are easily assessed. Protein structure studies possible with a real-time PCR instrument address core topics in biochemistry and have valuable high-throughput applications in the fields of drug discovery and protein engineering. Protein analysis using real-time PCR instrumentation has been incorporated in an undergraduate laboratory project based on previously described projects. Students express, purify, and characterize a protein. Based on literature research and analysis using bioinformatics tools, they select a specific mutation to investigate. They then attempt to express, purify, and characterize their mutated protein. Thermal denaturation using a real-time PCR instrument is the primary tool used to compare the wild-type and mutated proteins. Alternative means for incorporation of protein analysis by real-time PCR instrumentation into laboratory experiences and additional modes of analysis are also described.
van den Bogert, Antonie J; Geijtenbeek, Thomas; Even-Zohar, Oshri; Steenbrink, Frans; Hardin, Elizabeth C
Mechanical analysis of movement plays an important role in clinical management of neurological and orthopedic conditions. There has been increasing interest in performing movement analysis in real-time, to provide immediate feedback to both therapist and patient. However, such work to date has been limited to single-joint kinematics and kinetics. Here we present a software system, named human body model (HBM), to compute joint kinematics and kinetics for a full body model with 44 degrees of freedom, in real-time, and to estimate length changes and forces in 300 muscle elements. HBM was used to analyze lower extremity function during gait in 12 able-bodied subjects. Processing speed exceeded 120 samples per second on standard PC hardware. Joint angles and moments were consistent within the group, and consistent with other studies in the literature. Estimated muscle force patterns were consistent among subjects and agreed qualitatively with electromyography, to the extent that can be expected from a biomechanical model. The real-time analysis was integrated into the D-Flow system for development of custom real-time feedback applications and into the gait real-time analysis interactive lab system for gait analysis and gait retraining.
Background Teak (Tectona grandis L.f.) is currently the preferred choice of the timber trade for fabrication of woody products due to its extraordinary qualities and is widely grown around the world. Gene expression studies are essential to explore wood formation of vascular plants, and quantitative real-time reverse transcription PCR (qRT-PCR) is a sensitive technique employed for quantifying gene expression levels. One or more appropriate reference genes are crucial to accurately compare mRNA transcripts through different tissues/organs and experimental conditions. Despite being the focus of some genetic studies, a lack of molecular information has hindered genetic exploration of teak. To date, qRT-PCR reference genes have not been identified and validated for teak. Results Identification and cloning of nine commonly used qRT-PCR reference genes from teak, including ribosomal protein 60s (rp60s), clathrin adaptor complexes medium subunit family (Cac), actin (Act), histone 3 (His3), sand family (Sand), β-Tubulin (Β-Tub), ubiquitin (Ubq), elongation factor 1-α (Ef-1α), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Expression profiles of these genes were evaluated by qRT-PCR in six tissue and organ samples (leaf, flower, seedling, root, stem and branch secondary xylem) of teak. Appropriate gene cloning and sequencing, primer specificity and amplification efficiency was verified for each gene. Their stability as reference genes was validated by NormFinder, BestKeeper, geNorm and Delta Ct programs. Results obtained from all programs showed that TgUbq and TgEf-1α are the most stable genes to use as qRT-PCR reference genes and TgAct is the most unstable gene in teak. The relative expression of the teak cinnamyl alcohol dehydrogenase (TgCAD) gene in lignified tissues at different ages was assessed by qRT-PCR, using TgUbq and TgEf-1α as internal controls. These analyses exposed a consistent expression pattern with both reference genes. Conclusion This study
Niu, Xiaoping; Qi, Jianmin; Zhang, Gaoyang; Xu, Jiantang; Tao, Aifen; Fang, Pingping; Su, Jianguang
To accurately measure gene expression using quantitative reverse transcription PCR (qRT-PCR), reliable reference gene(s) are required for data normalization. Corchorus capsularis, an annual herbaceous fiber crop with predominant biodegradability and renewability, has not been investigated for the stability of reference genes with qRT-PCR. In this study, 11 candidate reference genes were selected and their expression levels were assessed using qRT-PCR. To account for the influence of experimental approach and tissue type, 22 different jute samples were selected from abiotic and biotic stress conditions as well as three different tissue types. The stability of the candidate reference genes was evaluated using geNorm, NormFinder, and BestKeeper programs, and the comprehensive rankings of gene stability were generated by aggregate analysis. For the biotic stress and NaCl stress subsets, ACT7 and RAN were suitable as stable reference genes for gene expression normalization. For the PEG stress subset, UBC, and DnaJ were sufficient for accurate normalization. For the tissues subset, four reference genes TUBβ, UBI, EF1α, and RAN were sufficient for accurate normalization. The selected genes were further validated by comparing expression profiles of WRKY15 in various samples, and two stable reference genes were recommended for accurate normalization of qRT-PCR data. Our results provide researchers with appropriate reference genes for qRT-PCR in C. capsularis, and will facilitate gene expression study under these conditions.
Niu, Xiaoping; Qi, Jianmin; Zhang, Gaoyang; Xu, Jiantang; Tao, Aifen; Fang, Pingping; Su, Jianguang
To accurately measure gene expression using quantitative reverse transcription PCR (qRT-PCR), reliable reference gene(s) are required for data normalization. Corchorus capsularis, an annual herbaceous fiber crop with predominant biodegradability and renewability, has not been investigated for the stability of reference genes with qRT-PCR. In this study, 11 candidate reference genes were selected and their expression levels were assessed using qRT-PCR. To account for the influence of experimental approach and tissue type, 22 different jute samples were selected from abiotic and biotic stress conditions as well as three different tissue types. The stability of the candidate reference genes was evaluated using geNorm, NormFinder, and BestKeeper programs, and the comprehensive rankings of gene stability were generated by aggregate analysis. For the biotic stress and NaCl stress subsets, ACT7 and RAN were suitable as stable reference genes for gene expression normalization. For the PEG stress subset, UBC, and DnaJ were sufficient for accurate normalization. For the tissues subset, four reference genes TUBβ, UBI, EF1α, and RAN were sufficient for accurate normalization. The selected genes were further validated by comparing expression profiles of WRKY15 in various samples, and two stable reference genes were recommended for accurate normalization of qRT-PCR data. Our results provide researchers with appropriate reference genes for qRT-PCR in C. capsularis, and will facilitate gene expression study under these conditions. PMID:26528312
Raynor, Michael P; Stephenson, Sally-Anne; Pittman, Kenneth B; Walsh, David CA; Henderson, Michael A; Dobrovic, Alexander
Introduction The ability to screen blood of early stage operable breast cancer patients for circulating tumour cells is of potential importance for identifying patients at risk of developing distant relapse. We present the results of a study of the efficacy of the immunobead RT-PCR method in identifying patients with circulating tumour cells. Results Immunomagnetic enrichment of circulating tumour cells followed by RT-PCR (immunobead RT-PCR) with a panel of five epithelial specific markers (ELF3, EPHB4, EGFR, MGB1 and TACSTD1) was used to screen for circulating tumour cells in the peripheral blood of 56 breast cancer patients. Twenty patients were positive for two or more RT-PCR markers, including seven patients who were node negative by conventional techniques. Significant increases in the frequency of marker positivity was seen in lymph node positive patients, in patients with high grade tumours and in patients with lymphovascular invasion. A strong trend towards improved disease free survival was seen for marker negative patients although it did not reach significance (p = 0.08). Conclusion Multi-marker immunobead RT-PCR analysis of peripheral blood is a robust assay that is capable of detecting circulating tumour cells in early stage breast cancer patients. PMID:19500345
Liu, Peng; Yeung, Stephanie H I; Crenshaw, Karin A; Crouse, Cecelia A; Scherer, James R; Mathies, Richard A
An integrated lab-on-a-chip system has been developed and successfully utilized for real-time forensic short tandem repeat (STR) analysis. The microdevice comprises a 160-nL polymerase chain reaction reactor with an on-chip heater and a temperature sensor for thermal cycling, microvalves for fluidic manipulation, a co-injector for sizing standard injection, and a 7-cm-long separation channel for capillary electrophoretic analysis. A 9-plex autosomal STR typing system consisting of amelogenin and eight combined DNA index system (CODIS) core STR loci has been constructed and optimized for this real-time human identification study. Reproducible STR profiles of control DNA samples are obtained in 2h and 30min with
Baumgart, C.W.; Creager, J.; Mathes, J.; Pounds, T.; VanDeusen, A.; Warthen, B.
This paper describes current efforts at AlliedSignal Federal Manufacturing and Technologies (FM and T) to develop and field an in-situ, data analysis platform to acquire, process, and display site survey data in near real-time. In past years, FM and T has performed a number of site survey tasks. Each of these surveys was unique in application as well as in the type of data processing and analysis that was required to extract and visualize useful site characterization information. However, common to each of these surveys were the following specific computational and operational requirements: (1) a capability to acquire, process, and visualize the site survey data in the field; (2) a capability to perform all processing in a timely fashion (ideally real-time); and (3) a technique for correlating (or fusing) data streams from multiple sensors. Two more general, but no less important, requirements include system architecture modularity and positioning capability. Potential applications include: survey, evaluation, and remediation of numerous Department of Defense and Department of Energy waste sites; real-time detection and characterization of unexploded ordnance and landmines; survey, evaluation, and remediation of industrial waste sites; location of underground utility lines; and providing law enforcement agencies with real-time surveys of crime scenes. The paper describes an integrated data acquisition, processing, and visualization platform that is capable of performing in-situ data processing, interpretation, and visualization in real-time.
Maes, Michaël; Willebrords, Joost; Crespo Yanguas, Sara; Cogliati, Bruno; Vinken, Mathieu
Summary Although connexin production is mainly regulated at the protein level, altered connexin gene expression has been identified as the underlying mechanism of several pathologies. When studying the latter, appropriate methods to quantify connexin mRNA levels are required. The present chapter describes a well-established reverse transcription quantitative real-time polymerase chain reaction procedure optimized for analysis of hepatic connexins. The method includes RNA extraction and subsequent quantification, generation of complementary DNA, quantitative real-time polymerase chain reaction and data analysis. PMID:27207283
Liu, Hua-Kuang; Diep, J.; Huang, K.
Viewgraphs on multi-channel holographic bifurcative neural network system for real-time adaptive Earth Observing System (EOS) data analysis are presented. The objective is to research and develop an optical bifurcating neuromorphic pattern recognition system for making optical data array comparisons and to evaluate the use of the system for EOS data classification, reduction, analysis, and other applications.
Journal of Chemical Education, 2005
Ocean Optics offers laser-induced breakdown spectrometer systems (LIBS) that can be used to identify light to heavy metals in a variety of sample types and geometries in environmental analysis applications. LIBS are versatile, real-time, high-resolution analyzers for qualitative analysis, in less than one second, of every element in solids,…
Chao, Jinquan; Yang, Shuguang; Chen, Yueyi; Tian, Wei-Min
Latex exploitation-caused latex flow is effective in enhancing latex regeneration in laticifer cells of rubber tree. It should be suitable for screening appropriate reference gene for analysis of the expression of latex regeneration-related genes by quantitative real-time PCR (qRT-PCR). In the present study, the expression stability of 23 candidate reference genes was evaluated on the basis of latex flow by using geNorm and NormFinder algorithms. Ubiquitin-protein ligase 2a (UBC2a) and ubiquitin-protein ligase 2b (UBC2b) were the two most stable genes among the selected candidate references in rubber tree clones with differential duration of latex flow. The two genes were also high-ranked in previous reference gene screening across different tissues and experimental conditions. By contrast, the transcripts of latex regeneration-related genes fluctuated significantly during latex flow. The results suggest that screening reference gene during latex flow should be an efficient and effective clue for selection of reference genes in qRT-PCR.
Chao, Jinquan; Yang, Shuguang; Chen, Yueyi; Tian, Wei-Min
Latex exploitation-caused latex flow is effective in enhancing latex regeneration in laticifer cells of rubber tree. It should be suitable for screening appropriate reference gene for analysis of the expression of latex regeneration-related genes by quantitative real-time PCR (qRT-PCR). In the present study, the expression stability of 23 candidate reference genes was evaluated on the basis of latex flow by using geNorm and NormFinder algorithms. Ubiquitin-protein ligase 2a (UBC2a) and ubiquitin-protein ligase 2b (UBC2b) were the two most stable genes among the selected candidate references in rubber tree clones with differential duration of latex flow. The two genes were also high-ranked in previous reference gene screening across different tissues and experimental conditions. By contrast, the transcripts of latex regeneration-related genes fluctuated significantly during latex flow. The results suggest that screening reference gene during latex flow should be an efficient and effective clue for selection of reference genes in qRT-PCR. PMID:27524995
Caselton, Dl; Arunga, G; Emukule, G; Muthoka, P; Mayieka, L; Kosgey, A; Ochola, R; Waiboci, Lw; Feikin, Dr; Mott, Ja; Breiman, Rf; Katz, Ma
In some influenza surveillance systems, timely transport to laboratories for reverse transcription-polymerase chain reaction (RT-PCR) testing is challenging.Guidelines suggest that samples can be stored at 4°Cfor up to 96 hours but the effect of longer storage times has not been systematically evaluated. We collected nasopharyngeal and oropharyngeal specimens from patients in Kenya and stored them in viral transport medium at 2 to 8°C before testing for influenza A and B using real-time RT-PCR. From April 2008 to November 2010, we collected 7,833 samples; 940 (12%) were positive for influenza. In multivariable analysis, specimens stored for six days were less likely to be influenza-positive compared to specimens stored between zero and one day (adjusted odds ratio (a OR): 0.49, 95%confidence interval (CI): 0.27–0.93). There was no statistically significant difference in influenza positivity of specimens stored for five days compared to zero to one day. There was no statistically significant relationship between days in refrigeration and cycle threshold(Ct) values for positive samples (p=0.31). We found that samples could remain in storage for at least five days without affecting the proportion-positive of samples,potentially increasing the feasibility of including influenza surveillance sites in remote areas.
Frank, M; Farquar, G; Adams, K; Bogan, M; Martin, A; Benner, H; Spadaccini, C; Steele, P; Davis, C; Loyola, B; Morgan, J; Sankaran, S
At LLNL and UC Davis, we are developing several techniques for the real-time sampling and analysis of trace gases, aerosols and exhaled breath that could be useful for a modular, integrated system for breath analysis. Those techniques include single-particle bioaerosol mass spectrometry (BAMS) for the analysis of exhaled aerosol particles or droplets as well as breath samplers integrated with gas chromatography mass spectrometry (GC-MS) or MEMS-based differential mobility spectrometry (DMS). We describe these techniques and present recent data obtained from human breath or breath condensate, in particular, addressing the question of how environmental exposure influences the composition of breath.
Zhang, Runkun; Huang, Wanting; Li, Gongke; Hu, Yufei
The development of noninvasive methods for real-time in vivo analysis is of great significant, which provides powerful tools for medical research and clinical diagnosis. In the present work, we described a new strategy based on cataluminescence (CTL) for real-time in vivo clinical breath analysis. To illustrate such strategy, a homemade real-time CTL monitoring system characterized by coupling an online sampling device with a CTL sensor for sevoflurane (SVF) was designed, and a real-time in vivo method for the monitoring of SVF in exhaled breath was proposed. The accuracy of the method was evaluated by analyzing the real exhaled breath samples, and the results were compared with those obtained by GC/MS. The measured data obtained by the two methods were in good agreement. Subsequently, the method was applied to real-time monitoring of SVF in exhaled breath from rat models of the control group to investigate elimination pharmacokinetics. In order to further probe the potential of the method for clinical application, the elimination pharmacokinetics of SVF from rat models of control group, liver fibrosis group alcohol liver group, and nonalcoholic fatty liver group were monitored by the method. The raw data of pharmacokinetics of different groups were normalized and subsequently subjected to linear discriminant analysis (LDA). These data were transformed to canonical scores which were visualized as well-clustered with the classification accuracy of 100%, and the overall accuracy of leave-one-out cross-validation procedure is 88%, thereby indicating the utility of the potential of the method for liver disease diagnosis. Our strategy undoubtedly opens up a new door for real-time clinical analysis in a pain-free and noninvasive way and also guides a promising development direction for CTL.
Park, Chang-Joon; Kim, Sung-Eun; Kim, Hong-Seok; Lee, In-Ho
This paper presents a real-time marker-free motion capture system which can reconstruct 3-dimensional human motions. The virtual character of the proposed system mimics the motion of an actor in real-time. The proposed system captures human motions by using three synchronized CCD cameras and detects the root and end-effectors of an actor such as a head, hands, and feet by exploiting the blob feature analysis. And then, the 3-dimensional positions of end-effectors are restored and tracked by using Kalman filter. At last, the positions of the intermediate joint are reconstructed by using anatomically constrained inverse kinematics algorithm. The proposed system was implemented under general lighting conditions and we confirmed that the proposed system could reconstruct motions of a lot of people wearing various clothes in real-time stably.
Zhang, Qican; Su, Xianyu; Chen, Wenjing; Cao, Yiping; Xiang, Liqun
Optical non-contact 3-D shape measurement provides a novel and useful tool for analysis of facial shape and movement in presurgical and postsurgical regular check. In this article we present a system, which allows a precise 3-D visualization of the patient's facial before and after craniofacial surgery. We discussed, in this paper, the real time 3-D image capture, processing and the 3-D phase unwrapping method to recover complex shape deformation when the movement of the mouth. The result of real-time measurement for facial shape and movement will be helpful for the more ideal effect in plastic surgery.
Tsohnlcs Re"s CLVWEI-e3-TR-34 ESC-TR40321 ___ _ai negie-Mellon University --- Software Engineering Institute AD-A279 014 ELECTE fMAY. 8(IS 4uB Il A...Taxonomy of Coordinai-tibon Mechanisms Used in Real-Time Software Based on Domain "Analysis / Jose L. Fernandez December 1993 / for ei)94-13600 94/\\,,5...0 8 Technical Report CMU/SEI-93-TR-34 ESC-TR-93-321 December 1993 A Taxonomy of Coordination Mechanisms Used in Real-Time Software Based on Domain
Dayakar, Seetha; Pillai, Heera R; Thulasi, Vineetha P; Nair, Radhakrishnan R
Human metapneumovirus (HMPV) and human respiratory syncytial virus (HRSV) are ubiquitous respiratory viral pathogens. They belong to the family Paramyxoviridae (subfamily Pneumovirinae) and is responsible for acute respiratory tract infections in children, elderly and immunocompromised patients. We designed and tested a multiplex reverse transcriptase polymerase chain reaction (mRT-PCR) as a cost-effective alternative to real-time PCR and cell culture based detection for HMPV and HRSV. The newly developed PCR was used to screen nasal/throat swab samples from 356 patients with suspected acute respiratory infection attending the Government Medical College, Thiruvananthapuram, Kerala, India. The method was compared with a commercially available kit employing real time PCR, for its sensitivity and specificity. 53 (14.9 %) samples were positive for at least one tested pathogen by mRT-PCR. All except one among the positive samples showed similar pathogen profile when tested using real time PCR. 8 (15.1 %) among these 53 were positive for HRSVA, 33 (62.3 %) positive for HRSVB and 12 (22.6 %) were positive for HMPV. 17 (32.7 %) samples showed co-infections in them. Sensitivity and specificity of the mRT-PCR was comparable to that of the commercial kit. Our findings indicate that this newly developed mRT-PCR can be used as a cost-effective alternative for laboratory diagnosis of HMPV/HRSV infection and will significantly reduce diagnostic costs for these viruses in clinical settings.
Wang, Yi; Ma, Xiang; Wen, Ya-Dong; Zou, Quan; Wang, Jun; Tu, Jia-Run; Cai, Wen-Sheng; Shao, Xue-Guang
Near infrared diffusive reflectance spectroscopy has been applied in on-site or on-line analysis due to its characteristics of fastness, non-destruction and the feasibility for real complex sample analysis. The present work reported a real-time monitoring method for industrial production by using near infrared spectroscopic technique and multivariate statistical process analysis. In the method, the real-time near infrared spectra of the materials are collected on the production line, and then the evaluation of the production process can be achieved by a statistic Hotelling T2 calculated with the established model. In this work, principal component analysis (PCA) is adopted for building the model, and the statistic is calculated by projecting the real-time spectra onto the PCA model. With an application of the method in a practical production, it was demonstrated that a real-time evaluation of the variations in the production can be realized by investigating the changes in the statistic, and the comparison of the products in different batches can be achieved by further statistics of the statistic. Therefore, the proposed method may provide a practical way for quality insurance of production processes.
Southard, Jonathan N.
Instrumentation for real-time PCR is used primarily for amplification and quantitation of nucleic acids. The capability to measure fluorescence while controlling temperature in multiple samples can also be applied to the analysis of proteins. Conformational stability and changes in stability due to ligand binding are easily assessed. Protein…
Müller, Oliver A.; Grau, Jan; Thieme, Sabine; Prochaska, Heike; Adlung, Norman; Sorgatz, Anika; Bonas, Ulla
The Gram-negative bacterium Xanthomonas campestris pv. vesicatoria (Xcv) causes bacterial spot disease of pepper and tomato by direct translocation of type III effector proteins into the plant cell cytosol. Once in the plant cell the effectors interfere with host cell processes and manipulate the plant transcriptome. Quantitative RT-PCR (qRT-PCR) is usually the method of choice to analyze transcriptional changes of selected plant genes. Reliable results depend, however, on measuring stably expressed reference genes that serve as internal normalization controls. We identified the most stably expressed tomato genes based on microarray analyses of Xcv-infected tomato leaves and evaluated the reliability of 11 genes for qRT-PCR studies in comparison to four traditionally employed reference genes. Three different statistical algorithms, geNorm, NormFinder and BestKeeper, concordantly determined the superiority of the newly identified reference genes. The most suitable reference genes encode proteins with homology to PHD finger family proteins and the U6 snRNA-associated protein LSm7. In addition, we identified pepper orthologs and validated several genes as reliable normalization controls for qRT-PCR analysis of Xcv-infected pepper plants. The newly identified reference genes will be beneficial for future qRT-PCR studies of the Xcv-tomato and Xcv-pepper pathosystems, as well as for the identification of suitable normalization controls for qRT-PCR studies of other plant-pathogen interactions, especially, if related plant species are used in combination with bacterial pathogens. PMID:26313760
Müller, Oliver A; Grau, Jan; Thieme, Sabine; Prochaska, Heike; Adlung, Norman; Sorgatz, Anika; Bonas, Ulla
The Gram-negative bacterium Xanthomonas campestris pv. vesicatoria (Xcv) causes bacterial spot disease of pepper and tomato by direct translocation of type III effector proteins into the plant cell cytosol. Once in the plant cell the effectors interfere with host cell processes and manipulate the plant transcriptome. Quantitative RT-PCR (qRT-PCR) is usually the method of choice to analyze transcriptional changes of selected plant genes. Reliable results depend, however, on measuring stably expressed reference genes that serve as internal normalization controls. We identified the most stably expressed tomato genes based on microarray analyses of Xcv-infected tomato leaves and evaluated the reliability of 11 genes for qRT-PCR studies in comparison to four traditionally employed reference genes. Three different statistical algorithms, geNorm, NormFinder and BestKeeper, concordantly determined the superiority of the newly identified reference genes. The most suitable reference genes encode proteins with homology to PHD finger family proteins and the U6 snRNA-associated protein LSm7. In addition, we identified pepper orthologs and validated several genes as reliable normalization controls for qRT-PCR analysis of Xcv-infected pepper plants. The newly identified reference genes will be beneficial for future qRT-PCR studies of the Xcv-tomato and Xcv-pepper pathosystems, as well as for the identification of suitable normalization controls for qRT-PCR studies of other plant-pathogen interactions, especially, if related plant species are used in combination with bacterial pathogens.
Larson, Richard R.; Millard, D. Edward
An approach to automate the real-time analysis of flight critical health monitoring and system status is being developed and evaluated at the NASA Dryden Flight Research Facility. A software package was developed in-house and installed as part of the extended aircraft interrogation and display system. This design features a knowledge-base structure in the form of rules to formulate interpretation and decision logic of real-time data. This technique has been applied for ground verification and validation testing and flight testing monitoring where quick, real-time, safety-of-flight decisions can be very critical. In many cases post processing and manual analysis of flight system data are not required. The processing is described of real-time data for analysis along with the output format which features a message stack display. The development, construction, and testing of the rule-driven knowledge base, along with an application using the X-31A flight test program, are presented.
Moench, Tobias; Hollmann, Maurice; Bernarding, Johannes
The real-time analysis of brain activation using functional MRI data offers a wide range of new experiments such as investigating self-regulation or learning strategies. However, besides special data acquisition and real-time data analysing techniques such examination requires dynamic and adaptive stimulus paradigms and self-optimising MRI-sequences. This paper presents an approach that enables the unified handling of parameters influencing the different software systems involved in the acquisition and analysing process. By developing a custom-made Experiment Description Language (EDL) this concept is used for a fast and flexible software environment which treats aspects like extraction and analysis of activation as well as the modification of the stimulus presentation. We describe how extracted real-time activation is subsequently evaluated by comparing activation patterns to previous acquired templates representing activated regions of interest for different predefined conditions. According to those results the stimulus presentation is adapted. The results showed that the developed system in combination with EDL is able to reliably detect and evaluate activation patterns in real-time. With a processing time for data analysis of about one second the approach is only limited by the natural time course of the hemodynamic response function of the brain activation.
Schoeffler, James D.
Successful application of software engineering methodologies requires an integrated analysis and design life-cycle in which the various phases flow smoothly 'seamlessly' from analysis through design to implementation. Furthermore, different analysis methodologies often lead to different structuring of the system so that the transition from analysis to design may be awkward depending on the design methodology to be used. This is especially important when object-oriented programming is to be used for implementation when the original specification and perhaps high-level design is non-object oriented. Two approaches to real-time systems analysis which can lead to an object-oriented design are contrasted: (1) modeling the system using structured analysis with real-time extensions which emphasizes data and control flows followed by the abstraction of objects where the operations or methods of the objects correspond to processes in the data flow diagrams and then design in terms of these objects; and (2) modeling the system from the beginning as a set of naturally occurring concurrent entities (objects) each having its own time-behavior defined by a set of states and state-transition rules and seamlessly transforming the analysis models into high-level design models. A new concept of a 'real-time systems-analysis object' is introduced and becomes the basic building block of a series of seamlessly-connected models which progress from the object-oriented real-time systems analysis and design system analysis logical models through the physical architectural models and the high-level design stages. The methodology is appropriate to the overall specification including hardware and software modules. In software modules, the systems analysis objects are transformed into software objects.
Mikel, P; Vasickova, P; Kralik, P
RNA viruses are pathogenic agents of many serious infectious diseases affecting humans and animals. The detection of pathogenic RNA viruses is based on modern molecular methods, of which the most widely used methods are the reverse transcription polymerase chain reaction (RT-PCR) and the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). All steps of RT-PCR and qRT-PCR should be strictly controlled to ensure the validity of obtained results. False-negative results may be caused not only by inhibition of RT or/and PCR steps but also by failure of the nucleic acid extraction step, particularly in the case of viral RNA extraction. The control of nucleic acid extraction generally involves the utilization of a non-pathogenic virus (process control virus) of similar structural properties to those of the target virus. Although in clinical samples the use of such process control virus is only recommended, in other kinds of settings such as food matrices its use is necessary. Currently, several different process control viruses are used for these purposes. Process control viruses can also be constructed artificially using technology for production of MS2 phage-like particles, which have many advantages in comparison with other used controls and are especially suited for controlling the detection and quantification of certain types of RNA viruses. The technology for production of MS2 phage-like particles is theoretically well established, uses the knowledge gained from the study of the familiar bacteriophage MS2 and utilizes many different approaches for the construction of the various process control viruses. Nevertheless, the practical use of MS2 phage-like particles in routine diagnostics is relatively uncommon. The current situation with regard to the use of MS2 phage-like particles as process control viruses in detection of RNA viruses and different methods of their construction, purification and use are summarized and discussed in this
Deng, Jikui; Ma, Zhuoya; Huang, Wenbo; Li, Chengrong; Wang, Heping; Zheng, Yuejie; Zhou, Rong; Tang, Yi-Wei
Multiplex RT-PCR assays have been widely used tools for detection and differentiation of a panel of respiratory viral pathogens. In this study, we evaluated the Qiagen ResPlex II V2.0 kit and explored factors influencing its sensitivity. Nasopharyngeal swab (NPS) specimens were prospectively collected from pediatric inpatients with lower respiratory tract infections at the time of admission in the Shenzhen Children's Hospital from May 2009 to April 2010. Total nucleic acids were extracted using the EZ1 system (Qiagen, Germany) and 17 respiratory viruses and genotypes including influenza A virus (FluA), FluB, parainfluenza virus 1 (PIV1), PIV2, PIV3, PIV4, respiratory syncytial virus (RSV), human metapneumovirus (hMPV), rhinoviruses (RhV), enteroviruses (EnV), human bocaviruses (hBoV), adenoviruses (AdV), four coronaviruses (229E, OC43, NL63 and HKU1), and FluA 2009 pandemic H1N1(H1N1-p) were detected and identified by the ResPlex II kit. In parallel, 16 real-time TaqMan quantitative RT-PCR assays were used to quantitatively detect each virus except for RhV. Influenza and parainfluenza viral cultures were also performed. Among the total 438 NPS specimens collected during the study period, one or more viral pathogens were detected in 274 (62.6%) and 201(45.9%) specimens by monoplex TaqMan RT-PCR and multiplex ResPlex, respectively. When results from monoplex PCR or cell culture were used as the reference standard, the multiplex PCR possessed specificities of 92.9-100.0%. The sensitivity of multiplex PCR for PIV3, hMPV, PIV1 and BoV were 73.1%, 70%, 66.7% and 55.6%, respectively, while low sensitivities (11.1%-40.0%) were observed for FluA, EnV, OC43, RSV and H1N1. Among the seven viruses/genotypes detected with higher frequencies, multiplex PCR sensitivities were correlated significantly with viral loads determined by the TaqMan RT-PCR in FluA, H1N1-p and RSV (p=0.011-0.000). The Qiagen ResPlex II multiplex RT-PCR kit possesses excellent specificity for simultaneous
Chen, Chien-Hsun; Lin, Ziqing; Tian, Ran; Shi, Riyi; Cooks, R. Graham; Ouyang, Zheng
A miniature mass spectrometry system with a sampling probe has been developed for real-time analysis of chemicals from sample surfaces. The sampling probe is 1.5m in length and is comprised of one channel for introducing the spray and the other channel for transferring the charged species back to the Mini MS. This system provides a solution to the problem of real-time mass spectrometry analysis of a three-dimensional object in the field and is successful with compounds including those in inks, agrochemicals, explosives, and animal tissues. This system can be implemented in the form of a backpack MS with a sampling probe for forensic analysis or in the form of a compact MS with an intra-surgical probe for tissue analysis. PMID:26237577
Liu, Changyu; Jia, Jianbo; Dong, Shaojun
We reported a biofilm reactor (BFR) based analytical system for real-time biochemical oxygen demand (BOD) monitoring. It does not need a blank solution and other chemical reagents to operate. The initial dissolved oxygen (DO) in sample solution was measured as blank, while DO in the BFR effluent was measured as response. The DO difference obtained before and after the sample solution flowed through the BFR was regarded as an indicator of real-time BOD. The analytical performance of this reagent-free BFR system was equal to the previous BFR system operated using phosphate buffer saline (PBS) and high purity deionized water in reproducibility, accuracy and long-term stability. Besides, this method embraces many notable advantages, such as no secondary pollution. Additionally, the sample solutions are free from temperature controlling and air-saturation before injection. Significantly, this is a real-time BOD analysis method. This method was successfully carried out in a simulated emergency, and the obtained results agreed well with conventional BOD₅. These advantages, coupled with simplicity in device, convenience in operation and minimal maintenance, make such a reagent-free BFR analytical system promising for practical BOD real-time warning.
Angelini, Claudia; Carfora, Maria Francesca; Carriero, Maria Vincenza; Natalini, Roberto
Experiments of cell migration and chemotaxis assays have been classically performed in the so-called Boyden Chambers. A recent technology, xCELLigence Real Time Cell Analysis, is now allowing to monitor the cell migration in real time. This technology measures impedance changes caused by the gradual increase of electrode surface occupation by cells during the course of time and provide a Cell Index which is proportional to cellular morphology, spreading, ruffling and adhesion quality as well as cell number. In this paper we propose a macroscopic mathematical model, based on advection-reaction-diffusion partial differential equations, describing the cell migration assay using the real-time technology. We carried out numerical simulations to compare simulated model dynamics with data of observed biological experiments on three different cell lines and in two experimental settings: absence of chemotactic signals (basal migration) and presence of a chemoattractant. Overall we conclude that our minimal mathematical model is able to describe the phenomenon in the real time scale and numerical results show a good agreement with the experimental evidences. PMID:27680883
Di Costanzo, Ezio; Ingangi, Vincenzo; Angelini, Claudia; Carfora, Maria Francesca; Carriero, Maria Vincenza; Natalini, Roberto
Experiments of cell migration and chemotaxis assays have been classically performed in the so-called Boyden Chambers. A recent technology, xCELLigence Real Time Cell Analysis, is now allowing to monitor the cell migration in real time. This technology measures impedance changes caused by the gradual increase of electrode surface occupation by cells during the course of time and provide a Cell Index which is proportional to cellular morphology, spreading, ruffling and adhesion quality as well as cell number. In this paper we propose a macroscopic mathematical model, based on advection-reaction-diffusion partial differential equations, describing the cell migration assay using the real-time technology. We carried out numerical simulations to compare simulated model dynamics with data of observed biological experiments on three different cell lines and in two experimental settings: absence of chemotactic signals (basal migration) and presence of a chemoattractant. Overall we conclude that our minimal mathematical model is able to describe the phenomenon in the real time scale and numerical results show a good agreement with the experimental evidences.
Oka, Kentaro; Iizuka, Atsushi; Inoue, Yasuo; Mizukoshi, Atsushi; Noguchi, Miyuki; Yamasaki, Akihiro; Yanagisawa, Yukio
A combined integration analysis and real time monitoring (Peak Capture System) system was developed for volatile organic compounds (VOCs). Individual integration analysis and real time monitoring can be used to qualitatively and quantitatively analyze VOCs in the atmosphere and in indoor environments and determine the variation in total VOC (TVOC) concentration with time, respectively. In the Peak Capture System, real time monitoring was used to predict future elevations in the TVOC concentration (peak), and this was used an indicator of when to collect (capture) ambient air samples for integration analysis. This enabled qualitative and quantitative analysis of VOCs when the TVOC concentration was high. We developed an algorithm to predict variation in the TVOC concentration, and constructed an automatic system to initiate air sampling for integration analysis. With the system, auto-sampling and analysis of VOCs in a conventional house were conducted. In comparison with background concentrations, the results of peak analysis enabled identification of compounds whose concentration rose. This also enabled an evaluation of possible VOC emission sources. PMID:21317996
Ciraci, Selim; Yin, Jian
The smart grid applications requires real time analysis, response within the order of milliseconds and high-reliability because of the mission critical structure of the power grid system. The only way to satisfy these requirements is in network data analysis and build-in redundancy routing for failures. To achieve this, we propose a data dissemination system that builds routes using network flow algorithms, have in network processing of the data and utilize data encoding to cope with high latencies.
Yang, Qi; Yin, Jiajia; Li, Gao; Qi, Liwang; Yang, Feiyun; Wang, Ruigang; Li, Guojing
Caragana korshinskii Kom., which is widely distributed in the northwest China and Mongolia, is an important forage bush belonging to the legume family with high economic and ecological value. Strong tolerance ability to various stresses makes C. korshinskii Kom. a valuable species for plant stress research. In this study, suitable reference genes for quantitative real-time reverse transcription PCR (qRT-PCR) were screened from 11 candidate reference genes, including ACT, GAPDH, EF1α, UBQ, TUA, CAP, TUB, TUB3, SKIP1, SKIP5-1 and SKIP5-2. A total of 129 samples under drought, heat, cold, salt, ABA and high pH treatment were profiled, and software such as geNORM, NormFinder and BestKeeper were used for reference gene evaluation and selection. Different suitable reference genes were selected under different stresses. Across all 129 samples, GAPDH, EF1α and SKIP5-1 were found to be the most stable reference genes, and EF1α+SKIP5-1 is the most stable reference gene combination. Conversely, TUA, TUB and SKIP1 were not suitable for using as reference genes owing to their great expression variation under some stress conditions. The relative expression levels of CkWRKY1 were detected using the stable and unstable reference genes and their applicability was confirmed. These results provide some stable reference genes and reference gene combinations for qRT-PCR under different stresses in C. korshinskii Kom. for future research work, and indicate that CkWRKY1 plays essential roles in response to stresses in C. korshinskii.
Jones, Roger W; McClelland, John F
Ink analysis is central to questioned document examination. We applied direct analysis in real time mass spectrometry (DART MS) to ballpoint, gel, and fluid writing ink analysis. DART MS acquires the mass spectrum of an ink while it is still on a document without altering the appearance of the document. Spectra were acquired from ink on a variety of papers, and the spectrum of the blank paper could be subtracted out to produce a cleanly isolated ink spectrum in most cases. Only certain heavy or heavily processed papers interfered. The time since an ink is written on paper has a large effect on its spectrum. DART spectra change radically during the first few months after an ink is written as the more volatile components evaporate, but the spectra stabilize after that. A library-search study involving 166 well-aged inks assessed the ability to identify inks from their DART spectra. The aggregate success rate was 92%.
Tafinine, Farid; Mokrani, Karim
A proposed signal processing technique for incipient real time bearing fault detection based on kurtogram analysis is presented in this paper. The kurtogram is a fourth-order spectral analysis tool introduced for detecting and characterizing non-stationarities in a signal. This technique starts from investigating the resonance signatures over selected frequency bands to extract the representative features. The traditional spectral analysis is not appropriate for non-stationary vibration signal and for real time diagnosis. The performance of the proposed technique is examined by a series of experimental tests corresponding to different bearing conditions. Test results show that this signal processing technique is an effective bearing fault automatic detection method and gives a good basis for an integrated induction machine condition monitor.
Cymbalyuk, Gennady; Shilnikov, Andrey
Neurons exhibit various activity regimes and transitions in between. The central pattern generator controlling the leech's heartbeat contains identified pairs of mutually inhibitory neurons (Calabrese et al. 1995). We describe real time systems approaches to the analysis of their activity. The hybrid system consists of a living neuron and a model neuron (or an artificial silicon neuron) interacting in the real time. Dynamic clamp is used to implement artificial ionic currents and synapses in the system (Sharp et al. 1993). Our study determines the mechanisms underlying and regulating bursting activity, based on intrinsic membrane dynamics and network interactions. The complexity of endogenous dynamics originates from the diversity of ionic currents operating on different time scales. Hybrid system analysis and slow-fast dynamical systems analysis have been combined in our studies of bursting, its origin and transformations in heart interneurons both as single cells and in the mutually inhibitory configuration.
Zhang, Jianhua; Nie, Xianzhou; Boquel, Sébastien; Al-Daoud, Fadi; Pelletier, Yvan
The sensitivity of reverse transcription-polymerase chain reaction (RT-PCR) for virus detection is influenced by many factors such as specificity of primers and quality of templates. These factors become extremely important for successful detection when virus concentration is low. Total RNA isolated from Potato virus Y (PVY)-infected potato plants using the sodium sulfite RNA isolation method or RNeasy plant mini kit contains a high proportion of host RNA and may also contain trace amount of phenolic and polysaccharide residues, which may inhibit RT-PCR. The goal of this study was to enhance the sensitivity of PVY detection by reducing host RNA in the extract by differential centrifugation followed by extraction using an RNeasy mini kit (DCR method). One-step RT-PCR had relatively low amplification efficiency for PVY RNA when a high proportion of plant RNA was present. SYBR Green-based real time RT-PCR showed that the RNA isolated by the DCR method had a higher cycle threshold value (Ct) for the elongation factor 1-α mRNA (Ef1α) of potato than the Ct value of the RNA extracted using the RNeasy plant mini kit, indicating that the DCR method significantly reduced the proportion of potato RNA in the extract. The detectable amount of RNA extracted using the DCR method was <0.001ng when plant sap from 10 PVY-infected and PVY-free potato leaflets in a 1.5:100 fresh weight ratio was extracted, compared with 0.01 and 0.02ng of RNA using the RNeasy plant mini kit and sodium sulfite RNA isolation methods, respectively.
L'Huillier, Arnaud G; Lombos, Ernesto; Tang, Elaine; Perusini, Stephen; Eshaghi, Alireza; Nagra, Sandeep; Frantz, Christine; Olsha, Romy; Kristjanson, Erik; Dimitrova, Kristina; Safronetz, David; Drebot, Mike; Gubbay, Jonathan B
Background: With the emerging ZIKA virus (ZIKV) epidemic, accessible real-time reverse-transcription PCR (rRT-PCR) assays are needed to streamline testing. The commercial Altona Diagnostics RealStar ZIKV rRT-PCR Test Kit has been approved for Emergency Use Authorization by the FDA. Our aim was to verify Altona-PCR, by comparing it to the CDC-designed dual target ZIKV virus rRT-PCR reference assay (Reference-PCR), and describe demographics of patients tested for ZIKV by rRT-PCR in Ontario, Canada.Methods: A large set of clinical specimens were tested for ZIKV by Altona-PCR and Reference-PCR. Positive or equivocal specimens underwent PCR and Sanger sequencing targeting ZIKV NS5 gene.Results: 671 serum specimens were tested by Reference-PCR: 58 (8.6%) were positive, 193 (28.8%) equivocal and 420 (62.6%) negative. Ninety percent of Reference-PCR positive patients were tested in the first 5 days after symptom onset. Altona-PCR was performed on 284/671 tested specimens by Reference-PCR. Altona-PCR was positive in 53/58 (91%) Reference-PCR positive and 16/193 (8%) Reference-PCR equivocal specimens; ZIKV NS5 PCR was positive in all 68 Altona-PCR positive specimens, and negative in all 181 Altona-PCR negative specimens that underwent NS5 PCR.Conclusion: Most ZIKV PCR positive cases are detected in the first five days of illness. Altona-PCR has very good sensitivity (91%) and specificity (97%) compared to Reference-PCR. Altona-PCR can be used for ZIKV diagnostic testing, with less extensive verification requirements compared to a laboratory developed test.
Techathuvanan, Chayapa; Draughon, Frances Ann; D'Souza, Doris Helen
Reverse transcriptase PCR (RT-PCR) detects the presence of mRNA and has a greater potential for detecting viable pathogens than do DNA-based PCR assays, with improved speed and sensitivity compared with traditional methods. Our objective was to rapidly and sensitively detect Salmonella Typhimurium from pork within two 8-h work shifts using a SYBR Green I real-time RT-PCR (rt-RT-PCR) assay. Pork chop and sausage samples (25 g) were inoculated with 10(8) to 10(0) CFU of Salmonella Typhimurium and stomached in 225 ml of tetrathionate broth. Serial dilutions were spread plated on xylose lysine Tergitol 4 agar either immediately or after 10 h of selective preenrichment or preenrichment followed by 12 h of selective enrichment (for stressed cells) at 37 degrees C for standard cultural enumeration. RNA was extracted using the TRIzol method. The rt-RT-PCR assay was carried out in a Bio-Rad iCycler using a SYBR Green I one-step RT-PCR kit and Salmonella specific invA gene primers with an internal amplification control (IAC). The PCR was followed by melting temperature (T(m)) analysis to determine specific Salmonella invA (T(m) = 87.5 degrees C) and IAC (T(m) = 82 degrees C) products. Improved Salmonella detection up to 10(1) CFU/25 g of pork and 10(0) CFU/25 g of sausages was obtained after 10 h of enrichment within approximately 24 h. Even without enrichment, Salmonella could be detected from both pork chop and sausage at 10(6) CFU/25 g within 1 day. This robust rt-RT-PCR detects and confirms Salmonella in pork within approximately 24 h and thus is significantly faster than traditional methods that take >/=1 week. This assay shows promise for routine testing and monitoring of Salmonella by the pork industry.
Delnatte, Pauline; Mak, Matthew; Ojkic, Davor; Raghav, Raj; DeLay, Josepha; Smith, Dale A
Avian bornavirus (ABV), the cause of proventricular dilation disease in psittacine birds, has been detected in multiple tissues of infected birds using immunohistochemical staining (IHC) and reverse transcription polymerase chain reaction (RT-PCR). In the current study, real-time RT-PCR, using primers targeting the ABV matrix gene, was used to detect ABV in 146 tissues from 7 ABV-infected psittacine birds. Eighty-six percent of the samples tested positive, with crossing point values ranging from 13.82 to 37.82 and a mean of 22.3. These results were compared to the findings of a previous study using gel-based RT-PCR and IHC on the same samples. The agreement between the 2 RT-PCR techniques was 91%; when tests disagreed it was because samples were negative using gel-based RT-PCR but positive on real-time RT-PCR. Agreement with IHC was 77%; 16 out of 74 samples were negative using IHC but positive on real-time RT-PCR. The results suggest that real-time RT-PCR is a more sensitive technique than gel-based RT-PCR and IHC to detect ABV in tissues. The tissues that were ranked most frequently as having a high amount of viral RNA were proventriculus, kidney, colon, cerebrum, and cerebellum. Skeletal muscle, on the other hand, was found to have a consistently low amount of viral RNA.
Sheng, X G; Zhao, Z Q; Yu, H F; Wang, J S; Zheng, C F; Gu, H H
Quantitative reverse-transcription PCR (qRT-PCR) is a versatile technique for the analysis of gene expression. The selection of stable reference genes is essential for the application of this technique. Cauliflower (Brassica oleracea L. var. botrytis) is a commonly consumed vegetable that is rich in vitamin, calcium, and iron. Thus far, to our knowledge, there have been no reports on the validation of suitable reference genes for the data normalization of qRT-PCR in cauliflower. In the present study, we analyzed 12 candidate housekeeping genes in cauliflower subjected to different abiotic stresses, hormone treatment conditions, and accessions. geNorm and NormFinder algorithms were used to assess the expression stability of these genes. ACT2 and TIP41 were selected as suitable reference genes across all experimental samples in this study. When different accessions were compared, ACT2 and UNK3 were found to be the most suitable reference genes. In the hormone and abiotic stress treatments, ACT2, TIP41, and UNK2 were the most stably expressed. Our study also provided guidelines for selecting the best reference genes under various experimental conditions.
Notaro, Sara; Reimer, Daniel; Duggan-Peer, Michaela; Fiegl, Heidi; Wiedermair, Annamarie; Rössler, Julia; Altevogt, Peter; Marth, Christian; Zeimet, Alain Gustave
Background Management of endometrial carcinoma (EC) still needs improvement of risk assessment. Recently, L1CAM immunohistochemical (IHC) evaluation showed a unique value to predict the outcome of early EC. However IHC results are often conflicting for lack of inter-laboratory standardisation. Methods Here, as a proof of concept and to increase reproducibility we assayed eighty-two EC and 26 normal endometrium samples for L1CAM expression (L1CAMEXP) via qRT-PCR. The IHC evaluation was performed in 50 cancer samples. Moreover, we aimed to substantiate the in-vitro findings of L1CAM regulation through its promoter methylation (L1CAMMET), miR-34a expression and miR-34a promoter methylation. DNA methylation was assessed with MethyLight PCR technique. Results High overall concordant results between IHC and RT-PCR evaluations were found. L1CAMEXP was detected in 11% of cancer specimens. These positive cancers exhibited a worse DFS (p=0.032) and OS (p=0.016) in a multivariate COX-regression model. L1CAMEXP predicted distant failure (p=0.007) and L1CAMMET predicted risk-reduction of lymph-node involvement (p=0.005). Inverse correlations between L1CAMEXP and L1CAMMET (p=0.004) and between L1CAMEXP and miR-34a expression (p=0.002) were found. Conclusions In conclusion qRT-PCR analysis is a reliable approach to evaluate L1CAM status in EC and L1CAMEXP was highly predictive for distant failure and poor outcome, confirming the large IHC-based studies. Interestingly, L1CAMMET was able to assess the risk of pelvic lymph-node involvement. Especially the latter finding has to be confirmed in larger prospective series. PMID:27233077
Bosworth, J. T.; West, J. C.
A technique has been developed to compare the open-loop frequency response of a flight test aircraft real time with linear analysis predictions. The result is direct feedback to the flight control systems engineer on the validity of predictions and adds confidence for proceeding with envelope expansion. Further, gain and phase margins can be tracked for trends in a manner similar to the techniques used by structural dynamics engineers in tracking structural modal damping.
Sharma, Shashi; Dash, Paban Kumar; Santhosh, S R; Shukla, Jyoti; Parida, Manmohan; Rao, P V Lakshmana
Chikungunya is one of the most important emerging arboviral infections of public health significance. Due to lack of a licensed vaccine, rapid diagnosis plays an important role in early management of patients. In this study, a QC-RT-PCR assay was developed to quantify Chikungunya virus (CHIKV) RNA by targeting the conserved region of E1 gene. A competitor molecule containing an internal insertion was generated, which provided a stringent control of the quantification process. The introduction of 10-fold serially diluted competitor in each reaction was further used to determine sensitivity. The applicability of this assay for quantification of CHIKV RNA was evaluated with human clinical samples, and the results were compared with real-time quantitative RT-PCR. The sensitivity of this assay was estimated to be 100 RNA copies per reaction with a dynamic detection range of 10(2) to 10(10) copies. Specificity was confirmed using closely related alpha and flaviviruses. The comparison of QC-RT-PCR result with real-time RT-PCR revealed 100% concordance for the detection of CHIKV in clinical samples. These findings demonstrated that the reported assay is convenient, sensitive and accurate method and has the potential usefulness for clinical diagnosis due to simultaneous detection and quantification of CHIKV in acute-phase serum samples.
Takekawa, John Y; Iverson, Samuel A; Schultz, Annie K; Hill, Nichola J; Cardona, Carol J; Boyce, Walter M; Dudley, Joseph P
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((R)), 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.
Takekawa, John Y.; Iverson, Samuel A.; Schultz, Annie K.; Hill, Nichola J.; Cardona, Carol J.; Boyce, Walter M.; Dudley, Joseph P.
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.
Goodell, Christa K.; Zhang, Jianqiang; Strait, Erin; Harmon, Karen; Patnayak, Devi; Otterson, Tracy; Culhane, Marie; Christopher-Hennings, Jane; Clement, Travis; Leslie-Steen, Pamela; Hesse, Richard; Anderson, Joe; Skarbek, Kevin; Vincent, Amy; Kitikoon, Pravina; Swenson, Sabrina; Jenkins-Moore, Melinda; McGill, Jodi; Rauh, Rolf; Nelson, William; O’Connell, Catherine; Shah, Rohan; Wang, Chong; Main, Rodger; Zimmerman, Jeffrey J.
The probability of detecting influenza A virus (IAV) in oral fluid (OF) specimens was calculated for each of 13 assays based on real-time reverse-transcription polymerase chain reaction (rRT-PCR) and 7 assays based on virus isolation (VI). The OF specimens were inoculated with H1N1 or H3N2 IAV and serially diluted 10-fold (10−1 to 10−8). Eight participating laboratories received 180 randomized OF samples (10 replicates × 8 dilutions × 2 IAV subtypes plus 20 IAV-negative samples) and performed the rRT-PCR and VI procedure(s) of their choice. Analysis of the results with a mixed-effect logistic-regression model identified dilution and assay as variables significant (P < 0.0001) for IAV detection in OF by rRT-PCR or VI. Virus subtype was not significant for IAV detection by either rRT-PCR (P = 0.457) or VI (P = 0.101). For rRT-PCR the cycle threshold (Ct) values increased consistently with dilution but varied widely. Therefore, it was not possible to predict VI success on the basis of Ct values. The success of VI was inversely related to the dilution of the sample; the assay was generally unsuccessful at lower virus concentrations. Successful swine health monitoring and disease surveillance require assays with consistent performance, but significant differences in reproducibility were observed among the assays evaluated. PMID:26733728
Goodell, Christa K; Zhang, Jianqiang; Strait, Erin; Harmon, Karen; Patnayak, Devi; Otterson, Tracy; Culhane, Marie; Christopher-Hennings, Jane; Clement, Travis; Leslie-Steen, Pamela; Hesse, Richard; Anderson, Joe; Skarbek, Kevin; Vincent, Amy; Kitikoon, Pravina; Swenson, Sabrina; Jenkins-Moore, Melinda; McGill, Jodi; Rauh, Rolf; Nelson, William; O'Connell, Catherine; Shah, Rohan; Wang, Chong; Main, Rodger; Zimmerman, Jeffrey J
The probability of detecting influenza A virus (IAV) in oral fluid (OF) specimens was calculated for each of 13 assays based on real-time reverse-transcription polymerase chain reaction (rRT-PCR) and 7 assays based on virus isolation (VI). The OF specimens were inoculated with H1N1 or H3N2 IAV and serially diluted 10-fold (10(-1) to 10(-8)). Eight participating laboratories received 180 randomized OF samples (10 replicates × 8 dilutions × 2 IAV subtypes plus 20 IAV-negative samples) and performed the rRT-PCR and VI procedure(s) of their choice. Analysis of the results with a mixed-effect logistic-regression model identified dilution and assay as variables significant (P < 0.0001) for IAV detection in OF by rRT-PCR or VI. Virus subtype was not significant for IAV detection by either rRT-PCR (P = 0.457) or VI (P = 0.101). For rRT-PCR the cycle threshold (Ct) values increased consistently with dilution but varied widely. Therefore, it was not possible to predict VI success on the basis of Ct values. The success of VI was inversely related to the dilution of the sample; the assay was generally unsuccessful at lower virus concentrations. Successful swine health monitoring and disease surveillance require assays with consistent performance, but significant differences in reproducibility were observed among the assays evaluated.
Zhong, D. H.; Yan, F. G.; Li, M. C.; Huang, C. X.; Fan, K.; Tang, J. F.
Real-time analysis and feedback systems play a vital role in obtaining good results from grouting processes. However, when there are intense construction schedules and complex geological structures, it is difficult for existing systems to provide to site engineers, prior to the borehole construction, prompt and accurate feedback, such as detailed geological information about grouting boreholes, which limits the use of such systems in practical applications. This paper proposes combining a three-dimensional (3D) geological model with real-time data collection technology in a system for both monitoring grouting, and providing analysis and feedback. This integrated grouting model, which comprises the geological model, the grouting borehole model and the grouting parameter database set, can be coupled and associated dynamically with grouting data. Additionally, the following methods are applied in this system: real-time grouting data processing and a monitoring alarm, prediction and visualization of geological conditions, forecasting of rock uplift, and visualization analysis of grouting parameters. The application of this system in Hydropower Project A, China is used as a case study. The predictions of geological conditions are closely matched with the actual situation, and this system can be used to monitor construction processes remotely and to help site engineers to design reasonable construction plans, optimize layouts for grouting boreholes and adjust construction measures.
Swider, Joseph R
The use of a direct analysis in real time (DART) mass spectrometer (MS) instrument was optimized for 22 compounds of organic explosive residues to provide a guide for DART-MS users in rapid screening of explosive compounds. Samples were introduced as neat solutions and sequential dilutions to determine optimal instrument conditions and lowest concentration detectable. Most compounds were optimized to 250°C in the negative ion mode, and several compounds benefited from the addition of a chloride dopant from methylene chloride (amino-dinitrotoluenes, RDX, EGDN, and PETN). Few compounds were more sensitive in the positive ion mode (TEGDN, DEGDN, HNS, and DMNB). Mixtures of compounds were detected using clean room wipes, directly from their surfaces and from subsequent extractions. Compounds from the mixtures were also successfully detected in soil and from swipes of spiked surfaces. The instrument showed merit in detection of pg/μL solutions for most of the compounds and among the substrates tested.
Liu, Hua-Kuang; Huang, K. S.; Diep, J.
Optical data processing techniques have the inherent advantage of high data throughout, low weight and low power requirements. These features are particularly desirable for onboard spacecraft in-situ real-time data analysis and data compression applications. the proposed multi-layer optical holographic neural net pattern recognition technique will utilize the nonlinear photorefractive devices for real-time adaptive learning to classify input data content and recognize unexpected features. Information can be stored either in analog or digital form in a nonlinear photofractive device. The recording can be accomplished in time scales ranging from milliseconds to microseconds. When a system consisting of these devices is organized in a multi-layer structure, a feedforward neural net with bifurcating data classification capability is formed. The interdisciplinary research will involve the collaboration with top digital computer architecture experts at the University of Southern California.
Liu, Hua-Kuang; Huang, K.; Diep, J.
Optical data processing techniques have the inherent advantage of high data throughout, low weight and low power requirements. These features are particularly desirable for onboard spacecraft in-situ real-time data analysis and data compression applications. The proposed multi-layer optical holographic neural net pattern recognition technique will utilize the nonlinear photorefractive devices for real-time adaptive learning to classify input data content and recognize unexpected features. Information can be stored either in analog or digital form in a nonlinear photorefractive device. The recording can be accomplished in time scales ranging from milliseconds to microseconds. When a system consisting of these devices is organized in a multi-layer structure, a feed forward neural net with bifurcating data classification capability is formed. The interdisciplinary research will involve the collaboration with top digital computer architecture experts at the University of Southern California.
Wieseman, Carol D.; Hoadley, Sherwood T.
This paper provides an overview of a versatile software package developed for time- and frequency-domain analyses of experimental wind-tunnel data. This package, originally developed for analyzing data in the NASA Langley Transonic Dynamics Tunnel (TDT), is applicable for analyzing any time-domain data. A Matlab-based software package, TDT-analyzer, provides a compendium of commonly-required dynamic analysis functions in a user-friendly interactive and batch processing environment. TDT-analyzer has been used extensively to provide on-line near real-time and post-test examination and reduction of measured data acquired during wind tunnel tests of aeroelastically-scaled models of aircraft and rotorcraft as well as a flight test of the NASA High Alpha Research Vehicle (HARV) F-18. The package provides near real-time results in an informative and timely manner far exceeding prior methods of data reduction at the TDT.
Bosworth, John T.; Stachowiak, Susan J.
A method has been developed for real-time stability margin measurement calculations. The method relies on a tailored-forced excitation targeted to a specific frequency range. Computation of the frequency response is matched to the specific frequencies contained in the excitation. A recursive Fourier transformation is used to make the method compatible with real-time calculation. The method was incorporated into the X-38 nonlinear simulation and applied to an X-38 robustness test. X-38 stability margins were calculated for different variations in aerodynamic and mass properties over the vehicle flight trajectory. The new method showed results comparable to more traditional stability analysis techniques, and at the same time, this new method provided coverage that is more complete and increased efficiency.
Pauli, Guido F; Pro, Samuel M; Chadwick, Lucas R; Burdick, Thomas; Pro, Luke; Friedl, Warren; Novak, Nick; Maltby, John; Qiu, Feng; Friesen, J Brent
Countercurrent separation (CCS) utilizes the differential partitioning behavior of analytes between two immiscible liquid phases. We introduce the first platform ("CherryOne") capable of real-time monitoring, metering, and control of the dynamic liquid-liquid CCS process. Automated phase monitoring and volumetrics are made possible with an array of sensors, including the new permittivity-based phase metering apparatus (PMA). Volumetric data for each liquid phase are converted into a dynamic real-time display of stationary phase retention (Sf) and eluent partition coefficients (K), which represent critical parameters of CCS reproducibility. When coupled with the elution-extrusion operational mode (EECCC), automated Sf and K determination empowers untargeted and targeted applications ranging from metabolomic analysis to preparative purifications.
Sánchez, Sergio; Plaza, Antonio
Hyperspectral image compression is an important task in remotely sensed Earth Observation as the dimensionality of this kind of image data is ever increasing. This requires on-board compression in order to optimize the donwlink connection when sending the data to Earth. A successful algorithm to perform lossy compression of remotely sensed hyperspectral data is the iterative error analysis (IEA) algorithm, which applies an iterative process which allows controlling the amount of information loss and compression ratio depending on the number of iterations. This algorithm, which is based on spectral unmixing concepts, can be computationally expensive for hyperspectral images with high dimensionality. In this paper, we develop a new parallel implementation of the IEA algorithm for hyperspectral image compression on graphics processing units (GPUs). The proposed implementation is tested on several different GPUs from NVidia, and is shown to exhibit real-time performance in the analysis of an Airborne Visible Infra-Red Imaging Spectrometer (AVIRIS) data sets collected over different locations. The proposed algorithm and its parallel GPU implementation represent a significant advance towards real-time onboard (lossy) compression of hyperspectral data where the quality of the compression can be also adjusted in real-time.
Saengseesom, Wachiraporn; Mitmoonpitak, Channarong; Kasempimolporn, Songsri; Sitprija, Visith
The use of a 10-day observation to determine whether a dog is rabid is standard practice. This study was conducted in order to look for evidence of rabies vius in saliva and cerebrospinal fluid (CSF) of suspected live rabid dogs at the time of quarantine by using a SYBR Green real-time RT-PCR based assay for the detection of rabies virus RNA. Saliva and CSF of dogs were collected once on the day of admission for the 10-day quarantine. All test dogs were or became ill and died of rabies within the observation period. Thirteen of 15 dogs (87%) had saliva samples that were positive for rabies RNA. Two dogs with furious rabies had negative saliva samples. Positive CSF samples were found in 4 of 15 dogs (27%) whose saliva samples were positive. The time from sample collection to result was less than 5 hours. Because virus may be absent or present at very low level in both clinical fluids, samples taken for ante-mortem diagnosis cannot definitively rule out rabies.
Cook, Linda; Sullivan, KaWing; Krantz, Elizabeth M; Bagabag, Arthur; Jerome, Keith R
A variety of methods have been used to determine hepatitis C virus (HCV) genotypes. Because therapeutic decisions for chronic HCV-related hepatitis are made on the basis of genotype, it is important that genotype be accurately determined by clinical laboratories. Existing methods are often subjective, inaccurate, manual, time-consuming, and contamination prone. We therefore evaluated real-time reverse transcription-PCR (RT-PCR) reagents that have recently become commercially available (Abbott HCV Genotype ASR). The assay developed by our laboratory starts with purified RNA and can be performed in 4 to 5 h. An initial evaluation of 479 samples was done with a restriction fragment length polymorphism (RFLP) method and the RT-PCR assay, and discrepant samples were sequenced. An additional 1,200 samples were then tested, and data from all assays were used to evaluate the efficiency and specificity of each genotype-specific reaction. Good correlation between results by the two methods was seen. Discrepant samples included those indeterminate by the RT-PCR assay (n = 110) and a subset that were incorrectly called 2a by the RFLP method (n = 75). The real-time RT-PCR assay performed well with genotype 1, 2, and 3 samples. Inadequate numbers of samples were available to evaluate fully genotypes 4, 5, and 6. Analysis of each primer-probe set demonstrated that weak cross-reactive amplifications were common but usually did not interfere with the genotype determination. However, in about 1% of samples, two or more genotypes amplified at roughly equivalent amounts. Further studies are necessary to determine whether these mixed-genotype samples are true mixtures or a reflection of occasional cross-reactive amplifications.
Quantitative analysis of the risk for reservoir real-time operation is a hard task owing to the difficulty of accurate description of inflow uncertainties. The ensemble-based probabilistic hydrologic forecasting, which outputs a lot of inflow scenarios or traces, does well in depicting the inflow not only the marginal distribution but also their corrections. This motivates us to analyze the reservoir operating risk by inputting probabilistic hydrologic forecasting into reservoir real-time operation. The proposed procedure involves: (1) based upon the Bayesian inference, two alternative techniques, the generalized likelihood uncertainty estimation (GLUE) and Markov chain Monte Carlo (MCMC), are implemented for producing probabilistic hydrologic forecasting, respectively, (2) the reservoir risk is defined as the ratio of the number of traces that excessive (or below) the critical value to the total number of traces, and (3) a multipurpose reservoir operation model is build to produce Pareto solutions for trade-offs between risks and profits with the inputted probabilistic hydrologic forecasting. With a case study of the China's Three Gorges Reservoir, it is found that the reservoir real-time operation risks can be estimated and minimized based on the proposed methods, and this is great potential benefit in decision and choosing the most realistic one.
Arkin, C.; Gillespie, Stacey; Ratzel, Christopher
A portable instrument incorporates both mass spectrometry and dew point measurement to provide real-time, quantitative gas measurements of helium, nitrogen, oxygen, argon, and carbon dioxide, along with real-time, quantitative moisture analysis. The Portable Dew Point Mass Spectrometry (PDP-MS) system comprises a single quadrupole mass spectrometer and a high vacuum system consisting of a turbopump and a diaphragm-backing pump. A capacitive membrane dew point sensor was placed upstream of the MS, but still within the pressure-flow control pneumatic region. Pressure-flow control was achieved with an upstream precision metering valve, a capacitance diaphragm gauge, and a downstream mass flow controller. User configurable LabVIEW software was developed to provide real-time concentration data for the MS, dew point monitor, and sample delivery system pressure control, pressure and flow monitoring, and recording. The system has been designed to include in situ, NIST-traceable calibration. Certain sample tubing retains sufficient water that even if the sample is dry, the sample tube will desorb water to an amount resulting in moisture concentration errors up to 500 ppm for as long as 10 minutes. It was determined that Bev-A-Line IV was the best sample line to use. As a result of this issue, it is prudent to add a high-level humidity sensor to PDP-MS so such events can be prevented in the future.
Martinez-Lozano Sinues, Pablo; Tarokh, Leila; Li, Xue; Kohler, Malcolm; Brown, Steven A.; Zenobi, Renato; Dallmann, Robert
Circadian clocks play a significant role in the correct timing of physiological metabolism, and clock disruption might lead to pathological changes of metabolism. One interesting method to assess the current state of metabolism is metabolomics. Metabolomics tries to capture the entirety of small molecules, i.e. the building blocks of metabolism, in a given matrix, such as blood, saliva or urine. Using mass spectrometric approaches we and others have shown that a significant portion of the human metabolome in saliva and blood exhibits circadian modulation; independent of food intake or sleep/wake rhythms. Recent advances in mass spectrometry techniques have introduced completely non-invasive breathprinting; a method to instantaneously assess small metabolites in human breath. In this proof-of-principle study, we extend these findings about the impact of circadian clocks on metabolomics to exhaled breath. As previously established, our method allows for real-time analysis of a rich matrix during frequent non-invasive sampling. We sampled the breath of three healthy, non-smoking human volunteers in hourly intervals for 24 hours during total sleep deprivation, and found 111 features in the breath of all individuals, 36–49% of which showed significant circadian variation in at least one individual. Our data suggest that real-time mass spectrometric "breathprinting" has high potential to become a useful tool to understand circadian metabolism, and develop new biomarkers to easily and in real-time assess circadian clock phase and function in experimental and clinical settings. PMID:25545545
Aytekin, I.; Aksit, H.; Sait, A.; Kaya, F.; Aksit, D.; Gokmen, M.; Baca, A. Unsal
Introduction Bluetongue (BT) is a non-contagious infectious disease of ruminants. The disease agent bluetongue virus (BTV) is classified in the Reoviridae family Orbivirus. Aims and objectives The aim of this study was to determine serum malondialdehyde (MDA), total antioxidative stres (TAS), total sialic acid (TSA), ceruloplasmin, triglyceride, alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyltransferase (GGT), cholesterol, creatinine, albumin, and total protein levels in sheep with and without bluetongue (BT). Materials and Methods The study included 13 Sakiz crossbreed sheep, aged 1–4 years and usually in the last stage of pregnancy, as the BT group and a control group consisting of 10 healthy sheep. All sheep were clinically examined before collecting blood samples. Serum ALT, AST, cholesterol, triglyceride, albumin, GGT, total protein, creatinine and TAS levels were measured using commercially available kits as per manufacturer's recommendations using a Biochemistry Auto Analyzer (Sinnowa D280, China). Serum lipid peroxidation was estimated through a previously described method in which MDA reacts with thiobarbituric acid (TBA) to form a coloured complex at a maximum absorbance of 535 nm. The TSA value was measured at 549 nm using the method described by Warren (1959): sialic acid was oxidised to formyl-pyruvic acid, which reacts with TBA to form a pink product. The ceruloplasmin concentration was measured according to Sunderman and Nomoto (1970): ceruloplasmin and p-phenylenediamine formed a coloured oxidation product that was proportional to the concentration of serum ceruloplasmin. Real time RT-PCR and conventional RT-PCR were performed as described by Shaw and others (2007). Results Biochemistry analysis of serum showed that in the BT group, TSA, MDA, triglyceride and ALT and AST were higher and that ceruloplasmin and TAS were lower than in the control group. Serum albumin, cholesterol, creatinine, total protein and GGT did
Wegener, Steven; Chan, K. Roland; Pfister, Leonhard; Arvesen, John
Microscale and some mesoscale scientific targets are often too small and fast moving in a dynamic atmosphere. In this environment the ER-2 may spend only a small time sampling the phenomena of interest. The ER-2 scientists currently rely on post-flight analysis to determine the success of the mission and to provide input for planning the next flight. Real time analysis, combined with realistic modeling, can provide important clues to significantly enhance the probability of success in the investigation of specific scientific targets.
Schwuttke, Ursula M.; Quan, A. G.; Angelino, R.; Veregge, J. R.
Real-time artificial intelligence is gaining increasing attention for applications in which conventional software methods are unable to meet technology needs. One such application area is the monitoring and analysis of complex systems. MARVEL, a distributed monitoring and analysis tool with multiple expert systems, was developed and successfully applied to the automation of interplanetary spacecraft operations at NASA's Jet Propulsion Laboratory. MARVEL implementation and verification approaches, the MARVEL architecture, and the specific benefits that were realized by using MARVEL in operations are described.
Kono, Tomoya; Takayama, Hiroaki; Nagamine, Ryusuke; Korenaga, Hiroki; Sakai, Masahiro
To monitor the expression of cytokine genes in Japanese pufferfish, a novel platform for quantitative multiplexed analysis was developed. This custom-designed multiplex RT-PCR assay was used to analyze the expression profiles of 19 cytokine genes, including pro-inflammatory (IL-1β, IL-6, IL-17A/F3, IL-18, TNF-α, TNF-N), anti-inflammatory (IL-4/13A, IL-4/13B, IL-10), T-cell proliferation/differentiation (IL-2, IL-15, IL-21, TGF-β1), B-cell activation/differentiation (IL-7, IL-6, IL-4/13A, IL-4/13B), NK cell stimulation (IL-12p35 and IL-12p40), induction of anti-viral activity (I-IFN-1 and IFN-γ), and monocyte/macrophage progenitor cell proliferation (M-CSF1b) cytokines in head kidney cells under immune stimulatory conditions. The expression profiles were dissimilar in the unstimulated control and immune-stimulated cells. Moreover, increased expression profile was observed due to different stimulations for IL-1β, IL-6, IL-10, IL-12p35, IL-12p40, IL-21, TNF-α, TNF-N, I-IFN-1 and IFN-γ genes. These results suggest that cytokine genes could be used as biomarkers to know the immune status of fish. The constructed multiplex RT-PCR assay will enhance understanding on immune regulation by cytokines in fish.
Vongsouvath, Manivanh; Phommasone, Koukeo; Sengvilaipaseuth, Onanong; Kosoltanapiwat, Nathamon; Chantratita, Narisara; Blacksell, Stuart D.; Lee, Sue J.; de Lamballerie, Xavier; Mayxay, Mayfong; Keomany, Sommay; Newton, Paul N.; Dubot-Pérès, Audrey
Background Dengue virus infection causes major public health problems in tropical and subtropical areas. In many endemic areas, including the Lao PDR, inadequate access to laboratory facilities is a major obstacle to surveillance and study of dengue epidemiology. Filter paper is widely used for blood collection for subsequent laboratory testing for antibody and nucleic acid detection. For the first time, we demonstrate that dengue viral RNA can be extracted from dengue rapid diagnostic tests (RDT) and then submitted to real-time RT-PCR for serotyping. Methodology/Principal Findings We evaluated the Standard Diagnostics (SD) Bioline Dengue Duo RDT, a commonly used test in dengue endemic areas. First, using the QIAamp RNA kit, dengue RNA was purified from the sample pad of the NS1 RDT loaded with virus isolates of the four serotypes, then quantified by RT-PCR. We observed greater recovery of virus, with a mean of 27 times more RNA recovered from RDT, than from filter paper. Second, we evaluated dengue NS1 RDTs from patients at Mahosot Hospital, Vientiane, (99 patients) and from rural Salavan Provincial Hospital (362 patients). There was good agreement between dengue RT-PCR from NS1 RDT with RT-PCR performed on RNA extracted from patient sera, either using RDT loaded with blood (82.8% and 91.4%, in Vientiane and Salavan, respectively) or serum (91.9% and 93.9%). There was 100% concordance between RDT and serum RT-PCR of infecting dengue serotype. Conclusions/Significance Therefore, the collection of NS1 positive RDTs, which do not require cold storage, may be a novel approach for dengue serotyping by RT-PCR and offers promising prospects for the collection of epidemiological data from previously inaccessible tropical areas to aid surveillance and public health interventions. PMID:27159058
D'Souza, Doris H; Critzer, Faith J; Golden, David A
Recent outbreaks of Salmonella linked to fresh produce emphasize the need for rapid detection methods to help control the spread of disease. Reverse-transcriptase polymerase chain reaction (RT-PCR) can detect the presence of mRNA (shorter half-life than DNA) with greater potential for detecting viable pathogens. The chromosomally located invA gene required for host invasion by Salmonella is widely used for detection of this pathogen by PCR. Detection of Salmonella was undertaken by real-time RT-PCR (rt-RT-PCR) using newly designed invA gene primers to develop a sensitive and specific assay. Salmonella serovars Typhimurium and Enteritidis were grown (7.68 log(10) CFU/mL) in Luria-Bertani broth overnight at 37 degrees C, and RNA was extracted, followed by rt-RT-PCR with and without SYBR green I and agarose gel electrophoresis. All experiments were replicated at least thrice. Detection for both serovars using traditional RT-PCR was lower ( approximately 10(5) CFU/mL) than rt-RT-PCR (10(3) CFU/mL) by gel electrophoresis. Melt curve analysis showed melt temperatures at 87.5 degrees C with Ct values from 12 to 15 for up to 10(3) CFU/mL and improved to 10(2) CFU/mL after further optimization. Further, addition of RNA internal amplification control constructed using in vitro transcription with a T7 RNA polymerase promoter, to the RT-PCR assay also gave detection limits of 10(2) CFU/mL. Cross-reactivity was not observed against a panel of 21 non-Salmonella bacteria. Heat-inactivated (autoclaved) Salmonella showed faint or no detection by rt-RT-PCR or gel electrophoresis. This method has potential to be applied for the detection of Salmonella serovars in fresh produce and the simultaneous detection of foodborne viral (RNA viruses) and bacterial pathogens in a multiplex format.
Jaiswal, Devina; Rad, Armin Tahmasbi; Nieh, Mu-Ping; Claffey, Kevin P.; Hoshino, Kazunori
Understanding the interaction of live cells with macromolecules is crucial for designing efficient therapies. Considering the functional heterogeneity found in cancer cells, real-time single cell analysis is necessary to characterize responses. In this study, we have designed and fabricated a microfluidic channel with patterned micromagnets which can temporarily immobilize the cells during analysis and release them after measurements. The microchannel is composed of plain coverslip top and bottom panels to facilitate easy microscopic observation and undisturbed application of analytes to the cells. Cells labeled with functionalized magnetic beads were immobilized in the device with an efficiency of 90.8±3.6%. Since the micromagnets are made of soft magnetic material (Ni), they released cells when external magnetic field was turned off from the channel. This allows the reuse of the channel for a new sample. As a model drug analysis, the immobilized breast cancer cells (MCF7) were exposed to fluorescent lipid nanoparticles and association and dissociation were measured through fluorescence analysis. Two concentrations of nanoparticles, 0.06 μg/ml and 0.08 μg/ml were tested and time lapse images were recorded and analyzed. The microfluidic device was able to provide a microenvironment for sample analysis, making it an efficient platform for real-time analysis.
Costafreda, M. Isabel; Bosch, Albert; Pintó, Rosa M.
A standardized real-time reverse transcription-PCR (RT-PCR) assay has been developed for an accurate estimation of the number of genome copies of hepatitis A virus (HAV) in clinical and shellfish samples. Real-time procedures were based on the amplification of a fragment of the highly conserved 5′ noncoding region and detection through an internal fluorescent probe, including TaqMan and beacon chemistries, in one- and two-step RT-PCR formats. The best performance in terms of sensitivity and reproducibility was achieved by a one-step TaqMan RT-PCR, with a sensitivity enabling the detection of 0.05 infectious unit and 10 copies of a single-stranded RNA (ssRNA) synthetic transcript. Standard reagents, such as a mengovirus strain and an ssRNA transcript, were employed as controls of nucleic acid extraction and RT-PCR, respectively. The test proved to be highly specific after a broad panel of enteric viruses was tested. Sequence alignment of target regions of the primers and probe proved them to be adequate for the quantification of all HAV genotypes. In addition, a quasispecies analysis of the mutant spectrum indicated that these regions are not prone to variability, thus confirming their robustness. PMID:16751488
Yang, Ji-Rong; Kuo, Chuan-Yi; Huang, Hsiang-Yi; Wu, Fu-Ting; Huang, Yi-Lung; Cheng, Chieh-Yu; Su, Yu-Ting; Chang, Feng-Yee; Wu, Ho-Sheng; Liu, Ming-Tsan
New variants of the influenza A(H1N1)pdm09 and A(H3N2) viruses were detected in Taiwan between 2012 and 2013. Some of these variants were not detected in clinical specimens using a common real-time reverse transcription-PCR (RT-PCR) assay that targeted the conserved regions of the viral matrix (M) genes. An analysis of the M gene sequences of the new variants revealed that several newly emerging mutations were located in the regions where the primers or probes of the real-time RT-PCR assay bind; these included three mutations (G225A, T228C, and G238A) in the A(H1N1)pdm09 virus, as well as one mutation (C163T) in the A(H3N2) virus. These accumulated mismatch mutations, together with the previously identified C154T mutation of the A(H1N1)pdm09 virus and the C153T and G189T mutations of the A(H3N2) virus, result in a reduced detection sensitivity for the real-time RT-PCR assay. To overcome the loss of assay sensitivity due to mismatch mutations, we established a real-time RT-PCR assay using degenerate nucleotide bases in both the primers and probe and successfully increased the sensitivity of the assay to detect circulating variants of the human influenza A viruses. Our observations highlight the importance of the simultaneous use of different gene-targeting real-time RT-PCR assays for the clinical diagnosis of influenza.
Kern, Rolf; Diels, Anna; Pettenpohl, Johanna; Kablau, Micha; Brade, Joachim; Hennerici, Michael G; Meairs, Stephen
Real-time ultrasound perfusion imaging (rt-UPI) allows visualization of microbubbles flowing through the cerebral microvasculature. We hypothesized that analysis of microbubble tissue replenishment would enable for characterization of perfusion deficits in acute middle cerebral artery (MCA) territory stroke. Twenty-three patients (mean age 70.2 ± 13.2 years, 9 weeks) were included. Sequential images of bubble replenishment were acquired by transcranial rt-UPI at low mechanical index immediately after microbubble destruction. Different parameters were calculated from regions of interest (ROIs): real-time time to peak (rt-TTP), rise rate (β), and plateau (A) of acoustic intensity, and A × β was used as an index of blood flow. Results were compared with diffusion-weighted and perfusion magnetic resonance imaging. Parameters of rt-UPI had lower values in ROIs of ischemic as compared with normal tissue (β=0.58 ± 0.40 versus 1.25 ± 0.83; P=0.001; A=1.44 ± 1.75 versus 2.63 ± 2.31; P=0.05; A × β=1.14 ± 2.25 versus 2.98 ± 2.70; P=0.01). Real-time time to peak was delayed in ischemic tissue (11.43 ± 2.67 versus 8.88 ± 1.66 seconds; P<0.001). From the analysis of receiver operating characteristic curves, β and A × β had the largest areas under the curve with optimal cutoff values of β<0.76 and A × β<1.91. We conclude that rt-UPI with analysis of microbubble replenishment correctly identifies ischemic brain tissue in acute MCA stroke.
direct analysis in real time mass spectrometry. The release of adenine from the inhomo- geneous substrate herring sperm DNA by ricin was determined to...chain catalyzes cleavage at adenosine 4324 (in rat RNA) of 28S rRNA to release adenine.10 This action inhibits protein synthesis, leading to cell...death. In addition to RNA, herring sperm DNA (hsDNA) is a substrate for ricin.11 We chose to employ hsDNA for this assay because it is relatively stable
D.T. Kendrick; Steven Saggese
Science & Engineering Associates, Inc. (SEA), under contract No. DE-AC26-00NT40768, was tasked by the US Department of Energy--National Energy Technology Laboratory to develop and test a near real-time beryllium monitor for airborne and surface measurements. Recent public awareness of the health risks associated with exposure to beryllium has underscored the need for better, faster beryllium monitoring capabilities within the DOE. A near real-time beryllium monitor will offer significant improvements over the baseline monitoring technology currently in use. Whereas the baseline technology relies upon collecting an air sample on a filter and the subsequent analysis of the filter by an analytical laboratory, this effort developed a monitor that offers near real-time measurement results while work is in progress. Since the baseline typically only offers after-the-fact documentation of exposure levels, the near real-time capability provides a significant increase in worker protection. The beryllium monitor developed utilizes laser induced breakdown spectroscopy, or LIBS as the fundamental measurement technology. LIBS has been used in a variety of laboratory and field based instrumentation to provide real-time, and near-real-time elemental analysis capabilities. LIBS is an analytical technique where a pulsed high energy laser beam is focused to a point on the sample to be interrogated. The high energy density produces a small high temperature plasma plume, sometimes called a spark. The conditions within this plasma plume result in the constituent atoms becoming excited and emitting their characteristic optical emissions. The emission light is collected and routed to an optical spectrometer for quantitative spectral analysis. Each element has optical emissions, or lines, of a specific wavelength that can be used to uniquely identify that element. In this application, the intensity of the beryllium emission is used to provide a quantitative measure of the abundance of the
Andersen, U V; Haakansson, I T; Roust, T; Rhod, M; Baptiste, K E; Nielsen, M K
Strongyle and trichostrongyle parasites are ubiquitous nematodes of grazing livestock. Several molecular diagnostic tests are based upon measuring and quantifying DNA obtained from parasite eggs. It is well known that such eggs undergo development during storage, but it remains unknown to which extent developmental stages can affect the variation of diagnostic test results. This study investigated the influence of developmental stages of strongyle eggs on the variation real-time polymerase chain reaction (PCR) results. Mixed species strongyle eggs were obtained from the faeces of a naturally infected horse. Eggs were isolated and placed in microtiter plates with demineralized water. A total of 25 wells containing 100 eggs each were set up and kept refrigerated for up to five days. Once daily, five wells were examined on an inverted microscope at 100× magnification, where the developmental stages of the eggs were noted, and then eggs harvested for DNA extraction. The protocol was repeated three times. Genomic DNA was extracted using a commercial kit previously validated for strongyle type eggs. PCR reactions were performed with a primer set specific for the ribosomal DNA region for all strongyle type parasites (NC1, NC2). SYBR Green Real-Time PCRs were performed in triplicates. Results revealed a statistically significant increase in PCR yield after three days, which was statistically associated with beginning embryonation of the eggs. In conclusion, storage time and developmental stage of strongyle eggs are significant sources of error in studies based on quantitative real-time PCR analysis. This study suggests that for refrigerated storage of more than three days, eggs should be inactivated and preserved for further analysis.
Kropivnitskaya, Yelena; Tiampo, Kristy F.; Qin, Jinhui; Bauer, Michael A.
Earthquake intensity is one of the key components of the decision-making process for disaster response and emergency services. Accurate and rapid intensity calculations can help to reduce total loss and the number of casualties after an earthquake. Modern intensity assessment procedures handle a variety of information sources, which can be divided into two main categories. The first type of data is that derived from physical sensors, such as seismographs and accelerometers, while the second type consists of data obtained from social sensors, such as witness observations of the consequences of the earthquake itself. Estimation approaches using additional data sources or that combine sources from both data types tend to increase intensity uncertainty due to human factors and inadequate procedures for temporal and spatial estimation, resulting in precision errors in both time and space. Here we present a processing approach for the real-time analysis of streams of data from both source types. The physical sensor data is acquired from the U.S. Geological Survey (USGS) seismic network in California and the social sensor data is based on Twitter user observations. First, empirical relationships between tweet rate and observed Modified Mercalli Intensity (MMI) are developed using data from the M6.0 South Napa, CAF earthquake that occurred on August 24, 2014. Second, the streams of both data types are analyzed together in simulated real-time to produce one intensity map. The second implementation is based on IBM InfoSphere Streams, a cloud platform for real-time analytics of big data. To handle large processing workloads for data from various sources, it is deployed and run on a cloud-based cluster of virtual machines. We compare the quality and evolution of intensity maps from different data sources over 10-min time intervals immediately following the earthquake. Results from the joint analysis shows that it provides more complete coverage, with better accuracy and higher
Hicks, John W.; Petersen, Kevin L.
The X-29A advanced technology demonstrator flight envelope expansion program and the subsequent flight research phase gave impetus to the development of several innovative real-time analysis and display techniques. These new techniques produced significant improvements in flight test productivity, flight research capabilities, and flight safety. These techniques include real-time measurement and display of in-flight structural loads, dynamic structural mode frequency and damping, flight control system dynamic stability and control response, aeroperformance drag polars, and aircraft specific excess power. Several of these analysis techniques also provided for direct comparisons of flight-measured results with analytical predictions. The aeroperformance technique was made possible by the concurrent development of a new simplified in-flight net thrust computation method. To achieve these levels of on-line flight test analysis, integration of ground and airborne systems was required. The capability of NASA Ames Research Center, Dryden Flight Research Facility's Western Aeronautical Test Range was a key factor to enable implementation of these methods.
Hicks, John W.; Petersen, Kevin L.
The X-29A advanced technology demonstrator flight envelope expansion program and the subsequent flight research phase gave impetus to the development of several innovative real-time analysis and display techniques. These new techniques produced significant improvements in flight test productivity, flight research capabilities, and flight safety. These techniques include real-time measurement and display of in-flight structural loads, dynamic structural mode frequency and damping, flight control system dynamic stability and control response, aeroperformance drag polars, and aircraft specific excess power. Several of these analysis techniques also provided for direct comparisons of flight-measured results with analytical predictions. The aeroperformance technique was made possible by the concurrent development of a new simplified in-flight net thrust computation method. To achieve these levels of on-line flight test analysis, integration of ground and airborne systems was required. The capability of NASA Ames Research Center, Dryden Flight Research Facility's Western Aeronautical Test Range was a key factor in enabling implementation of these methods.
Dong, Biqin; Chen, Siyu; Zhou, Fan; Chan, Christina H. Y.; Yi, Ji; Zhang, Hao F.; Sun, Cheng
We report the application of spectral-domain optical coherence tomography (SD-OCT) technology that enables real-time functional analysis of sorting microparticles and cells in an inertial microfluidic device. We demonstrated high-speed, high-resolution acquisition of cross-sectional images at a frame rate of 350 Hz, with a lateral resolution of 3 μm and an axial resolution of 1 μm within the microfluidic channel filled with water. We analyzed the temporal sequence of cross-sectional SD-OCT images to determine the position and diameter of microspheres in a spiral microfluidic channel under various flow rates. We used microspheres with known diameters to validate the sub-micrometer precision of the particle size analysis based on a scattering model of spherical microparticles. An additional investigation of sorting live HT-29 cells in the spiral microfluidic channel indicated that the distribution of cells within in the microchannel has a close correspondence with the cells’ size distribution. The label-free real-time imaging and analysis of microscale particles in flow offers robustness for practical applications with live cells and allows us to better understand the mechanisms of particle separations in microfluidic sorting systems. PMID:27619202
Charoenvilaisiri, Saengsoon; Seepiban, Channarong; Bhunchoth, Anjana; Warin, Nuchnard; Luxananil, Plearnpis; Gajanandana, Oraprapai
In this study, a multiplex RT-PCR-ELISA was developed to detect and differentiate four tospovirus species found in Thailand, namely Capsicum chlorosis virus (CaCV), Melon yellow spot virus (MYSV), Tomato necrotic ringspot virus (TNRV), and Watermelon silver mottle virus (WSMoV). In this system, nucleocapsid (N) gene fragments of four tospoviruses were simultaneously amplified and labeled with digoxigenin (DIG) in a single RT-PCR reaction using a pair of degenerate primers binding to the same conserved regions in all four tospovirus N genes. The DIG-labeled amplicons were distinguished into species by four parallel hybridizations to species-specific biotinylated probes in streptavidin-coated microtiter wells followed by ELISA detection using a peroxidase-conjugated anti-DIG antibody. Results indicated that the multiplex RT-PCR-ELISA assay could specifically identify each of these four tospoviruses without cross-reactivity between species or reactivity to healthy plant negative controls. Assay sensitivity was 10- to 1000-fold higher than conventional RT-PCR. When applied to naturally infected plants, all samples yielded concordant results between RT-PCR-ELISA and the reference RT-PCR. In conclusion, the multiplex RT-PCR-ELISA developed in this study has superior specificity, sensitivity, and high-throughput capacity compared to conventional RT-PCR and is an attractive alternative for the identification of different tospovirus species.
Domanski, Marian M.; Straub, Timothy D.; Wood, Molly S.; Landers, Mark N.; Wall, Gary R.; Brady, Steven J.
The use of acoustic and other parameters as surrogates for suspended-sediment concentrations (SSC) in rivers has been successful in multiple applications across the Nation. Critical to advancing the operational use of surrogates are tools to process and evaluate the data along with the subsequent development of regression models from which real-time sediment concentrations can be made available to the public. Recent developments in both areas are having an immediate impact on surrogate research, and on surrogate monitoring sites currently in operation. The Surrogate Analysis and Index Developer (SAID) standalone tool, under development by the U.S. Geological Survey (USGS), assists in the creation of regression models that relate response and explanatory variables by providing visual and quantitative diagnostics to the user. SAID also processes acoustic parameters to be used as explanatory variables for suspended-sediment concentrations. The sediment acoustic method utilizes acoustic parameters from fixed-mount stationary equipment. The background theory and method used by the tool have been described in recent publications, and the tool also serves to support sediment-acoustic-index methods being drafted by the multi-agency Sediment Acoustic Leadership Team (SALT), and other surrogate guidelines like USGS Techniques and Methods 3-C4 for turbidity and SSC. The regression models in SAID can be used in utilities that have been developed to work with the USGS National Water Information System (NWIS) and for the USGS National Real-Time Water Quality (NRTWQ) Web site. The real-time dissemination of predicted SSC and prediction intervals for each time step has substantial potential to improve understanding of sediment-related water-quality and associated engineering and ecological management decisions.
Wormhoudt, J.; Nelson, D. D.; Annen, K.; Locke, R. J.; Wernet, M.
Raman scattering has long been used as a non-intrusive diagnostic of temperatures in combustion exhaust flows, using a variety of spectral analysis techniques. As part of their ongoing program of experiments to support development of computer codes that calculate exhaust flow fields and predict jet noise, NASA Glenn Research Center is developing a laser Raman diagnostic system that will measure mean temperatures and temperature fluctuations in hot and cold jet flows. We describe a software package, ART (Analysis for Raman Temperatures), that analyzes Raman spectra of air for temperature and density using vibrational or resolved or unresolved rotational bands, presenting results in a variety of real-time displays. Each analysis technique presents its own challenges in obtaining the most precise and accurate values, and we will comment on these issues by exhibiting example spectra of each type. The ART program is closely related to another Aerodyne software package (TDLWintel) which automates the acquisition and analysis of tunable laser absorption spectra.
The hsp 16 gene of the probiotic Lactobacillus acidophilus is differently regulated by salt, high temperature and acidic stresses, as revealed by reverse transcription quantitative PCR (qRT-PCR) analysis.
Capozzi, Vittorio; Arena, Mattia Pia; Crisetti, Elisabetta; Spano, Giuseppe; Fiocco, Daniela
Small heat shock proteins (sHsps) are ubiquitous conserved chaperone-like proteins involved in cellular proteins protection under stressful conditions. In this study, a reverse transcription quantitative PCR (RT-qPCR) procedure was developed and used to quantify the transcript level of a small heat shock gene (shs) in the probiotic bacterium Lactobacillus acidophilus NCFM, under stress conditions such as heat (45 °C and 53 °C), bile (0.3% w/v), hyperosmosis (1 M and 2.5 M NaCl), and low pH value (pH 4). The shs gene of L. acidophilus NCFM was induced by salt, high temperature and acidic stress, while repression was observed upon bile stress. Analysis of the 5' noncoding region of the hsp16 gene reveals the presence of an inverted repeat (IR) sequence (TTAGCACTC-N9-GAGTGCTAA) homologue to the controlling IR of chaperone expression (CIRCE) elements found in the upstream regulatory region of Gram-positive heat shock operons, suggesting that the hsp16 gene of L. acidophilus might be transcriptionally controlled by HrcA. In addition, the alignment of several small heat shock proteins identified so far in lactic acid bacteria, reveals that the Hsp16 of L. acidophilus exhibits a strong evolutionary relationship with members of the Lactobacillus acidophilus group.
Khan, F; Gokhale, M; Chuah, C N
The problem of Network Traffic Classification (NTC) has attracted significant amount of interest in the research community, offering a wide range of solutions at various levels. The core challenge is in addressing high amounts of traffic diversity found in today's networks. The problem becomes more challenging if a quick detection is required as in the case of identifying malicious network behavior or new applications like peer-to-peer traffic that have potential to quickly throttle the network bandwidth or cause significant damage. Recently, Traffic Dispersion Graphs (TDGs) have been introduced as a viable candidate for NTC. The TDGs work by forming a network wide communication graphs that embed characteristic patterns of underlying network applications. However, these patterns need to be quickly evaluated for mounting real-time response against them. This paper addresses these concerns and presents a novel solution for real-time analysis of Traffic Dispersion Metrics (TDMs) in the TDGs. We evaluate the dispersion metrics of interest and present a dedicated solution on an FPGA for their analysis. We also present analytical measures and empirically evaluate operating effectiveness of our design. The mapped design on Virtex-5 device can process 7.4 million packets/second for a TDG comprising of 10k flows at very high accuracies of over 96%.
Zagelbaum, Jennifer; Shimazaki, Noriko; Esguerra, Zitadel Anne; Watanabe, Go; Lieber, Michael R; Rothenberg, Eli
Single-molecule FRET (smFRET) and single-molecule colocalization (smCL) assays have allowed us to observe the recombination-activating gene (RAG) complex reaction mechanism in real time. Our smFRET data have revealed distinct bending modes at recombination signal sequence (RSS)-conserved regions before nicking and synapsis. We show that high mobility group box 1 (HMGB1) acts as a cofactor in stabilizing conformational changes at the 12RSS heptamer and increasing RAG1/2 binding affinity for 23RSS. Using smCL analysis, we have quantitatively measured RAG1/2 dwell time on 12RSS, 23RSS, and non-RSS DNA, confirming a strict RSS molecular specificity that was enhanced in the presence of a partner RSS in solution. Our studies also provide single-molecule determination of rate constants that were previously only possible by indirect methods, allowing us to conclude that RAG binding, bending, and synapsis precede catalysis. Our real-time analysis offers insight into the requirements for RSS-RSS pairing, architecture of the synaptic complex, and dynamics of the paired RSS substrates. We show that the synaptic complex is extremely stable and that heptamer regions of the 12RSS and 23RSS substrates in the synaptic complex are closely associated in a stable conformational state, whereas nonamer regions are perpendicular. Our data provide an enhanced and comprehensive mechanistic description of the structural dynamics and associated enzyme kinetics of variable, diversity, and joining [V(D)J] recombination.
Meyer-Baese, Uwe; Odom, Crispin; Botella, Guillermo; Meyer-Baese, Anke
The conditions that arise in the Cocktail Party Problem prevail across many fields creating a need for of Blind Source Separation. The need for BSS has become prevalent in several fields of work. These fields include array processing, communications, medical signal processing, and speech processing, wireless communication, audio, acoustics and biomedical engineering. The concept of the cocktail party problem and BSS led to the development of Independent Component Analysis (ICA) algorithms. ICA proves useful for applications needing real time signal processing. The goal of this research was to perform an extensive study on ability and efficiency of Independent Component Analysis algorithms to perform blind source separation on mixed signals in software and implementation in hardware with a Field Programmable Gate Array (FPGA). The Algebraic ICA (A-ICA), Fast ICA, and Equivariant Adaptive Separation via Independence (EASI) ICA were examined and compared. The best algorithm required the least complexity and fewest resources while effectively separating mixed sources. The best algorithm was the EASI algorithm. The EASI ICA was implemented on hardware with Field Programmable Gate Arrays (FPGA) to perform and analyze its performance in real time.
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
Background 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. Objectives 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. Patients and Methods 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. Results 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. Conclusions 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. PMID:27099688
Hasanpour, Mojtaba; Najafi, Akram
Uropathogenic Escherichia coli (UPEC) is among major pathogens causing 80-90% of all episodes of urinary tract infections (UTIs). Recently, E. coli strains are divided into eight main phylogenetic groups including A, B1, B2, C, D, E, F, and clade I. This study was aimed to develop a rapid, sensitive, and specific multiplex real time PCR method capable of detecting phylogenetic groups of E. coli strains. This study was carried out on E. coli strains (isolated from the patient with UTI) in which the presence of all seven target genes had been confirmed in our previous phylogenetic study. An EvaGreen-based singleplex and multiplex real-time PCR with melting curve analysis was designed for simultaneous detection and differentiation of these genes. The primers were selected mainly based on the production of amplicons with melting temperatures (Tm) ranging from 82°C to 93°C and temperature difference of more than 1.5°C between each peak.The multiplex real-time PCR assays that have been developed in the present study were successful in detecting the eight main phylogenetic groups. Seven distinct melting peaks were discriminated, with Tm value of 93±0.8 for arpA, 89.2±0.1for chuA, 86.5±0.1 for yjaA, 82.3±0.2 for TspE4C2, 87.8±0.1for trpAgpC, 85.4±0.6 for arpAgpE genes, and 91±0.5 for the internal control. To our knowledge, this study is the first melting curve-based real-time PCR assay developed for simultaneous and discrete detection of these seven target genes. Our findings showed that this assay has the potential to be a rapid, reliable and cost-effective alternative for routine phylotyping of E. coli strains.
Wang, Xia; Feng, Jianhua; Huang, Aiyou; He, Linwen; Niu, Jianfeng; Wang, Guangce
Pyropia haitanensis has prominent stress-resistance characteristics and is endemic to China. Studies into the stress responses in these algae could provide valuable information on the stress-response mechanisms in the intertidal Rhodophyta. Here, the effects of salinity and light intensity on the quantum yield of photosystem II in Py. haitanensis were investigated using pulse-amplitude-modulation fluorometry. Total RNA and genomic DNA of the samples under different stress conditions were isolated. By normalizing to the genomic DNA quantity, the RNA content in each sample was evaluated. The cDNA was synthesized and the expression levels of seven potential internal control genes were evaluated using qRT-PCR method. Then, we used geNorm, a common statistical algorithm, to analyze the qRT-PCR data of seven reference genes. Potential genes that may constantly be expressed under different conditions were selected, and these genes showed stable expression levels in samples under a salinity treatment, while tubulin, glyceraldehyde-3-phosphate dehydrogenase and actin showed stability in samples stressed by strong light. Based on the results of the pulse amplitude-modulation fluorometry, an absolute quantification was performed to obtain gene copy numbers in certain stress-treated samples. The stably expressed genes as determined by the absolute quantification in certain samples conformed to the results of the geNorm screening. Based on the results of the software analysis and absolute quantification, we proposed that elongation factor 3 and 18S ribosomal RNA could be used as internal control genes when the Py. haitanensis blades were subjected to salinity stress, and that α-tubulin and 18S ribosomal RNA could be used as the internal control genes when the stress was from strong light. In general, our findings provide a convenient reference for the selection of internal control genes when designing experiments related to stress responses in Py. haitanensis.
Nebenfuhr, A.; Lomax, T. L.
We have developed an improved method for determination of gene expression levels with RT-PCR. The procedure is rapid and does not require extensive optimization or densitometric analysis. Since the detection of individual transcripts is PCR-based, small amounts of tissue samples are sufficient for the analysis of expression patterns in large gene families. Using this method, we were able to rapidly screen nine members of the Aux/IAA family of auxin-responsive genes and identify those genes which vary in message abundance in a tissue- and light-specific manner. While not offering the accuracy of conventional semi-quantitative or competitive RT-PCR, our method allows quick screening of large numbers of genes in a wide range of RNA samples with just a thermal cycler and standard gel analysis equipment.
Barr, N B; Ledezma, L A; Farris, R E; Epstein, M E; Gilligan, T M
A molecular assay for diagnosis of light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), in North America is reported. The assay multiplexes two TaqMan real-time polymerase chain reaction (RT-PCR) probe systems that are designed to target DNA segments of the internal transcribed spacer region 2 (ITS2) and 18S rRNA gene. The RT-PCR probe designed for the 18S target recognizes a DNA sequence conserved in all of the moths included in the study and functions as a control in the assay. The second probe recognizes a segment of the ITS2 specifically found in E. postvittana and not found in the other moths included in the study, i.e., this segment is not conserved. Inclusion of the two markers in a single multiplex reaction did not affect assay performance. The assay was tested against 637 moths representing > 90 taxa in 15 tribes in all three subfamilies in the Tortricidae. The assay generated no false negatives based on analysis of 355 E. postvittana collected from California, Hawaii, England, New Zealand, and Australia. Analysis of a data set including 282 moths representing 41 genera generated no false positives. Only three inconclusive results were generated from the 637 samples. Spike experiments demonstrated that DNA contamination in the assay can affect samples differently. Contaminated samples analyzed with the ITS2 RT-PCR assay and DNA barcode methodology by using the cytochrome oxidase I gene can generate contradictory diagnoses.
Ma, Jun-Xin; Wang, Lin-Nong; Zhou, Ru-Xia; Yu, Yang; Du, Tong-Xin
AIM To design, optimize and validate a rapid, internally controlled real-time polymerase chain reaction (RT-PCR) test for herpes simplex virus (HSV) in the diagnosis of necrotizing herpes stromal keratitis. METHODS Tears alone or together with corneal epithelium scrapings from 30 patients (30 eyes) suspected of necrotizing herpes stromal keratitis were tested for HSV DNA by RT-PCR. The samples were collected during the first visit and then on the subsequent 7, 14, 28, 42, and 56d. The symptoms of the patients were scored before treatment to determine the correlation between HSV concentration in the corneal epithelium scrapings and clinical scores. RESULTS The positive rate (46.4%) in the corneal epithelium group before the therapy was significantly higher than that (13.3%) in the tears group (P=0.006). There were 13 positive HSV patients before the therapy, the concentration of HSV DNA in corneal epithelium scrapings group was significantly higher than that in the tears group (paired t-test, P=0.0397). Multilevel mixed-effects model analysis showed that the difference between the corneal epithelium scrapings group and the tears group was statistically significant (P=0.0049). The Spearman rank correlation analysis indicated a positive correlation between the HSV concentration in the corneal epithelium scrapings and clinical scores before the treatment (r=0.844, P<0.0001). CONCLUSION RT-PCR appears to be a powerful molecular tool for the diagnosis of necrotizing herpes stromal keratitis. PMID:27275421
Chan, Jasper Fuk-Woo; Choi, Garnet Kwan-Yue; Tsang, Alan Ka-Lun; Tee, Kah-Meng; Lam, Ho-Yin; Yip, Cyril Chik-Yan; To, Kelvin Kai-Wang; Cheng, Vincent Chi-Chung; Yeung, Man-Lung; Lau, Susanna Kar-Pui; Woo, Patrick Chiu-Yat; Chan, Kwok-Hung; Tang, Bone Siu-Fai; Yuen, Kwok-Yung
Based on findings in small RNA-sequencing (Seq) data analysis, we developed highly sensitive and specific real-time reverse transcription (RT)-PCR assays with locked nucleic acid probes targeting the abundantly expressed leader sequences of Middle East respiratory syndrome coronavirus (MERS-CoV) and other human coronaviruses. Analytical and clinical evaluations showed their noninferiority to a commercial multiplex PCR test for the detection of these coronaviruses.
Bajpai, Vikas; Sharma, Deepty; Kumar, Brijesh; Madhusudanan, K P
Piper betle Linn. is a traditional plant associated with the Asian and southeast Asian cultures. Its use is also recorded in folk medicines in these regions. Several of its medicinal properties have recently been proven. Phytochemical analysis showed the presence of mainly terpenes and phenols in betel leaves. These constituents vary in the different cultivars of Piper betle. In this paper we have attempted to profile eight locally available betel cultivars using the recently developed mass spectral ionization technique of direct analysis in real time (DART). Principal component analysis has also been employed to analyze the DART MS data of these betel cultivars. The results show that the cultivars of Piper betle could be differentiated using DART MS data.
Solinsky, James C.; Budge, Scott E.; Majors, Paul D.; Rex, Bruce B.
This paper reports on the Pacific Northwest National Laboratory (PNNL) DOE Initiative in Image Science and Technology (ISAT) research, which is developing algorithms and software tool sets for remote sensing and biological applications. In particular, the PNNL ISAT work is applying these research results to the automated analysis of real-time cellular biology imagery to assist the biologist in determining the correct data collection region for the current state of a conglomerate of living cells in three-dimensional motion. The real-time computation of the typical 120 MB/sec multi-spectral data sets is executed in a Field Programmable Gate Array (FPGA) technology, which has very high processing rates due to large-scale parallelism. The outcome of this artificial vision work will allow the biologist to work with imagery as a creditable set of dye-tagged chemistry measurements in formats for individual cell tracking through regional feature extraction, and animation visualization through individual object isolation/characterization of the microscopy imagery.
Kodama, Takashi; Kurosawa, Yasunori; Kitta, Kazumi; Naito, Shigehiro
The Horwitz curve estimates interlaboratory precision as a function only of concentration, and is frequently used as a method performance criterion in food analysis with chemical methods. The quantitative biochemical methods based on real-time PCR require an analogous criterion to progressively promote method validation. We analyzed the tendency of precision using a simplex real-time PCR technique in 53 collaborative studies of seven genetically modified (GM) crops. Reproducibility standard deviation (SR) and repeatability standard deviation (Sr) of the genetically modified organism (GMO) amount (%) was more or less independent of GM crops (i.e., maize, soybean, cotton, oilseed rape, potato, sugar beet, and rice) and evaluation procedure steps. Some studies evaluated whole steps consisting of DNA extraction and PCR quantitation, whereas others focused only on the PCR quantitation step by using DNA extraction solutions. Therefore, SR and Sr for GMO amount (%) are functions only of concentration similar to the Horwitz curve. We proposed S(R) = 0.1971C 0.8685 and S(r) = 0.1478C 0.8424, where C is the GMO amount (%). We also proposed a method performance index in GMO quantitative methods that is analogous to the Horwitz Ratio.
Pollmeier, Vincent M.
XRTD is a graphical user interface (GUI) based tool for monitoring real time radiometric spacecraft data. The tool is designed to allow the navigation analyst to both view and analyze the characteristics of Doppler and ranging data. This capability is critical if ground personnel wish to verify the correct performance of ongoing maneuvers. The raw tracking data is transferred from Deep Space Network (DSN) computers to a local workstation, where the predicted value for the observable is subtracted from the actual observed value to create a residual. The tool then allows the navigation analyst to rescale and replot the data using simple GUI techniques. The navigator may then perform a number of data analysis and modeling techniques on the resulting residuals to allow for the real time characterization of spacecraft events. These techniques include the modeling of maneuvers, the compression and differencing of data, and Fast Fourier transforms of the data. This tool has shortened the amount of time required for initial characterization of spacecraft maneuvers from several hours to a few minutes.
Tanner, Carol E; Sun, Nan; Deatsch, Alison; Li, Frank; Ruggiero, Steven T
This paper describes light transmission spectroscopy (LTS), a technique for eliminating spectral noise and systematic effects in real-time spectroscopic measurements. In our work, we combine LTS with spectral inversion for the purpose of nanoparticle analysis. This work employs a wideband multi-wavelength light source and grating spectrometers coupled to CCD detectors. The light source ranges from 210 to 2000 nm, the wavelength-dependent light detection system ranges from 200 to 1100 nm with ≤1 nm resolution, and the nanoparticle diameters range from 1 to 3000 nm. The nanoparticles are suspended in pure water or water-based buffer solutions. For testing and calibration purposes, results are presented for nanoparticles composed of polystyrene and gold. Mie theory is used to model the total extinction cross section, and spectral inversion is employed to obtain quantitative particle size distributions, from which information on the size, shape, and number of nanoparticles can be derived. Discussed are the precision, accuracy, resolution, and sensitivity of our results. The LTS technique is quite versatile and can be applied to spectroscopic investigations where wideband, accurate, low-noise, real-time spectra are desired.
Hsu, Sheng-Hsiou; Mullen, Tim R; Jung, Tzyy-Ping; Cauwenberghs, Gert
Independent component analysis (ICA) has been widely applied to electroencephalographic (EEG) biosignal processing and brain-computer interfaces. The practical use of ICA, however, is limited by its computational complexity, data requirements for convergence, and assumption of data stationarity, especially for high-density data. Here we study and validate an optimized online recursive ICA algorithm (ORICA) with online recursive least squares (RLS) whitening for blind source separation of high-density EEG data, which offers instantaneous incremental convergence upon presentation of new data. Empirical results of this study demonstrate the algorithm's: 1) suitability for accurate and efficient source identification in high-density (64-channel) realistically-simulated EEG data; 2) capability to detect and adapt to nonstationarity in 64-ch simulated EEG data; and 3) utility for rapidly extracting principal brain and artifact sources in real 61-channel EEG data recorded by a dry and wearable EEG system in a cognitive experiment. ORICA was implemented as functions in BCILAB and EEGLAB and was integrated in an open-source Real-time EEG Source-mapping Toolbox (REST), supporting applications in ICA-based online artifact rejection, feature extraction for real-time biosignal monitoring in clinical environments, and adaptable classifications in brain-computer interfaces.
Chen, Szi-Wen; Liaw, Jiunn-Woei; Chan, Hsiao-Lung; Chang, Ya-Ju; Ku, Chia-Hao
A real-time muscle fatigue monitoring system was developed to quantitatively detect the muscle fatigue of subjects during cycling movement, where a fatigue progression measure (FPM) was built-in. During the cycling movement, the electromyogram (EMG) signals of the vastus lateralis and gastrocnemius muscles in one leg as well as cycling speed are synchronously measured in a real-time fashion. In addition, the heart rate (HR) and the Borg rating of perceived exertion scale value are recorded per minute. Using the EMG signals, the electrical activity and median frequency (MF) are calculated per cycle. Moreover, the updated FPM, based on the percentage of reduced MF counts during cycling movement, is calculated to measure the onset time and the progressive process of muscle fatigue. To demonstrate the performance of our system, five young healthy subjects were recruited. Each subject was asked to maintain a fixed speed of 60 RPM, as best he/she could, under a constant load during the pedaling. When the speed reached 20 RPM or the HR reached the maximal training HR, the experiment was then terminated immediately. The experimental results show that the proposed system may provide an on-line fatigue monitoring and analysis for the lower extremity muscles during cycling movement. PMID:25014101
Sanford, Lindsay N; Wittwer, Carl T
Accurate control of the sample temperature during thermal cycling is critical for successful polymerase chain reaction (PCR). Direct sensor contact with the reaction is problematic, forcing measurements external to the sample and compromising accuracy during rapid temperature transitions. The widespread use of fluorescence in real-time PCR and melting analysis suggests another measure of temperature, the intrinsic fluorescence of temperature-sensitive passive dyes. Calibration curves correlating sulforhodamine B fluorescence to temperature on nine real-time PCR instruments were obtained by heating at 0.018-0.1 °C/s between 50 and 95 °C, with a twofold change in fluorescence. After instrument stabilization for 20 min, no dye photobleaching was observed and thermal degradation was 2.2%/h at 80 °C. During cycling, solution temperatures derived from fluorescence were well matched to thermocouples placed within samples, but not to temperatures recorded by the instrument. Solution temperatures lagged instrument temperatures by up to 8 °C during cycling, often requiring 5-10 s at target temperatures for equilibration. Melting curves were displaced by 0.2-1.1 °C. Temperature inaccuracies were dependent on the instrument, the ramp rate, and the sample volume. The fluorescence of passive dyes can be used to accurately assess solution temperatures during PCR and should be particularly useful at fast cycling speeds.
Hsu, Sheng-Hsiou; Mullen, Tim; Jung, Tzyy-Ping; Cauwenberghs, Gert
Independent Component Analysis (ICA) has been widely applied to electroencephalographic (EEG) biosignal processing and brain-computer interfaces. The practical use of ICA, however, is limited by its computational complexity, data requirements for convergence, and assumption of data stationarity, especially for high-density data. Here we study and validate an optimized online recursive ICA algorithm (ORICA) with online recursive least squares (RLS) whitening for blind source separation of high-density EEG data, which offers instantaneous incremental convergence upon presentation of new data. Empirical results of this study demonstrate the algorithm's: (a) suitability for accurate and efficient source identification in high-density (64-channel) realistically-simulated EEG data; (b) capability to detect and adapt to non-stationarity in 64-ch simulated EEG data; and (c) utility for rapidly extracting principal brain and artifact sources in real 61-channel EEG data recorded by a dry and wearable EEG system in a cognitive experiment. ORICA was implemented as functions in BCILAB and EEGLAB and was integrated in an open-source Real-time EEG Source-mapping Toolbox (REST), supporting applications in ICA-based online artifact rejection, feature extraction for real-time biosignal monitoring in clinical environments, and adaptable classifications in brain-computer interfaces. PMID:26685257
Nakafuji, Dora; Gouveia, Lauren
This project supports development of the next generation, integrated energy management infrastructure (EMS) able to incorporate advance visualization of behind-the-meter distributed resource information and probabilistic renewable energy generation forecasts to inform real-time operational decisions. The project involves end-users and active feedback from an Utility Advisory Team (UAT) to help inform how information can be used to enhance operational functions (e.g. unit commitment, load forecasting, Automatic Generation Control (AGC) reserve monitoring, ramp alerts) within two major EMS platforms. Objectives include: Engaging utility operations personnel to develop user input on displays, set expectations, test and review; Developing ease of use and timeliness metrics for measuring enhancements; Developing prototype integrated capabilities within two operational EMS environments; Demonstrating an integrated decision analysis platform with real-time wind and solar forecasting information and timely distributed resource information; Seamlessly integrating new 4-dimensional information into operations without increasing workload and complexities; Developing sufficient analytics to inform and confidently transform and adopt new operating practices and procedures; Disseminating project lessons learned through industry sponsored workshops and conferences;Building on collaborative utility-vendor partnership and industry capabilities
Ibrahim, Sofi M; Aitichou, Mohamed; Hardick, Justin; Blow, Jamie; O'Guinn, Monica L; Schmaljohn, Connie
The development of sensitive and specific nucleic acid diagnostic assays for viral pathogens is essential for proper medical intervention. This chapter describes four fluorescence-based PCR assays to detect the Crimean-Congo Hemorrhagic Fever (CCHFV), Andes (ANDV), Hantaan (HANV), and Sandfly Fever Sicilian (SFSV) Viruses. These assays are based on species-specific hydrolysis probes targeting the nucleocapsid protein gene for CCHFV and SFSV and the glycoprotein gene for ANDV and HANV. All four assays were optimized for LightCycler 2.0 (Roche Diagnostics, Indianapolis, IN) or Ruggedized Advanced Pathogen Identification Device (R.A.P.I.D.; Idaho Technology Inc., Salt Lake City, UT). The assays were evaluated using the protocols described in the Subheading 3. The limits of detection were approximately 5, 2, 2, and 5 plaque-forming units (PFUs) for CCHFV, ANDV, HTNV, and SFSV assays, respectively. The sensitivity and specificity of the assays were evaluated with test panels that consisted of 20-60 known positive and 30-135 known negative samples, representing 7-34 genetically diverse bacterial and viral species. The CCHFV assay detected 59 out of the 60 positive samples and no false positives, resulting in 98.3% sensitivity at LOD of 5 PFU and 100% specificity. The ANDV and HTNV assays correctly identified all the positive samples with no false positive reactions; therefore, the sensitivity and specificity of these assays were determined to be 100% at LOD of 2 PFU. The SFSV assay missed three positive samples and cross-reacted with one of 48 negative samples, resulting in 95% sensitivity at LOD of 5 PFU and 98% specificity.
Grapevine leafroll-associated virus 7 (GLRaV-7) is an unassigned member in the Closteroviridae family that was first recorded in an asymptomatic white-berried grapevine cultivar from Albania. In California, the virus has been detected in several cultivars including Chardonnay, Merlot, Pinot Noir, Em...
Wilson, William C; Romito, Marco; Jasperson, Dane C; Weingartl, Hana; Binepal, Yatinder S; Maluleke, Moabi R; Wallace, David B; van Vuren, Petrus Jansen; Paweska, Janusz T
Outbreaks of Rift Valley fever in Kenya, Madagascar, Mauritania, and South Africa had devastating effects on livestock and human health. In addition, this disease is a food security issue for endemic countries. There is growing concern for the potential introduction of RVF into non-endemic countries. A number of single-gene target amplification assays have been developed for the rapid detection of RVF viral RNA. This paper describes the development of an improved amplification assay that includes two confirmatory target RNA segments (L and M) and a third target gene, NSs, which is deleted in the Clone 13 commercial vaccine and other candidate vaccines. The assay also contains an exogenous RNA control added during the PCR setup for detection of amplification inhibitors. The assay was evaluated initially with samples from experimentally infected animals, after which clinical veterinary and human samples from endemic countries were tested for further evaluation. The assay has a sensitivity range of 66.7-100% and a specificity of 92.0-100% depending on the comparison. The assay has an overall sensitivity of 92.5%, specificity of 95% and a positive predictive value of 98.7%. The single-tube assay provides confirmation of the presence of RVFV RNA for improved confidence in diagnostic results and a "differentiate infected from vaccinated animals" (DIVA)--compatible marker for RVFV NSs--deleted vaccines, which is useful for RVF endemic countries, but especially important in non-endemic countries.
Outbreaks of Rift Valley fever in Kenya, Madagascar, Mauritania, and South Africa had devastating effects on livestock and human health. In addition, this disease is a food security issue for endemic countries. There is growing concern for the potential introduction of RVF into non-endemic countries...
Shigella flexneriATCC12022 1 Negative Shigella sonnei ATCC25931 1 Negative Vibrio cholera (NAG) (Culture) 2 Negative Vibrio cholera (Ogawa...Culture) 1 Negative Vibrio cholera (Inaga) (Culture) 1 Negative Sapovivus (Known specimen extract) 2 Negative Rotavirus (Known specimen extract) 2
Popova, A G; Oreshkova, S F; Zhchelkunov, I S; Rudakova, S L; Zhchelkunova, T I; Tikunova, N V; Blinova, N N; Il'ichev, A A
A RT-PCR method has been developed to diagnose infectious hemopoietic necrosis virus (IHNV) in salmons. The authors show it possible to use the method for viral shedding in both a cell culture and a clinical sample from infected fishes. Genotyping of IHNV strains originating from North America, Europe, and Russia, by using the restriction fragment length polymerase analysis, has revealed that 10 of them belong to 3 existing genogroups (U, M, and L). Three Russian isolates are assigned into a separate subgroup. Phylogenetic analysis of several isolates has confirmed that viral strains from Katchatka belong to the North American U-genogroup whereas 3 Russian isolates from the continental zone of the country make up a separate subgroup within the same genogroup.
Hild, Jutta; Krüger, Wolfgang; Brüstle, Stefan; Trantelle, Patrick; Unmüßig, Gabriel; Heinze, Norbert; Peinsipp-Byma, Elisabeth; Beyerer, Jürgen
Motion video analysis is a challenging task, especially in real-time applications. In most safety and security critical applications, a human observer is an obligatory part of the overall analysis system. Over the last years, substantial progress has been made in the development of automated image exploitation algorithms. Hence, we investigate how the benefits of automated video analysis can be integrated suitably into the current video exploitation systems. In this paper, a system design is introduced which strives to combine both the qualities of the human observer's perception and the automated algorithms, thus aiming to improve the overall performance of a real-time video analysis system. The system design builds on prior work where we showed the benefits for the human observer by means of a user interface which utilizes the human visual focus of attention revealed by the eye gaze direction for interaction with the image exploitation system; eye tracker-based interaction allows much faster, more convenient, and equally precise moving target acquisition in video images than traditional computer mouse selection. The system design also builds on prior work we did on automated target detection, segmentation, and tracking algorithms. Beside the system design, a first pilot study is presented, where we investigated how the participants (all non-experts in video analysis) performed in initializing an object tracking subsystem by selecting a target for tracking. Preliminary results show that the gaze + key press technique is an effective, efficient, and easy to use interaction technique when performing selection operations on moving targets in videos in order to initialize an object tracking function.
Disasters, such as major storm events or earthquakes, trigger an immediate response by the disaster management system of the nation in question. The quality of this response is a large factor in its ability to limit the impacts on the local population. Improving the quality of disaster response therefore reduces disaster impacts. Studying past disasters is a valuable exercise to understand what went wrong, identify measures which could have mitigated these issues, and make recommendations to improve future disaster planning and response. While such ex post evaluations can lead to improvements in the disaster management system, there are limitations. The main limitation that has influenced this research is that ex post evaluations do not have the ability to inform the disaster response being assessed for the obvious reason that they are carried out long after the response phase is over. The result is that lessons learned can only be applied to future disasters. In the field of humanitarian relief, this limitation has led to the development of real time evaluations. The key aspect of real time humanitarian evaluations is that they are completed while the operation is still underway. This results in findings being delivered at a time when they can still make a difference to the humanitarian response. Applying such an approach to the immediate disaster response phase requires an even shorter time-frame, as well as a shift in focus from international actors to the nation in question's government. As such, a pilot study was started and methodology developed, to analyze disaster response in near real-time. The analysis uses the information provided by the disaster management system within the first 0 - 5 days of the response. The data is collected from publicly available sources such as ReliefWeb and sorted under various categories which represent each aspect of disaster response. This process was carried out for 12 disasters. The quantity and timeliness of information
Kirby, Sarah; Lauritsen, Janet; Pack, Ginger; Ha, Anhhoang; Jowers, Steven; Mcnenny, Robert; Truong, The; Dell, James
A system which is to provide real-time failure analysis support to controllers at the NASA Johnson Space Center Control Center Complex (CCC) for both Space Station and Space Shuttle on-orbit operations is described. The system employs monitored systems' models of failure behavior and model evaluation algorithms which are domain-independent. These failure models are viewed as a stepping stone to more robust algorithms operating over models of intended function. The described system is designed to meet two sets of requirements. It must provide a useful failure analysis capability enhancement to the mission controller. It must satisfy CCC operational environment constraints such as cost, computer resource requirements, verification, and validation. The underlying technology and how it may be used to support operations is also discussed.
This thesis provides design and analysis of techniques for global load balancing on ensemble architectures running soft-real-time object-oriented applications with statistically periodic loads. It focuses on estimating the instantaneous average load over all the processing elements. The major contribution is the use of explicit stochastic process models for both the loading and the averaging itself. These models are exploited via statistical time-series analysis and Bayesian inference to provide improved average load estimates, and thus to facilitate global load balancing. This thesis explains the distributed algorithms used and provides some optimality results. It also describes the algorithms' implementation and gives performance results from simulation. These results show that our techniques allow more accurate estimation of the global system load ing, resulting in fewer object migration than local methods. Our method is shown to provide superior performance, relative not only to static load-balancing schemes but also to many adaptive methods.
Fitzgerald, Timothy L; McQualter, Richard B
The quantitative real-time polymerase chain reaction is used to simultaneously amplify and quantify a targeted DNA molecule. It can be used to determine exact copy number of a molecule within a sample and/or to compare the quantity of a molecule between samples. When combined with reverse transcription, it is a powerful tool for the analysis of gene expression, and it is widely used for this purpose in plant species. Here we provide an introduction to fundamental concepts relevant for the analysis of gene expression in plants using this technique and a protocol for quantification of the relative expression of a sucrose phosphate synthase gene along the maturation gradient of a sugarcane leaf.
Bessaud, Maël; Autret, Arnaud; Jegouic, Sophie; Balanant, Jean; Joffret, Marie-Line; Delpeyroux, Francis
Human enteroviruses are among the most common viruses infecting humans. These viruses are known to be able to infect a wide range of tissues and are believed to establish persistent infections. Enteroviruses are positive-sense single-stranded RNA viruses whose replication involves the synthesis of negative strand intermediates. Therefore, the specific detection of negatively stranded viral RNA in tissues or cells is a reliable marker of active enteroviral replication. The present report presents the development of a real-time RT-PCR allowing the specific detection and quantification of negatively stranded viral RNA. Since it was known that specific amplification of single-stranded RNA can be made difficult by false-priming events leading to false-positive or overestimated results, the assay was developed by using a tagged RT primer. This tagged RT-PCR was shown to be able to amplify specifically negative RNA of enteroviruses grown in cell cultures by preventing the amplification of cDNAs generated by false-priming.
Wang, Lu; Zeng, Shanshan; Chen, Teng; Qu, Haibin
A promising process analytical technology (PAT) tool has been introduced for batch processes monitoring. Direct analysis in real time mass spectrometry (DART-MS), a means of rapid fingerprint analysis, was applied to a percolation process with multi-constituent substances for an anti-cancer botanical preparation. Fifteen batches were carried out, including ten normal operations and five abnormal batches with artificial variations. The obtained multivariate data were analyzed by a multi-way partial least squares (MPLS) model. Control trajectories were derived from eight normal batches, and the qualification was tested by R(2) and Q(2). Accuracy and diagnosis capability of the batch model were then validated by the remaining batches. Assisted with high performance liquid chromatography (HPLC) determination, process faults were explained by corresponding variable contributions. Furthermore, a batch level model was developed to compare and assess the model performance. The present study has demonstrated that DART-MS is very promising in process monitoring in botanical manufacturing. Compared with general PAT tools, DART-MS offers a particular account on effective compositions and can be potentially used to improve batch quality and process consistency of samples in complex matrices.
Love, David C; Casteel, Michael J; Meschke, John S; Sobsey, Mark D
Enteric viruses are important agents of foodborne disease. Unfortunately, robust, quantitative methods for sampling and analysis of enteric and other viruses in processed or complex foods are not well-established. As a result, epidemiologically determined etiologies or pathogen sources in foodborne outbreaks are rarely confirmed by virological analysis. In this study, an acid-adsorption elution concentration (AEC) method previously used to monitor virus occurrence and investigate enteric virus outbreaks in shellfish was adapted for examination of processed food items, namely tomato sauce and blended strawberries. Hepatitis A virus (HAV), poliovirus, and coliphage MS2 (MS2) were seeded in 10 or 30 g samples of tomato sauce or blended strawberries, recovered by AEC, and quantified by cell culture infectivity assay. In addition, nested reverse transcription-polymerase chain reaction (RT-PCR) and TaqMan RT-PCR assays were used to detect HAV RNA. Viruses were efficiently adsorbed to foods as an initial concentration step, with infectious HAV and MS2 adsorption of 67% and 93%, respectively, to tomato sauce, and 89% and 99%, respectively, to blended strawberries. Forty-three to 65% of HAV and poliovirus were subsequently eluted and recovered from tomato sauce using 0.5 M threonine, pH 7.2. The lower limits of HAV detection were at initial seeding levels of 14 PFU/g of tomato sauce and 33 PFU/g of blended strawberries. Unlike TaqMan RT-PCR, nested RT-PCR was not inhibited by undiluted final RNA extracts of tomato sauce or blended strawberries. The successful adaptation of the AEC method for enteric and other virus recovery, quantitation and detection in processed foods demonstrates its potential for use in the investigation of foodborne outbreaks of viral etiology and for validation of virus disinfection and sanitary processing procedures used by the food industry.
Samija, Ivan; Mateša, Neven; Lukač, Josip; Kusić, Zvonko
The aim of the study was to determine the diagnostic value of reverse transcriptase polymerase chain reaction (RT-PCR) analysis of galectin-3 and CD44v6 as markers for preoperative diagnosis of malignancy in lesions of the thyroid. RT-PCR analysis of galectin-3 and CD44v6 expression was performed on RNA isolated from fine-needle aspirates of thyroid lesions from 428 patients. The results were evaluated against the postoperative histopathological diagnosis or definitive cytological diagnosis in cases of nodular goiter and Hashimoto thyroiditis. A total of 57 (13%) samples were inadequate for RT-PCR. Galectin-3 and CD44v6 were positive in 167 (45%) and 158 (43%) out of 371 adequate samples, respectively. Galectin-3 and CD44v6 were positive in 56 (86%) and 54 (83%) out of 65 papillary carcinomas, in 16 (29%) and 18 (32%) out of 56 Hashimoto's thyroiditis, in 61 (34%) and 52 (29%) out of 181 nodular goiters, in 23 (43%) and 23 (43%) out of 53 follicular adenomas, in 3 (100%) and 3 (100%) out of 3 follicular carcinomas, and in 8 (62%) and 8 (62%) out of 13 Hurthle cell adenomas, respectively. Specificity, sensitivity, and positive and negative predictive values in discriminating between malignant and benign thyroid nodules were 64, 87, and 35 and 96% for galectin-3; 67, 84, and 36 and 95% for CD44v6; and 79, 82, and 47 and 95% for the analysis of both markers (considered positive only if both galectin-3 and CD44v6 were positive), respectively. Owing to relatively low specificity, the clinical value of galectin-3 and CD44v6 analysis by RT-PCR as a marker for preoperative diagnosis of malignancy in thyroid lesions is limited.
Direct analysis in real time (DART) ionization coupled to a high resolution mass spectrometer (MS) was used for screening of aflatoxins from a variety of surfaces and the rapid quantitative analysis of aflatoxins extracted from corn. Sample preparation procedure and instrument parameter settings wer...
Kaminska, Paulina S; Yernazarova, Aliya; Murawska, Emilia; Swiecicki, Jakub; Fiedoruk, Krzysztof; Bideshi, Dennis K; Swiecicka, Izabela
Entomopathogenic Bacillus thuringiensis is closely related to Bacillus cereus, a human pathogen known to cause emesis and diarrhea. Standard detection methods do not distinguish these bacilli. Hemolysin BL (hbl) and non-hemolytic enterotoxin (nhe) genes that encode, respectively, HBL and NHE enterotoxins, are known to be harbored in both bacterial species, suggesting that differentiation of these bacilli is clinically and epidemiologically relevant. In this study the reliability of quantitative reverse transcription real-time PCR (qRT-PCR) and enzyme immunoassays (EIAs) in detecting hbl and nhe transcripts and corresponding toxins in environmental B. thuringiensis isolates was assessed. At least one enterotoxin gene was present in each isolate, and nhe or hbl genes were found in 85% and 55% of the strains, respectively. Based on statistical analyses, both BCET-RPLA and Duopath detected HBL at similar levels, and TECRA and Duopath can be used interchangeably for the detection of NHE, although TECRA has significantly lower sensitivity than Duopath. Thus, as potential enterotoxic B. thuringiensis strains occur in the natural environment, and EIA results may not correspond with the presence of enterotoxin genes and their expression, we suggest that reliable interpretation will be significantly enhanced by including qRT-PCR to support inferences based on EIAs.
Pabinger, Stephan; Thallinger, Gerhard G; Snajder, René; Eichhorn, Heiko; Rader, Robert; Trajanoski, Zlatko
Background Since its introduction quantitative real-time polymerase chain reaction (qPCR) has become the standard method for quantification of gene expression. Its high sensitivity, large dynamic range, and accuracy led to the development of numerous applications with an increasing number of samples to be analyzed. Data analysis consists of a number of steps, which have to be carried out in several different applications. Currently, no single tool is available which incorporates storage, management, and multiple methods covering the complete analysis pipeline. Results QPCR is a versatile web-based Java application that allows to store, manage, and analyze data from relative quantification qPCR experiments. It comprises a parser to import generated data from qPCR instruments and includes a variety of analysis methods to calculate cycle-threshold and amplification efficiency values. The analysis pipeline includes technical and biological replicate handling, incorporation of sample or gene specific efficiency, normalization using single or multiple reference genes, inter-run calibration, and fold change calculation. Moreover, the application supports assessment of error propagation throughout all analysis steps and allows conducting statistical tests on biological replicates. Results can be visualized in customizable charts and exported for further investigation. Conclusion We have developed a web-based system designed to enhance and facilitate the analysis of qPCR experiments. It covers the complete analysis workflow combining parsing, analysis, and generation of charts into one single application. The system is freely available at PMID:19712446
Wargo, M. J.; Witt, A. F.
A real time thermal imaging system with temperature resolution better than +/- 0.5 C and spatial resolution of better than 0.5 mm has been developed. It has been applied to the analysis of melt surface thermal field distributions in both Czochralski and liquid encapsulated Czochralski growth configurations. The sensor can provide single/multiple point thermal information; a multi-pixel averaging algorithm has been developed which permits localized, low noise sensing and display of optical intensity variations at any location in the hot zone as a function of time. Temperature distributions are measured by extraction of data along a user selectable linear pixel array and are simultaneously displayed, as a graphic overlay, on the thermal image.
Lingerfelt, Eric J; Messer, Bronson; Desai, Sharvari S; Holt, Chastity A; Lentz, Eric J
We present an overview of a software system, Bellerophon, built to support a production-level HPC application called CHIMERA, which simulates core-collapse supernova events at the petascale. Developed over the last four years, Bellerophon enables CHIMERA s geographically dispersed team of collaborators to perform data analysis in near real-time. Its n-tier architecture provides an encapsulated, end-to-end software solution that enables the CHIMERA team to quickly and easily access highly customizable animated and static views of results from anywhere in the world via a web-deliverable, cross-platform desktop application. In addition, Bellerophon addresses software engineering tasks for the CHIMERA team by providing an automated mechanism for performing regression testing on a variety of supercomputing platforms. Elements of the team s workflow management needs are met with software tools that dynamically generate code repository statistics, access important online resources, and monitor the current status of several supercomputing resources.
Widergren, Steven E.; Subbarao, Krishnappa; Fuller, Jason C.; Chassin, David P.; Somani, Abhishek; Marinovici, Maria C.; Hammerstrom, Janelle L.
This report contributes initial findings from an analysis of significant aspects of the gridSMART® Real-Time Pricing (RTP) – Double Auction demonstration project. Over the course of four years, Pacific Northwest National Laboratory (PNNL) worked with American Electric Power (AEP), Ohio and Battelle Memorial Institute to design, build, and operate an innovative system to engage residential consumers and their end-use resources in a participatory approach to electric system operations, an incentive-based approach that has the promise of providing greater efficiency under normal operating conditions and greater flexibility to react under situations of system stress. The material contained in this report supplements the findings documented by AEP Ohio in the main body of the gridSMART report. It delves into three main areas: impacts on system operations, impacts on households, and observations about the sensitivity of load to price changes.
Zhang, Qi-Lin; Zhu, Qian-Hua; Liao, Xin; Wang, Xiu-Qiang; Chen, Tao; Xu, Han-Ting; Wang, Juan; Yuan, Ming-Long; Chen, Jun-Yuan
Amphioxus is a closest living proxy to the ancestor of cephalochordates with vertebrates, and key animal for novel understanding in the evolutionary origin of vertebrate body plan, genome, tissues and immune system. Reliable analyses using quantitative real-time PCR (qRT-PCR) for answering these scientific questions is heavily dependent on reliable reference genes (RGs). In this study, we evaluated stability of thirteen candidate RGs in qRT-PCR for different developmental stages and tissues of amphioxus by four independent (geNorm, NormFinder, BestKeeper and deltaCt) and one comparative algorithms (RefFinder). The results showed that the top two stable RGs were the following: (1) S20 and 18 S in thirteen developmental stages, (2) EF1A and ACT in seven normal tissues, (3) S20 and L13 in both intestine and hepatic caecum challenged with lipopolysaccharide (LPS), and (4) S20 and EF1A in gill challenged with LPS. The expression profiles of two target genes (EYA and HHEX) in thirteen developmental stages were used to confirm the reliability of chosen RGs. This study identified optimal RGs that can be used to accurately measure gene expression under these conditions, which will benefit evolutionary and functional genomics studies in amphioxus. PMID:27869224
Yan, Zhaoping; Gao, Jinhang; Lv, Xiuhe; Yang, Wenjuan; Wen, Shilei; Tong, Huan; Tang, Chengwei
The analysis of differences in gene expression is dependent on normalization using reference genes. However, the expression of many of these reference genes, as evaluated by quantitative RT-PCR, is upregulated in acute pancreatitis, so they cannot be used as the standard for gene expression in this condition. For this reason, we sought to identify a stable reference gene, or a suitable combination, for expression analysis in acute pancreatitis. The expression stability of 10 reference genes (ACTB, GAPDH, 18sRNA, TUBB, B2M, HPRT1, UBC, YWHAZ, EF-1α, and RPL-13A) was analyzed using geNorm, NormFinder, and BestKeeper software and evaluated according to variations in the raw Ct values. These reference genes were evaluated using a comprehensive method, which ranked the expression stability of these genes as follows (from most stable to least stable): RPL-13A, YWHAZ > HPRT1 > GAPDH > UBC > EF-1α > 18sRNA > B2M > TUBB > ACTB. RPL-13A was the most suitable reference gene, and the combination of RPL-13A and YWHAZ was the most stable group of reference genes in our experiments. The expression levels of ACTB, TUBB, and B2M were found to be significantly upregulated during acute pancreatitis, whereas the expression level of 18sRNA was downregulated. Thus, we recommend the use of RPL-13A or a combination of RPL-13A and YWHAZ for normalization in qRT-PCR analyses of gene expression in mouse models of acute pancreatitis. PMID:27069927
Abuzaghleh, Omar; Barkana, Buket D.
Melanoma spreads through metastasis, and therefore, it has been proved to be very fatal. Statistical evidence has revealed that the majority of deaths resulting from skin cancer are as a result of melanoma. Further investigations have shown that the survival rates in patients depend on the stage of the cancer; early detection and intervention of melanoma implicate higher chances of cure. Clinical diagnosis and prognosis of melanoma are challenging, since the processes are prone to misdiagnosis and inaccuracies due to doctors’ subjectivity. Malignant melanomas are asymmetrical, have irregular borders, notched edges, and color variations, so analyzing the shape, color, and texture of the skin lesion is important for the early detection and prevention of melanoma. This paper proposes the two major components of a noninvasive real-time automated skin lesion analysis system for the early detection and prevention of melanoma. The first component is a real-time alert to help users prevent skinburn caused by sunlight; a novel equation to compute the time for skin to burn is thereby introduced. The second component is an automated image analysis module, which contains image acquisition, hair detection and exclusion, lesion segmentation, feature extraction, and classification. The proposed system uses PH2 Dermoscopy image database from Pedro Hispano Hospital for the development and testing purposes. The image database contains a total of 200 dermoscopy images of lesions, including benign, atypical, and melanoma cases. The experimental results show that the proposed system is efficient, achieving classification of the benign, atypical, and melanoma images with accuracy of 96.3%, 95.7%, and 97.5%, respectively. PMID:27170906
Bulgarelli, Andrea; Fioretti, Valentina; Zoli, Andrea; Aboudan, Alessio; Rodríguez-Vázquez, Juan José; Maier, Gernot; Lyard, Etienne; Bastieri, Denis; Lombardi, Saverio; Tosti, Gino; De Rosa, Adriano; Bergamaschi, Sonia; Interlandi, Matteo; Beneventano, Domenico; Lamanna, Giovanni; Jacquemier, Jean; Kosack, Karl; Antonelli, Lucio Angelo; Boisson, Catherine; Burkowski, Jerzy; Buson, Sara; Carosi, Alessandro; Conforti, Vito; Contreras, Jose Luis; De Cesare, Giovanni; de los Reyes, Raquel; Dumm, Jon; Evans, Phil; Fortson, Lucy; Fuessling, Matthias; Graciani, Ricardo; Gianotti, Fulvio; Grandi, Paola; Hinton, Jim; Humensky, Brian; Knödlseder, Jürgen; Malaguti, Giuseppe; Marisaldi, Martino; Neyroud, Nadine; Nicastro, Luciano; Ohm, Stefan; Osborne, Julian; Rosen, Simon; Tacchini, Alessandro; Torresi, Eleonora; Testa, Vincenzo; Trifoglio, Massimo; Weinstein, Amanda
The Cherenkov Telescope Array (CTA) observatory will be one of the biggest ground-based very-high-energy (VHE) γ- ray observatory. CTA will achieve a factor of 10 improvement in sensitivity from some tens of GeV to beyond 100 TeV with respect to existing telescopes. The CTA observatory will be capable of issuing alerts on variable and transient sources to maximize the scientific return. To capture these phenomena during their evolution and for effective communication to the astrophysical community, speed is crucial. This requires a system with a reliable automated trigger that can issue alerts immediately upon detection of γ-ray flares. This will be accomplished by means of a Real-Time Analysis (RTA) pipeline, a key system of the CTA observatory. The latency and sensitivity requirements of the alarm system impose a challenge because of the anticipated large data rate, between 0.5 and 8 GB/s. As a consequence, substantial efforts toward the optimization of highthroughput computing service are envisioned. For these reasons our working group has started the development of a prototype of the Real-Time Analysis pipeline. The main goals of this prototype are to test: (i) a set of frameworks and design patterns useful for the inter-process communication between software processes running on memory; (ii) the sustainability of the foreseen CTA data rate in terms of data throughput with different hardware (e.g. accelerators) and software configurations, (iii) the reuse of nonreal- time algorithms or how much we need to simplify algorithms to be compliant with CTA requirements, (iv) interface issues between the different CTA systems. In this work we focus on goals (i) and (ii).
Park, Kwanghyun; Kinginger, Celeste
The advance of digital video technology in the past two decades facilitates empirical investigation of learning in real time. The focus of this paper is the combined use of real-time digital video and a networked linguistic corpus for exploring the ways in which these technologies enhance our capability to investigate the cognitive process of…
Use of dual priming oligonucleotide system-based multiplex RT-PCR combined with high performance liquid chromatography assay for simultaneous detection of five enteric viruses associated with acute enteritis.
Fan, Wen-Lu; Wang, Zi-Wei; Qin, Yue; Sun, Chao; Liu, Zhong-Mei; Jiang, Yan-Ping; Qiao, Xin-Yuan; Tang, Li-Jie; Li, Yi-Jing; Xu, Yi-Gang
In this study, a specific and sensitive method for simultaneous detection of human astrovirus, human rotavirus, norovirus, sapovirus and enteric adenovirus associated with acute enteritis was developed, based on the specific dual priming oligonucleotide (DPO) system and the sensitive high-performance liquid chromatography (HPLC) analysis. The DPO system-based multiplex reverse transcription-polymerase chain reaction (RT-PCR) combined with HPLC assay was more sensitive than agarose gel electrophoresis analysis and real-time SYBR Green PCR assay, and showed a specificity of 100% and sensitivity of 96%-100%. The high sensitivity and specificity of the assay indicates its great potential to be a useful tool for the accurate diagnosis of enteric virus infections.
Adler, Robert; Hong, Yang; Huffman, George
Satellite data acquired and processed in real time now have the potential to provide the spacetime information on rainfall needed to monitor flood and landslide events around the world. This can be achieved by integrating the satellite-derived forcing data with hydrological models and landslide algorithms. Progress in using the TRMM Multi-satellite Precipitation Analysis (TMPA) as input to flood and landslide forecasts is outlined, with a focus on understanding limitations of the rainfall data and impacts of those limitations on flood/landslide analyses. Case studies of both successes and failures will be shown, as well as comparison with ground comparison data sets-- both in terms of rainfall and in terms of flood/landslide events. In addition to potential uses in real-time, the nearly ten years of TMPA data allow retrospective running of the models to examine variations in extreme events. The flood determination algorithm consists of four major components: 1) multi-satellite precipitation estimation; 2) characterization of land surface including digital elevation from NASA SRTM (Shuttle Radar Terrain Mission), topography-derived hydrologic parameters such as flow direction, flow accumulation, basin, and river network etc.; 3) a hydrological model to infiltrate rainfall and route overland runoff; and 4) an implementation interface to relay the input data to the models and display the flood inundation results to potential users and decision-makers, In terms of landslides, the satellite rainfall information is combined with a global landslide susceptibility map, derived from a combination of global surface characteristics (digital elevation topography, slope, soil types, soil texture, and land cover classification etc.) using a weighted linear combination approach. In those areas identified as "susceptible" (based on the surface characteristics), landslides are forecast where and when a rainfall intensity/duration threshold is exceeded. Results are described
Sakai, Tatsuhiro; Tamura, Keiichi
Since social media started getting more attention from users on the Internet, social media has been one of the most important information source in the world. Especially, with the increasing popularity of social media, data posted on social media sites are rapidly becoming collective intelligence, which is a term used to refer to new media that is displacing traditional media. In this paper, we focus on geotagged tweets on the Twitter site. These geotagged tweets are referred to as georeferenced documents because they include not only a short text message, but also the documents' posting time and location. Many researchers have been tackling the development of new data mining techniques for georeferenced documents to identify and analyze emergency topics, such as natural disasters, weather, diseases, and other incidents. In particular, the utilization of geotagged tweets to identify and analyze natural disasters has received much attention from administrative agencies recently because some case studies have achieved compelling results. In this paper, we propose a novel real-time analysis application for identifying bursty local areas related to emergency topics. The aim of our new application is to provide new platforms that can identify and analyze the localities of emergency topics. The proposed application is composed of three core computational intelligence techniques: the Naive Bayes classifier technique, the spatiotemporal clustering technique, and the burst detection technique. Moreover, we have implemented two types of application interface: a Web application interface and an android application interface. To evaluate the proposed application, we have implemented a real-time weather observation system embedded the proposed application. we used actual crawling geotagged tweets posted on the Twitter site. The weather observation system successfully detected bursty local areas related to observed emergency weather topics.
Konig, Gerhardt; Waters, Jonathan H; Javidroozi, Mazyar; Philip, Bridget; Ting, Vicki; Abbi, Gaurav; Hsieh, Eric; Tully, Griffeth; Adams, Gregg
Monitoring blood loss is important for management of surgical patients. This study reviews a device (Triton) that uses computer analysis of a photograph to estimate hemoglobin (Hb) mass present on surgical sponges. The device essentially does what a clinician does when trying to make a visual estimation of blood loss by looking at a sponge, albeit with less subjective variation. The performance of the Triton system is reported upon in during real-time use in surgical procedures. The cumulative Hb losses estimated using the Triton system for 50 enrolled patients were compared with reference Hb measurements during the first quarter, half, three-quarters and full duration of the surgery. Additionally, the estimated blood loss (EBL) was calculated using the Triton measured Hb loss and compared with values obtained from both visual estimation and gravimetric measurements. Hb loss measured by Triton correlated with the reference method across the four measurement intervals. Bias remained low and increased from 0.1 g in the first quarter to 3.7 g at case completion. The limits of agreement remained narrow and increased proportionally from the beginning to the end of the cases, reaching a maximum range of -15.3 to 22.7 g. The median (IQR) difference of EBL derived from the Triton system, gravimetric method and visual estimation versus the reference value were 13 (74), 389 (287), and 4 (230) mL, respectively. Use of the Triton system to measure Hb loss in real-time during surgery is feasible and accurate.
Modroño, Sagrario; Soldado, Ana; Martínez-Fernández, Adela; de la Roza-Delgado, Begoña
Significant advances achieved in different sensor technologies and computer processing data have made possible to respond the needs of livestock sector, providing precise and rapid information on feed composition, being an alternative to real time quality control on compound feed the use of handheld NIRS sensors. This work aimed to evaluate two hand-held portable NIR spectrophotometers for on-site and real time analysis of nutritive parameters in raw compound feed: Phazir 1624 Polychromix Inc (PhIR) and MicroNIR(TM) 1700 by JDSU (MICRO). For computing data, different combinations of pre-treatments and multivariate statistical methods have been assayed to extract the valuable information of spectra data and to develop appropriate calibrations. The calibration models displayed greatest predictive capacity for Crude Protein (CP), Crude Fiber (CF) and Starch (STCH) and the determination coefficients of cross validation were 0.90-0.88 for CP, 0.85-0.91 for CF, 0.89-0.88 and 0.89-0.91 for STCH using PhIR and MICRO instruments respectively. Dry Matter showed the lowest determination coefficients of cross validation 0.67-0.73. Accuracy achieved 99-101% for both NIRS instruments and no differences were found when applying tstudent-test comparing reference and predicted data. Results obtained with both instruments were compared by using standard deviation and not significant differences were observed at the 5% level. Results so far have demonstrated the potential of these handheld NIRS instruments proposed here to estimate the individual compound feeds composition changes at farms level instantly, time avoiding the disadvantage of moving the samples to the lab.
Leitner, G; Merin, U; Lemberskiy-Kuzin, L; Bezman, D; Katz, G
The economical profitability of the dairy industry is based on the quality of the bulk milk collected in the farms, therefore it was based on the herd level rather than on the individual animals at real time. Udder infection and stage of lactation are directly related to the quality of milk produced on the herd level. However, improvement of milk quality requires testing each animal's milk separately and continuously. Recently, it was postulated that online equipment can estimate milk quality according to its clotting parameters, and thus result in better economical return for cheese making. This study further investigated the potential application of the AfiLab™ equipment to provide real-time analysis of milk-clotting parameters for cheese manufacture and cheese yield on quarter (1018) and individual cow (277) levels. Days in milk, lactose, log SCC and udder infection were found to have a significant effect on curd firmness and cheese properties and yield. The results clearly indicate that: (a) the parameter Afi-CF determined with the AfiLab™ is suitable for assessing milk quality for its clotting parameters, a value which is not provided by merely measuring fat and protein content on the gland and the cow levels; (b) bacterial type is the single major cause of reduced milk quality, with variations depending on the bacterial species; and (c) early and late lactation also had negative effects on milk-clotting parameters. Cheese made from the various milk samples that were determined by the Afilab™ to be of higher quality for cheese making resulted in higher yield and better texture, which were related mainly to the bacterial species and stage of lactation.
Wang, J; Deng, Y; Xu, G
Time Petri nets (TPNs) are a popular Petri net model for specification and verification of real-time systems. A fundamental and most widely applied method for analyzing Petri nets is reachability analysis. The existing technique for reachability analysis of TPNs, however, is not suitable for timing property verification because one cannot derive end-to-end delay in task execution, an important issue for time-critical systems, from the reachability tree constructed using the technique. In this paper, we present a new reachability based analysis technique for TPNs for timing property analysis and verification that effectively addresses the problem. Our technique is based on a concept called clock-stamped state class (CS-class). With the reachability tree generated based on CS-classes, we can directly compute the end-to-end time delay in task execution. Moreover, a CS-class can be uniquely mapped to a traditional state class based on which the conventional reachability tree is constructed. Therefore, our CS-class-based analysis technique is more general than the existing technique. We show how to apply this technique to timing property verification of the TPN model of a command and control (C2) system.
Wei, Haiyan; Zeng, Jing; Deng, Congliang; Zheng, Chengzhong; Zhang, Ximeng; Ma, Dan; Yi, Yong
A one-step, real-time reverse-transcription loop-mediated isothermal amplification (rRT-LAMP) method targeting the 5' end of the capsid gene for rapid and quantitative detection of human astrovirus serotype 1 (HAstV 1) was developed. The assay is highly sensitive and comparable to real-time RT-PCR (rRT-PCR), with a detection limit of ∼100 RNA copies per assay. The specificity of the method was validated by the absence of any cross-reaction with RNA samples of HAstV 2-8 and other gastroenteritis viruses, followed by nucleotide sequencing of the amplified product. Fecal specimens (n=120) obtained from children under five years of age with gastroenteritis were tested by rRT-LAMP, rRT-PCR and transmission electron microscopy (TEM). Six (5%) of these samples were determined to be positive by both rRT-LAMP and rRT-PCR assay, and these two nucleic acid amplification methods resulted in a 200% increase in detection rates for HAstV infection compared with TEM alone. Furthermore, the rRT-LAMP assay is much more rapid than rRT-PCR and generates results in less than 20min for positive samples. The quantitation of viral load in stool specimens was determined from the standard curve plot of time-of-positivity versus initial RNA concentration. Most viral loads were determined to be within the range of 10(5)-10(8) copies. The results highlight the significance of the rapid rRT-LAMP method as a diagnostic and routine screening tool for the analysis of stool samples in hospital laboratories.
Fernández-Serra, A.; Rubio, L.; Calatrava, A.; Rubio-Briones, J.; Salgado, R.; Gil-Benso, R.; Espinet, B.; García-Casado, Z.; López-Guerrero, J. A.
Prostate cancer (PCa) is a very heterogeneous disease, and there are constraints in its current diagnosis. Serum PSA levels, digital rectal examination (DRE), and histopathologic analysis often drive to overdiagnosis and overtreatment. Since 2005, the presence of the genetic rearrangement between transmembrane-serine protease gene (TMPRSS2) and the erythroblast transformation-specific (ETS) member ERG (v-ets erythroblastosis virus E26 oncogene homolog avian) has been demonstrated in almost half of PCa cases. Both FISH and RT-PCR are useful tools for detecting these rearrangements, but very few comparatives between both techniques have been published. In this study, we included FFPE tumors from 294 PCa patients treated with radical prostatectomy with more than 5 years of followup. We constructed a total of 20 tissue microarrays in order to perform break-apart and tricolor probe FISH approaches that were compared with RT-PCR, showing a concordance of 80.6% (P < 0.001). The presence of TMPRSS2-ERG rearrangement was observed in 56.6% of cases. No association between TMPRSS2-ERG status and clinicopathological parameters nor biochemical progression and clinical progression free survival was found. In conclusion, this study demonstrates that both FISH and RT-PCR are useful tools in the assessment of the TMPRSS2-ERG fusion gene status in PCa patients and that this genetic feature per se lacks prognostic value. PMID:23781502
Fernández-Serra, A; Rubio, L; Calatrava, A; Rubio-Briones, J; Salgado, R; Gil-Benso, R; Espinet, B; García-Casado, Z; López-Guerrero, J A
Prostate cancer (PCa) is a very heterogeneous disease, and there are constraints in its current diagnosis. Serum PSA levels, digital rectal examination (DRE), and histopathologic analysis often drive to overdiagnosis and overtreatment. Since 2005, the presence of the genetic rearrangement between transmembrane-serine protease gene (TMPRSS2) and the erythroblast transformation-specific (ETS) member ERG (v-ets erythroblastosis virus E26 oncogene homolog avian) has been demonstrated in almost half of PCa cases. Both FISH and RT-PCR are useful tools for detecting these rearrangements, but very few comparatives between both techniques have been published. In this study, we included FFPE tumors from 294 PCa patients treated with radical prostatectomy with more than 5 years of followup. We constructed a total of 20 tissue microarrays in order to perform break-apart and tricolor probe FISH approaches that were compared with RT-PCR, showing a concordance of 80.6% (P < 0.001). The presence of TMPRSS2-ERG rearrangement was observed in 56.6% of cases. No association between TMPRSS2-ERG status and clinicopathological parameters nor biochemical progression and clinical progression free survival was found. In conclusion, this study demonstrates that both FISH and RT-PCR are useful tools in the assessment of the TMPRSS2-ERG fusion gene status in PCa patients and that this genetic feature per se lacks prognostic value.
Huang, Zhenyu; Chen, Yousu; Chavarría-Miranda, Daniel
Power grids worldwide are undergoing an unprecedented transition as a result of grid evolution meeting information revolution. The grid evolution is largely driven by the desire for green energy. Emerging grid technologies such as renewable generation, smart loads, plug-in hybrid vehicles, and distributed generation provide opportunities to generate energy from green sources and to manage energy use for better system efficiency. With utility companies actively deploying these technologies, a high level of penetration of these new technologies is expected in the next 5-10 years, bringing in a level of intermittency, uncertainties, and complexity that the grid did not see nor design for. On the other hand, the information infrastructure in the power grid is being revolutionized with large-scale deployment of sensors and meters in both the transmission and distribution networks. The future grid will have two-way flows of both electrons and information. The challenge is how to take advantage of the information revolution: pull the large amount of data in, process it in real time, and put information out to manage grid evolution. Without addressing this challenge, the opportunities in grid evolution will remain unfulfilled. This transition poses grand challenges in grid modeling, simulation, and information presentation. The computational complexity of underlying power grid modeling and simulation will significantly increase in the next decade due to an increased model size and a decreased time window allowed to compute model solutions. High-performance computing is essential to enable this transition. The essential technical barrier is to vastly increase the computational speed so operation response time can be reduced from minutes to seconds and sub-seconds. The speed at which key functions such as state estimation and contingency analysis are conducted (typically every 3-5 minutes) needs to be dramatically increased so that the analysis of contingencies is both
Pavlovich, Matthew J; Musselman, Brian; Hall, Adam B
Over the last decade, direct analysis in real time (DART) has emerged as a viable method for fast, easy, and reliable "ambient ionization" for forensic analysis. The ability of DART to generate ions from chemicals that might be present at the scene of a criminal activity, whether they are in the gas, liquid, or solid phase, with limited sample preparation has made the technology a useful analytical tool in numerous forensic applications. This review paper summarizes many of those applications, ranging from the analysis of trace evidence to security applications, with a focus on providing the forensic scientist with a resource for developing their own applications. The most common uses for DART in forensics are in studying seized drugs, drugs of abuse and their metabolites, bulk and detonated explosives, toxic chemicals, chemical warfare agents, inks and dyes, and commercial plant and animal products that have been adulterated for economic gain. This review is meant to complement recent reviews that have described the fundamentals of the ionization mechanism and the general use of DART. We describe a wide range of forensic applications beyond the field of analyzing drugs of abuse, which dominates the literature, including common experimental and data analysis methods. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 9999: XX-XX, 2016.
Palmer, Peter T.; Wong, C. M.; Salmonson, J. D.; Yost, R. A.; Griffin, T. P.; Yates, N. A.; Lawless, James G. (Technical Monitor)
The utility of MS/MS for both target compound analysis and the structure elucidation of unknowns has been described in a number of references. A broader acceptance of this technique has not yet been realized as it requires large, complex, and costly instrumentation which has not been competitive with more conventional techniques. Recent advancements in ion trap mass spectrometry promise to change this situation. Although the ion trap's small size, sensitivity, and ability to perform multiple stages of mass spectrometry have made it eminently suitable for on-line, real-time monitoring applications, advance automation techniques are required to make these capabilities more accessible to non-experts. Towards this end we have developed custom software for the design and implementation of MS/MS experiments. This software allows the user to take full advantage of the ion trap's versatility with respect to ionization techniques, scan proxies, and ion accumulation/ejection methods. Additionally, expert system software has been developed for autonomous target compound analysis. This software has been linked to ion trap control software and a commercial data system to bring all of the steps in the analysis cycle under control of the expert system. These software development efforts and their utilization for a number of trace analysis applications will be described.
Wang, Lu; Chen, Teng; Zeng, Shanshan; Qu, Haibin
A mass spectrometry method has been developed using the Quality by Design (QbD) principle. Direct analysis in real time mass spectrometry (DART-MS) was adopted to analyze a pharmaceutical preparation. A fishbone diagram for DART-MS and the Plackett-Burman design were utilized to evaluate the impact of a number of factors on the method performance. Multivariate regression and Pareto ranking analysis indicated that the temperature, determined distance, and sampler speed were statistically significant (P < 0.05). Furthermore, the Box-Behnken design combined with response surface analysis was then employed to study the relationships between these three factors and the quality of the DART-MS analysis. The analytical design space of DART-MS was thus constructed and its robustness was validated. In this presented approach, method performance was mathematically described as a composite desirability function of the critical quality attributes (CQAs). Two terms of method validation, including analytical repeatability and method robustness, were carried out at an operating work point. Finally, the validated method was successfully applied to the pharmaceutical quality assurance in different manufacturing batches. These results revealed that the QbD concept was practical in DART-MS method development. Meanwhile, the determined quality was controlled by the analytical design space. This presented strategy provided a tutorial to the development of a robust QbD-compliant mass spectrometry method for industrial quality control.
Toogoshi, M.; Kano, S. S.; Zempo, Y.
The maximum entropy method (MEM) is one of the key techniques for spectral analysis. The major feature is that spectra in the low frequency part can be described by the short time-series data. Thus, we applied MEM to analyse the spectrum from the time dependent dipole moment obtained from the time-dependent density functional theory (TDDFT) calculation in real time. It is intensively studied for computing optical properties. In the MEM analysis, however, the maximum lag of the autocorrelation is restricted by the total number of time-series data. We proposed that, as an improved MEM analysis, we use the concatenated data set made from the several-times repeated raw data. We have applied this technique to the spectral analysis of the TDDFT dipole moment of ethylene and oligo-fluorene with n = 8. As a result, the higher resolution can be obtained, which is closer to that of FT with practically time-evoluted data as the same total number of time steps. The efficiency and the characteristic feature of this technique are presented in this paper.
Wang, Lu; Chen, Teng; Zeng, Shanshan; Qu, Haibin
A mass spectrometry method has been developed using the Quality by Design (QbD) principle. Direct analysis in real time mass spectrometry (DART-MS) was adopted to analyze a pharmaceutical preparation. A fishbone diagram for DART-MS and the Plackett-Burman design were utilized to evaluate the impact of a number of factors on the method performance. Multivariate regression and Pareto ranking analysis indicated that the temperature, determined distance, and sampler speed were statistically significant (P < 0.05). Furthermore, the Box-Behnken design combined with response surface analysis was then employed to study the relationships between these three factors and the quality of the DART-MS analysis. The analytical design space of DART-MS was thus constructed and its robustness was validated. In this presented approach, method performance was mathematically described as a composite desirability function of the critical quality attributes (CQAs). Two terms of method validation, including analytical repeatability and method robustness, were carried out at an operating work point. Finally, the validated method was successfully applied to the pharmaceutical quality assurance in different manufacturing batches. These results revealed that the QbD concept was practical in DART-MS method development. Meanwhile, the determined quality was controlled by the analytical design space. This presented strategy provided a tutorial to the development of a robust QbD-compliant mass spectrometry method for industrial quality control.
Hope, Kristine M; Casey, Rufina N; Groocock, Geoffrey H; Getchell, Rodman G; Bowser, Paul R; Casey, James W
Viral hemorrhagic septicemia virus (VHSV) is an important pathogen of cultured and wild fish in marine and freshwater environments. A new genotype, VHSV IVb, was isolated from a fish collected from the Great Lakes in 2003. Since the first isolation, VHSV IVb has been confirmed in 28 species, signaling the early invasion and continued spread of this Office International des Epizooties-reportable agent. For surveillance of this virus in both wild and experimental settings, we have developed a rapid and sensitive one-step quantitative real-time polymerase chain reaction (qRT-PCR) assay that amplifies a 100-base-pair conserved segment from both the genomic negative strand and the mRNA positive strand of the nucleoprotein (N) gene of VHSV IVb. This assay is linear over seven orders of magnitude, with an analytical capability of detecting a single copy of viral RNA and reproducibility at 100 copies. The assay is approximately linear with RNA input from 50 to 1000 ng per assay and works equally well with RNA prepared from a column-based or phenol-chloroform-based method. In wild-caught fish, 97% of the cases were found to be more than three orders of magnitude more sensitive using qRT-PCR than using cell culture. Of the 1,428 fish from the Great Lakes region tested in 2006 and 2007, 24% were positive by qRT-PCR whereas only 5% were positive by cell culture. All of the fish that were positive by cell culture were also positive by qRT-PCR. Importantly, qRT-PCR sensitivity is comparable to that of cell culture detection when comparing VHSV viral RNA levels with viral titer stocks, confirming that the high qRT-PCR signals obtained with diagnostic samples are due to the accumulation of N gene mRNA by transcriptional attenuation. The qRT-PCR assay is particularly valuable for rapid and high-throughput prescreening of fish before confirmatory testing by cell culture or sequencing tissue-derived amplicons and especially in detecting infection in fish that do not show clinical
Brault, Aaron C; Fang, Ying; Reisen, William K
Following the introduction of West Nile virus into California during the summer of 2003, public health and vector control programs expanded surveillance efforts and were in need of diagnostics capable of rapid, sensitive, and specific detection of arbovirus infections of mosquitoes to inform decision support for intervention. Development of a multiplex TaqMan or real-time semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay in which three virus specific primer-probe sets were used in the same reaction is described herein for the detection of western equine encephalomyelitis, St. Louis encephalitis and West Nile viral RNA. Laboratory validation and field data from 10 transmission seasons are reported. The comparative sensitivity and specificity of this multiplex assay to singleplex RT-PCR as well as an antigen detection (rapid analyte measurement platform) and standard plaque assays indicate this assay to be rapid and useful in providing mosquito infection data to estimate outbreak risk.
Chen, Kuan-Fu; Blyn, Lawrence; Rothman, Richard E.; Ramachandran, Padmini; Valsamakis, Alexandra; Ecker, David; Sampath, Rangarajan; Gaydos, Charlotte A.
Diagnosis of respiratory viruses traditionally relies on culture or antigen detection.We aimed to demonstrate capacity of the RT-PCR/ESI-MS platform to identify clinical relevant respiratory viruses in nasopharyngeal aspirate (NPA) samples and compare the diagnostic performance characteristics relative to conventional culture- and antigen-based methods. A RT-PCR/ESI-MS respiratory virus surveillance kit designed to detect respiratory syncytial virus, influenza A and B, parainfluenza types 1-4, adenoviridae types A-F, coronaviridae, human bocavirus, and human metapneumovirus was evaluated using both mock-ups and frozen archived NPA (N=280), 95 of which were positive by clinical virology methods. RT-PCR/ESI-MS detected 74/95 (77.9%) known positive samples and identified an additional 13/185 (7%) from culture negative samples. Viruses that are non-detectable with conventional methods were also identified. Viral load was semi-quantifiable and ranged from 2,400 to >320,000copies/ml. Time to results was 8hrs. RT-PCR/ESI-MS showed promise in rapid detection of respiratory viruses, merits further evaluation for use in clinical settings. PMID:21251562
Di Giuliomaria, C; Capponi, M; D'Alessio, T; Sacco, R; Zanette, E
In assessing the level of stenosis in extracranial Doppler analysis, spectral analysis has until now been used qualitatively, for the most part. Owing to the many variables affecting the measurements (mainly noise level and instrument setting made subjectively by the operator), the reliability of the inferences on the degree of stenosis is not clearly definable. Under such conditions the need arises for algorithms and systems that can estimate spectral parameters with a higher degree of accuracy, to verify whether reliable inferences can indeed by made or if this technique is only a qualitative one. In the paper a real-time spectral analysis system is described. The system relies on a new spectral estimation algorithm which gives estimates with good robustness with respect to noise. Moreover, a clear measurement procedure which eliminates the many subjective factors affecting the estimates has also been proposed and used. The system has been evaluated with simulated signals and in clinical trials and has shown better performance than the commonly used commercial analysers.
This thesis provides design and analysis of techniques for global load balancing on ensemble architectures running soft-real-time object-oriented applications with statistically periodic loads. It focuses on estimating the instantaneous average load over all the processing elements. The major contribution is the use of explicit stochastic process models for both the loading and the averaging itself. These models are exploited via statistical time-series analysis and Bayesian inference to provide improved average load estimates, and thus to facilitate global load balancing. This thesis explains the distributed algorithms used and provides some optimality results. It also describes the algorithms' implementation and gives performance results from simulation. These results show that the authors' techniques allow more accurate estimation of the global system loading, resulting in fewer object migrations than local methods. The authors' method is shown to provide superior performance, relative not only to static load-balancing schemes but also to many adaptive load-balancing methods. Results from a preliminary analysis of another system and from simulation with a synthetic load provide some evidence of more general applicability.
Yang, Yuanyuan; Pathirathna, Pavithra; Siriwardhane, Thushani; McElmurry, Shawn P; Hashemi, Parastoo
Lead (Pb) pollution is an important environmental and public health concern. Rapid Pb transport during stormwater runoff significantly impairs surface water quality. The ability to characterize and model Pb transport during these events is critical to mitigating its impact on the environment. However, Pb analysis is limited by the lack of analytical methods that can afford rapid, sensitive measurements in situ. While electrochemical methods have previously shown promise for rapid Pb analysis, they are currently limited in two ways. First, because of Pb's limited solubility, test solutions that are representative of environmental systems are not typically employed in laboratory characterizations. Second, concerns about traditional Hg electrode toxicity, stability, and low temporal resolution have dampened opportunities for in situ analyses with traditional electrochemical methods. In this paper, we describe two novel methodological advances that bypass these limitations. Using geochemical models, we first create an environmentally relevant test solution that can be used for electrochemical method development and characterization. Second, we develop a fast-scan cyclic voltammetry (FSCV) method for Pb detection on Hg-free carbon fiber microelectrodes. We assess the method's sensitivity and stability, taking into account Pb speciation, and utilize it to characterize rapid Pb fluctuations in real environmental samples. We thus present a novel real-time electrochemical tool for Pb analysis in both model and authentic environmental solutions.
Ewald, Andrew J.
A grand challenge in biology is to understand the cellular and molecular basis of tissue and organ level function in mammals. The ultimate goals of such efforts are to explain how organs arise in development from the coordinated actions of their constituent cells and to determine how molecularly regulated changes in cell behavior alter the structure and function of organs during disease processes. Two major barriers stand in the way of achieving these goals: the relative inaccessibility of cellular processes in mammals and the daunting complexity of the signaling environment inside an intact organ in vivo. To overcome these barriers, we have developed a suite of tissue isolation, three dimensional (3D) culture, genetic manipulation, nanobiomaterials, imaging, and molecular analysis techniques to enable the real-time study of cell biology within intact tissues in physiologically relevant 3D environments. This manuscript introduces the rationale for 3D culture, reviews challenges to optical imaging in these cultures, and identifies current limitations in the analysis of complex experimental designs that could be overcome with improved imaging, imaging analysis, and automated classification of the results of experimental interventions.
Silva, Samuel; Teixeira, António
In recent years, real-time Magnetic Resonance Imaging (RT-MRI) has been used to acquire vocal tract data to support articulatory studies. The large amount of images resulting from these acquisitions needs to be processed and the resulting data analysed to extract articulatory features. This analysis is often performed by linguists and phoneticists and requires not only tools providing a high level exploration of the data, to gather insight over the different aspects of speech, but also a set of features to compare different vocal tract configurations in static and dynamic scenarios. In order to make the data available in a faster and systematic fashion, without the continuous direct involvement of image processing specialists, a framework is being developed to bridge the gap between the more technical aspects of raw data and the higher level analysis required by speech researchers. In its current state it already includes segmentation of the vocal tract, allows users to explore the different aspects of the acquired data using coordinated views, and provides support for vocal tract configuration comparison. Beyond the traditional method of visual comparison of vocal tract profiles, a quantitative method is proposed, considering relevant anatomical features, supported by an abstract representation of the data both for static and dynamic analysis.
Davis, Russell O...; Katz, David F.
A new model of sperm swimming kinematics, which uses signal processing methods and multivariate statistical techniques to identify individual cell-motion parameters and unique cell populations, is presented. Swimming paths of individual cells are obtained using real-time, video-edge digitization. Raw paths are adaptively filtered to identify average paths, and measurements of space-time oscillations about average paths are made. Time-dependent frequency information is extracted from spatial variations about average paths using harmonic analysis. Raw-path and average-path measures such as curvature, curve length, and straight-line length, and measures of oscillations about average paths such as time-dependent amplitude and frequency variations, are used in a multivariate, cluster analysis to identify unique cell populations. The entire process, including digitization of sperm video images, is computer-automated. Preliminary results indicate that this method of tracking, digitization, and kinematic analysis accurately identifies unique cell subpopulations, including: the relative numbers of cells in each subpopulation, how subpopulations differ, and the extent and significance of such differences. With appropriate work, this approach may be useful for clinical discrimination between normal and abnormal semen specimens.
Taisne, B.; Brenguier, F.; Nercessian, A.; Beauducel, F.; Smith, P. J.
Seismic amplitude ratio analysis (SARA) has been used successfully to track the sub-surface migration of magma prior to an eruption at Piton de la Fournaise volcano, La Réunion. The methodology is based on the temporal analysis of the seismic amplitude ratio between different pairs of stations, along with a model of seismic wave attenuation. This method has already highlighted the complexity of magma migration in the shallower part of the volcanic edifice during a seismic crisis using continuous records. We will see that this method can also be applied to the localization of individual earthquakes triggered by monitoring systems, prior to human intervention such as phase picking. As examples, the analysis is performed on two kinds of seismic events observed at Soufrière Hills Volcano, Montserrat during the last 15 years, namely: Hybrids events and Volcano-Tectonic earthquakes. Finally, we present the implementation of a fully automatic SARA method for monitoring of Piton de la Fournaise volcano using continuous data in real-time.
Courboulay, Vincent; Perreira Da Silva, Matthieu
Providing real time analysis of the huge amount of data generated by computer vision algorithms in interactive applications is still an open problem. It promises great advances across a wide variety of fields. When using dynamics scene analysis algorithms for computer vision, a trade-off must be found between the quality of the results expected, and the amount of computer resources allocated for each task. It is usually a design time decision, implemented through the choice of pre-defined algorithms and parameters. However, this way of doing limits the generality of the system. Using an adaptive vision system provides a more flexible solution as its analysis strategy can be changed according to the new information available. As a consequence, such a system requires some kind of guiding mechanism to explore the scene faster and more efficiently. We propose a visual attention system that it adapts its processing according to the interest (or salience) of each element of the dynamic scene. Somewhere in between hierarchical salience based and competitive distributed, we propose a hierarchical yet competitive and non salience based model. Our original approach allows the generation of attentional focus points without the need of neither saliency map nor explicit inhibition of return mechanism. This new realtime computational model is based on a preys / predators system. The use of this kind of dynamical system is justified by an adjustable trade-off between nondeterministic attentional behavior and properties of stability, reproducibility and reactiveness.
Enteric viruses often contaminate water sources causing frequent outbreaks of gastroenteritis. Reverse transcription-polymerase chain reaction (RT-PCR) assays are commonly used for detection of human enteric viruses in environmental and drinking water samples. RT-PCR provides ...
Calcagno, Anna Maria; Chewning, Katherine J; Wu, Chung-Pu; Ambudkar, Suresh V
Background Although relative quantification of real-time RT-PCR data can provide valuable information, one limitation remains the selection of an appropriate reference gene. No one gene has emerged as a universal reference gene and much debate surrounds some of the more commonly used reference genes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). At this time, no gene encoding for a plasma membrane protein serves as a reference gene, and relative quantification of plasma membrane proteins is performed with genes encoding soluble proteins, which differ greatly in quantity and in targeting and trafficking from plasma membrane proteins. In this work, our aim was to identify a housekeeping gene, ideally one that codes for a plasma membrane protein, whose expression remains the same regardless of drug treatment and across a wide range of tissues to be used for relative quantification of real-time RT-PCR data for ATP binding cassette (ABC) plasma membrane transporters. Results In studies evaluating the expression levels of two commonly used reference genes coding for soluble proteins and two genes coding for membrane proteins, one plasma membrane protein, plasma membrane calcium-ATPase 4 (PMCA4), was comparable to the two reference genes already in use. In addition, PMCA4 expression shows little variation across eight drug-treated cell lines and was found to be superior to GAPDH and HPRT1, commonly used reference genes. Finally, we show PMCA4 used as a reference gene for normalizing ABC transporter expression in a drug-resistant lung carcinoma cell line. Conclusion We have found that PMCA4 is a good housekeeping gene for normalization of gene expression for polytopic membrane proteins including transporters and receptors. PMID:16978418
Ma, Xinyue; Zhang, Hang; Zhao, Xiaojie; Yao, Li; Long, Zhiying
Real-time functional magnetic resonance imaging (fMRI) is a type of neurofeedback tool that enables researchers to train individuals to actively gain control over their brain activation. Independent component analysis (ICA) based on data-driven model is seldom used in real-time fMRI studies due to large time cost, though it has been very popular to offline analysis of fMRI data. The feasibility of performing real-time ICA (rtICA) processing has been demonstrated by previous study. However, rtICA was only applied to analyze single-slice data rather than full-brain data. In order to improve the performance of rtICA, we proposed semi-blind real-time ICA (sb-rtICA) for our real-time fMRI system by adding regularization of certain estimated time courses using the experiment paradigm information to rtICA. Both simulated and real-time fMRI experiment were conducted to compare the two approaches. Results from simulated and real full-brain fMRI data demonstrate that sb-rtICA outperforms rtICA in robustness, computational time and spatial detection power. Moreover, in contrast to rtICA, the first component estimated by sb-rtICA tends to be the target component in more sliding windows.
Background The electroencephalography (EEG) signals are known to involve the firings of neurons in the brain. The P300 wave is a high potential caused by an event-related stimulus. The detection of P300s included in the measured EEG signals is widely investigated. The difficulties in detecting them are that they are mixed with other signals generated over a large brain area and their amplitudes are very small due to the distance and resistivity differences in their transmittance. Methods A novel real-time feature extraction method for detecting P300 waves by combining an adaptive nonlinear principal component analysis (ANPCA) and a multilayer neural network is proposed. The measured EEG signals are first filtered using a sixth-order band-pass filter with cut-off frequencies of 1 Hz and 12 Hz. The proposed ANPCA scheme consists of four steps: pre-separation, whitening, separation, and estimation. In the experiment, four different inter-stimulus intervals (ISIs) are utilized: 325 ms, 350 ms, 375 ms, and 400 ms. Results The developed multi-stage principal component analysis method applied at the pre-separation step has reduced the external noises and artifacts significantly. The introduced adaptive law in the whitening step has made the subsequent algorithm in the separation step to converge fast. The separation performance index has varied from -20 dB to -33 dB due to randomness of source signals. The robustness of the ANPCA against background noises has been evaluated by comparing the separation performance indices of the ANPCA with four algorithms (NPCA, NSS-JD, JADE, and SOBI), in which the ANPCA algorithm demonstrated the shortest iteration time with performance index about 0.03. Upon this, it is asserted that the ANPCA algorithm successfully separates mixed source signals. Conclusions The independent components produced from the observed data using the proposed method illustrated that the extracted signals were clearly the P300 components elicited by task
Vincart, Benoit; De Mendonça, Ricardo; Rottiers, Sylvianne; Vermeulen, Françoise; Struelens, Marc J; Denis, Olivier
A novel real-time PCR (RT-PCR) assay was developed for detection of Bordetella pertussis in respiratory specimens by targeting the pertactin gene. In vitro evaluation with reference strains and quality control samples showed analytical sensitivity equivalent to and specificity superior to those of PCR assays which target the IS481 element. The pertactin-based RT-PCR assay offers better discrimination between B. pertussis and other Bordetella species than previously described assays.
Chalak, Lina F.; Tian, Fenghua; Adams-Huet, Beverley; Vasil, Diana; Laptook, Abbot; Tarumi, Takashi; Zhang, Rong
Birth asphyxia constitutes a major global public health burden for millions of infants, despite hypothermia therapy. There is a critical need for real time surrogate markers of therapeutic success, to aid in patient selection and/or modification of interventions in neonatal encephalopathy (NE). This is a proof of concept study aiming to quantify neurovascular coupling (NVC) using wavelet analysis of the dynamic coherence between amplitude-integrated electroencephalography (aEEG) and near-infrared spectroscopy in NE. NVC coupling is assessed by a wavelet metric estimation of percent time of coherence between NIRS SctO2 and aEEG for 78 hours after birth. An abnormal outcome was predefined by a Bayley III score <85 by 18–24 m. We observed high coherence, intact NVC, between the oscillations of SctO2 and aEEG in the frequency range of 0.00025–0.001 Hz in the non-encephalopathic newborns. NVC coherence was significantly decreased in encephalopathic newborns who were cooled vs. non-encephalopathic controls (median IQR 3[2–9] vs.36 [33–39]; p < 0.01), and was significantly lower in those with abnormal 24 months outcomes relative to those with normal outcomes (median IQR 2[1–3] vs 28[19–26], p = 0.04). Wavelet coherence analysis of neurovascular coupling in NE may identify infants at risk for abnormal outcomes. PMID:28393884
von Laszeski, G.; Insley, J. A.; Foster, I.; Bresnahan, J.; Kesselman, C.; Su, M.; Thiebaux, M.; Rivers, M. L.; Wang, S.; Tieman, B., McNulty, I.
A new generation of specialized scientific instruments called synchrotron light sources allow the imaging of materials at very fine scales. However, in contrast to a traditional microscope, interactive use has not previously been possible because of the large amounts of data generated and the considerable computation required translating this data into a useful image. The authors describe a new software architecture that uses high-speed networks and supercomputers to enable quasi-real-time and hence interactive analysis of synchrotron light source data. This architecture uses technologies provided by the Globus computational grid toolkit to allow dynamic creation of a reconstruction pipeline that transfers data from a synchrotron source beamline to a preprocessing station, next to a parallel reconstruction system, and then to multiple visualization stations. Collaborative analysis tools allow multiple users to control data visualization. As a result, local and remote scientists can see and discuss preliminary results just minutes after data collection starts. The implications for more efficient use of this scarce resource and for more effective science appear tremendous.
Rao, Xiayu; Lai, Dejian; Huang, Xuelin
Quantitative real-time polymerase chain reaction (qPCR) is a sensitive gene quantification method that has been extensively used in biological and biomedical fields. The currently used methods for PCR data analysis, including the threshold cycle method and linear and nonlinear model-fitting methods, all require subtracting background fluorescence. However, the removal of background fluorescence can hardly be accurate and therefore can distort results. We propose a new method, the taking-difference linear regression method, to overcome this limitation. Briefly, for each two consecutive PCR cycles, we subtract the fluorescence in the former cycle from that in the latter cycle, transforming the n cycle raw data into n-1 cycle data. Then, linear regression is applied to the natural logarithm of the transformed data. Finally, PCR amplification efficiencies and the initial DNA molecular numbers are calculated for each reaction. This taking-difference method avoids the error in subtracting an unknown background, and thus it is more accurate and reliable. This method is easy to perform, and this strategy can be extended to all current methods for PCR data analysis.
Lesiak, Ashton D; Musah, Rabi A; Cody, Robert B; Domin, Marek A; Dane, A John; Shepard, Jason R E
Rapid and versatile direct analysis in real time mass spectrometry (DART-MS) methods were developed for detection and characterization of synthetic cathinone designer drugs, also known as "bath salts". The speed and efficiency associated with DART-MS testing of such highly unpredictable samples demonstrate the technique as an attractive alternative to conventional GC-MS and LC-MS methods. A series of isobaric and closely related synthetic cathinones, alone and in mixtures, were differentiated using high mass accuracy and in-source collision induced dissociation (CID). Crime laboratories have observed a dramatic rise in the use of these substances, which has caused sample testing backlogs, particularly since the myriad of structurally related compounds are challenging to efficiently differentiate. This challenge is compounded by the perpetual emergence of new structural variants as soon as older generation derivatives become scheduled. Because of the numerous chemical substances that fall into these categories, along with the varying composition and complexity of mixtures of these drugs, DART-MS CID has the potential to dramatically streamline sample analysis, minimize the number of sample preparation steps, and enable rapid characterization of emerging structural analogs.
Prasad, Brinda; Du, Shan; Badawy, Wael; Kaler, Karan V. I. S.
There is an increasing demand from biosciences to develop new and efficient techniques to assist in the preparation and analysis of biological samples such as cells in suspension. A dielectrophoresis (DEP)-based characterization and measurement technique on biological cells opens up a broader perspective for early diagnosis of diseases. An efficient real-time multiple-cell tracking platform coupled with DEP to capture and quantify the dynamics of cell motio