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Sample records for multiplex rt-pcr assay

  1. An improved multiplex IC-RT-PCR assay distinguishes nine strains of Potato virus Y

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A multiplex RT-PCR assay was previously developed to identify a group of PVY isolates with unusual recombinant structures, e.g. PVYNTN-NW and SYR-III, and to differentiate them from other PVY strains. In the present study, the efficiency of this multiplex RT-PCR assay was validated and extended cons...

  2. Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth disease virus and look-alike disease viruses

    SciTech Connect

    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

    2007-07-26

    A high-throughput multiplexed assay was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspect cases of foot-and-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.

  3. Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth and look-alike disease viruses

    SciTech Connect

    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

    2007-09-18

    A high-throughput multiplexed assay (Multiplex Version 1.0) was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from 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.

  4. A multiplex RT-PCR assay for detection and differentiation of avian H3, H5, and H9 subtype influenza viruses and Newcastle disease viruses.

    PubMed

    Tang, Qingdong; Wang, Jinliang; Bao, Jingnan; Sun, Honglei; Sun, Yipeng; Liu, Jinhua; Pu, Juan

    2012-05-01

    Avian influenza viruses (AIVs) and Newcastle disease viruses (NDVs) co-circulate in the poultry population in China. These viruses cause repeated disease outbreaks that exhibit similar clinical symptoms and epidemiological patterns. H5 and H9 influenza viruses are the major pathogens infecting poultry stocks. Recently, H3 AIV (one of the main subtypes in waterfowl) has become endemic in chickens. A multiplex reverse-transcriptase polymerase chain reaction (mRT-PCR) assay was designed for simultaneous detection and differentiation of avian H3, H5, H9 subtype AIVs and NDVs. Four primer sets were evaluated, three of which specifically targeted the hemagglutinin genes of H3, H5 and H9 AIVs, while the other targeted the NDV fusion gene. The sensitivity and specificity of the mRT-PCR assay was determined. The assay detected the major clades or genotypes of all of the reference AIVs and NDVs currently circulating in China. In addition, the mRT-PCR results obtained from screening 380 clinical swabs and 12 experimental tracheal samples were consistent with those obtained using conventional virus isolation methods. The mRT-PCR assay was established successfully for the detection and differentiation of avian H3, H5, and H9 subtype AIVs and NDVs. The method should, therefore, provide a valuable diagnostic tool for these infections. PMID:22387341

  5. The current incidence of viral disease in korean sweet potatoes and development of multiplex rt-PCR assays for simultaneous detection of eight sweet potato viruses.

    PubMed

    Kwak, Hae-Ryun; Kim, Mi-Kyeong; Shin, Jun-Chul; Lee, Ye-Ji; Seo, Jang-Kyun; Lee, Hyeong-Un; Jung, Mi-Nam; Kim, Sun-Hyung; Choi, Hong-Soo

    2014-12-01

    Sweet potato is grown extensively from tropical to temperate regions and is an important food crop worldwide. In this study, we established detection methods for 17 major sweet potato viruses using single and multiplex RT-PCR assays. To investigate the current incidence of viral diseases, we collected 154 samples of various sweet potato cultivars showing virus-like symptoms from 40 fields in 10 Korean regions, and analyzed them by RT-PCR using specific primers for each of the 17 viruses. Of the 17 possible viruses, we detected eight in our samples. Sweet potato feathery mottle virus (SPFMV) and sweet potato virus C (SPVC) were most commonly detected, infecting approximately 87% and 85% of samples, respectively. Furthermore, Sweet potato symptomless virus 1 (SPSMV-1), Sweet potato virus G (SPVG), Sweet potato leaf curl virus (SPLCV), Sweet potato virus 2 ( SPV2), Sweet potato chlorotic fleck virus (SPCFV), and Sweet potato latent virus (SPLV) were detected in 67%, 58%, 47%, 41%, 31%, and 20% of samples, respectively. This study presents the first documented occurrence of four viruses (SPVC, SPV2, SPCFV, and SPSMV-1) in Korea. Based on the results of our survey, we developed multiplex RT-PCR assays for simple and simultaneous detection of the eight sweet potato viruses we recorded. PMID:25506306

  6. The Current Incidence of Viral Disease in Korean Sweet Potatoes and Development of Multiplex RT-PCR Assays for Simultaneous Detection of Eight Sweet Potato Viruses

    PubMed Central

    Kwak, Hae-Ryun; Kim, Mi-Kyeong; Shin, Jun-Chul; Lee, Ye-Ji; Seo, Jang-Kyun; Lee, Hyeong-Un; Jung, Mi-Nam; Kim, Sun-Hyung; Choi, Hong-Soo

    2014-01-01

    Sweet potato is grown extensively from tropical to temperate regions and is an important food crop worldwide. In this study, we established detection methods for 17 major sweet potato viruses using single and multiplex RT-PCR assays. To investigate the current incidence of viral diseases, we collected 154 samples of various sweet potato cultivars showing virus-like symptoms from 40 fields in 10 Korean regions, and analyzed them by RT-PCR using specific primers for each of the 17 viruses. Of the 17 possible viruses, we detected eight in our samples. Sweet potato feathery mottle virus (SPFMV) and sweet potato virus C (SPVC) were most commonly detected, infecting approximately 87% and 85% of samples, respectively. Furthermore, Sweet potato symptomless virus 1 (SPSMV-1), Sweet potato virus G (SPVG), Sweet potato leaf curl virus (SPLCV), Sweet potato virus 2 ( SPV2), Sweet potato chlorotic fleck virus (SPCFV), and Sweet potato latent virus (SPLV) were detected in 67%, 58%, 47%, 41%, 31%, and 20% of samples, respectively. This study presents the first documented occurrence of four viruses (SPVC, SPV2, SPCFV, and SPSMV-1) in Korea. Based on the results of our survey, we developed multiplex RT-PCR assays for simple and simultaneous detection of the eight sweet potato viruses we recorded. PMID:25506306

  7. Validation of an internally controlled one-step real-time multiplex RT-PCR assay for the detection and quantitation of dengue virus RNA in plasma.

    PubMed

    Hue, Kien Duong Thi; Tuan, Trung Vu; Thi, Hanh Tien Nguyen; Bich, Chau Tran Nguyen; Anh, Huy Huynh Le; Wills, Bridget A; Simmons, Cameron P

    2011-11-01

    Dengue is mosquito-borne virus infection that annually causes ~50 million clinically apparent cases worldwide. An internally controlled one-step real-time multiplex RT-PCR assay was developed for detection and quantitation of DENV RNA in plasma sample by using specific primers and fluorogenic TaqMan probes. All primers and probes targeted sequences near the 3' end of the NS5 gene. The method comprised two multiplex assays and was validated for sensitivity, specificity, linearity, reproducibility and precision. An internal control template was spiked into each clinical specimen to provide quality assurance for each experimental step. The assay allowed for detection of between 0.5 and 3 infectious particles per mL, is rapid and has been operationally characterized in 287 Vietnamese dengue patients from two therapeutic intervention trials at the Hospital for Tropical Diseases, Ho Chi Minh City, Viet Nam. PMID:21843553

  8. Multiplex real-time RT-PCR assay for bovine viral diarrhea virus type 1, type 2 and HoBi-like pestivirus.

    PubMed

    Mari, Viviana; Losurdo, Michele; Lucente, Maria Stella; Lorusso, Eleonora; Elia, Gabriella; Martella, Vito; Patruno, Giovanni; Buonavoglia, Domenico; Decaro, Nicola

    2016-03-01

    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. PMID:26709100

  9. Simultaneous Detection of CDC Category “A” DNA and RNA Bioterrorism Agents by Use of Multiplex PCR & RT-PCR Enzyme Hybridization Assays

    PubMed Central

    He, Jie; Kraft, Andrea J.; Fan, Jiang; Van Dyke, Meredith; Wang, Lihua; Bose, Michael E.; Khanna, Marilyn; Metallo, Jacob A.; Henrickson, Kelly J.

    2009-01-01

    Assays to simultaneously detect multiple potential agents of bioterrorism are limited. Two multiplex PCR and RT-PCR enzyme hybridization assays (mPCR-EHA, mRT-PCR-EHA) were developed to simultaneously detect many of the CDC category “A” bioterrorism agents. The “Bio T” DNA assay was developed to detect: Variola major (VM), Bacillus anthracis (BA), Yersinia pestis (YP), Francisella tularensis (FT) and Varicella zoster virus (VZV). The “Bio T” RNA assay (mRT-PCR-EHA) was developed to detect: Ebola virus (Ebola), Lassa fever virus (Lassa), Rift Valley fever (RVF), Hantavirus Sin Nombre species (HSN) and dengue virus (serotypes 1–4). Sensitivity and specificity of the 2 assays were tested by using genomic DNA, recombinant plasmid positive controls, RNA transcripts controls, surrogate (spiked) clinical samples and common respiratory pathogens. The analytical sensitivity (limit of detection (LOD)) of the DNA asssay for genomic DNA was 1×100∼1×102 copies/mL for BA, FT and YP. The LOD for VZV whole organism was 1×10−2 TCID50/mL. The LOD for recombinant controls ranged from 1×102∼1×103copies/mL for BA, FT, YP and VM. The RNA assay demonstrated LOD for RNA transcript controls of 1×104∼1×106 copies/mL without extraction and 1×105∼1×106 copies/mL with extraction for Ebola, RVF, Lassa and HSN. The LOD for dengue whole organisms was ∼1×10−4 dilution for dengue 1 and 2, 1×104 LD50/mL and 1×102 LD50/mL for dengue 3 and 4. The LOD without extraction for recombinant plasmid DNA controls was ∼1×103 copies/mL (1.5 input copies/reaction) for Ebola, RVF, Lassa and HSN. No cross-reactivity of primers and probes used in both assays was detected with common respiratory pathogens or between targeted analytes. Clinical sensitivity was estimated using 264 surrogate clinical samples tested with the BioT DNA assay and 549 samples tested with the BioT RNA assay. The clinical specificity is 99.6% and 99.8% for BioT DNA assay and BioT RNA assay

  10. Development and Validation of a Multiplex, Real-Time RT PCR Assay for the Simultaneous Detection of Classical and African Swine Fever Viruses

    PubMed Central

    Haines, Felicity J.; Hofmann, Martin A.; King, Donald P.; Drew, Trevor W.; Crooke, Helen R.

    2013-01-01

    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

  11. Development of a multiplex RT-PCR assay for the identification of recombination types at different genomic regions of vaccine-derived polioviruses.

    PubMed

    Dimitriou, T G; Kyriakopoulou, Z; Tsakogiannis, D; Fikatas, A; Gartzonika, C; Levidiotou-Stefanou, S; Markoulatos, P

    2016-08-01

    Polioviruses (PVs) are the causal agents of acute paralytic poliomyelitis. Since the 1960s, poliomyelitis has been effectively controlled by the use of two vaccines containing all three serotypes of PVs, the inactivated poliovirus vaccine and the live attenuated oral poliovirus vaccine (OPV). Despite the success of OPV in polio eradication programme, a significant disadvantage was revealed: the emergence of vaccine-associated paralytic poliomyelitis (VAPP). VAPP is the result of accumulated mutations and putative recombination events located at the genome of attenuated vaccine Sabin strains. In the present study, ten Sabin isolates derived from OPV vaccinees and environmental samples were studied in order to identify recombination types located from VP1 to 3D genomic regions of virus genome. The experimental procedure that was followed was virus RNA extraction, reverse transcription to convert the virus genome into cDNA, PCR and multiplex-PCR using specific designed primers able to localize and identify each recombination following agarose gel electrophoresis. This multiplex RT-PCR assay allows for the immediate detection and identification of multiple recombination types located at the viral genome of OPV derivatives. After the eradication of wild PVs, the remaining sources of poliovirus infection worldwide would be the OPV derivatives. As a consequence, the immediate detection and molecular characterization of recombinant derivatives are important to avoid epidemics due to the circulation of neurovirulent viral strains. PMID:27098645

  12. Development and Characterization of A Multiplexed RT-PCR Species Specific Assay for Bovine and one for Porcine Foot-and-Mouth Disease Virus Rule-Out

    SciTech Connect

    Smith, S M; Danganan, L; Tammero, L; Vitalis, B; Lenhoff, R; Naraghi-arani, P; Hindson, B

    2007-08-06

    Lawrence Livermore National Laboratory (LLNL), in collaboration with the Department of Homeland Security (DHS) and the United States Department of Agriculture (USDA), Animal and Plant Health Inspection Services (APHIS) has developed candidate multiplexed assays that may potentially be used within the National Animal Health Laboratory Network (NAHLN), the National Veterinary Services Laboratory (Ames, Iowa) and the Plum Island Animal Disease Center (PIADC). This effort has the ability to improve our nation's capability to discriminate between foreign animal diseases and those that are endemic using a single assay, thereby increasing our ability to protect food and agricultural resources with a diagnostic test which could enhance the nation's capabilities for early detection of a foreign animal disease. In FY2005 with funding from the DHS, LLNL developed the first version (Version 1.0) of a multiplexed (MUX) nucleic-acid-based RT-PCR assay that included signatures for foot-and-mouth disease virus (FMDV) detection with rule-out tests for two other foreign animal diseases (FADs) of swine, Vesicular Exanthema of Swine (VESV) and Swine Vesicular Disease Virus (SVDV), and four other domestic viral diseases Bovine Viral Diarrhea Virus (BVDV), Bovine Herpes Virus 1 (BHV-1), Bluetongue virus (BTV) and Parapox virus complex (which includes Bovine Papular Stomatitis Virus [BPSV], Orf of sheep, and Pseudocowpox). In FY06, LLNL has developed Bovine and Porcine species-specific panel which included existing signatures from Version 1.0 panel as well as new signatures. The MUX RT-PCR porcine assay for detection of FMDV includes the FADs, VESV and SVD in addition to vesicular stomatitis virus (VSV) and porcine reproductive and respiratory syndrome (PRRS). LLNL has also developed a MUX RT-PCR bovine assay for detection of FMDV with rule out tests for the two bovine FADs malignant catarrhal fever (MCF), rinderpest virus (RPV) and the domestic diseases vesicular stomatitis virus (VSV

  13. Multiplex RT-PCR detection of H3N2 influenza A virus in dogs.

    PubMed

    Lee, EunJung; Kim, Eun-Ju; Kim, Bo-Hye; Song, Jae-Young; Cho, In-Soo; Shin, Yeun-Kyung

    2016-02-01

    A multiplex RT-PCR (mRT-PCR) assay to detect H3N2 CIV genomic segments was developed as a rapid and cost-effective method. Its performance was evaluated with forty-six influenza A viruses from different hosts using three primer sets which amplify four segments of H3N2 CIV simultaneously. The mRT-PCR has been successful in detecting the viral segments, indicating that it can improve the speed of diagnosis for H3N2 CIV and its reassortants. PMID:26738688

  14. DEVELOPMENT OF MULTIPLEX REAL-TIME RT-PCR AS A DIAGNOSTIC TOOL FOR AVIAN INFLUENZA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A multiplex real-time RT-PCR (RRT-PCR) assay for the simultaneous detection of the H5 and H7 hemagglutinin (HA) subtypes was developed with hydrolysis type probes labeled with the FAM (H5 probe) and ROX (H7 probe) dyes. The sensitivity of the H5-H7 subtyping assay was determined, using in vitro tran...

  15. One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples.

    PubMed

    Gautam, Rashi; Mijatovic-Rustempasic, Slavica; Esona, Mathew D; Tam, Ka Ian; Quaye, Osbourne; Bowen, Michael D

    2016-01-01

    Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8-100% sensitivity, 99.7-100% specificity, 85-95% efficiency and a limit of detection of 4-60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81-92% efficiency and limit of detection of 150-600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98

  16. One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples

    PubMed Central

    Mijatovic-Rustempasic, Slavica; Esona, Mathew D.; Tam, Ka Ian; Quaye, Osbourne; Bowen, Michael D.

    2016-01-01

    Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8

  17. Simultaneous detection of influenza viruses A, B, and swine origin influenza A using multiplex one-step real-time RT-PCR assay.

    PubMed

    Monavari, S H R; Mollaie, H R; Fazlalipour, M

    2014-01-01

    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. PMID:24142356

  18. Detection of a broad range of class I and II Newcastle disease viruses using a multiplex real time RT-PCR assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Prompt detection of virulent strains of Newcastle disease virus (vNDV) using real time RT-PCR (rRT-PCR) is challenging due to the broad genetic variability across two clades comprising 18 recognized genotypes. A large proportion of class I low virulence Newcastle disease viruses (loNDV) recently id...

  19. Real-time RT-PCR Assay for Detection and Differentiation of Citrus Tristeza Virus Isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Multiplex one step real time RT-PCR assays using TaqMan probes were developed for detection and strain differentiation of Citrus tristeza virus (CTV). For broad spectrum CTV detection, a TaqMan primer and Cy5-labeled probe were designed using CP gene sequences. An internal control was developed us...

  20. A multiplex RT-PCR for the detection of astrovirus, rotavirus, and reovirus in turkeys.

    PubMed

    Jindal, Naresh; Chander, Yogesh; Patnayak, Devi P; Mor, Sunil K; Ziegler, Andre F; Goyal, Sagar M

    2012-09-01

    This study was undertaken to develop and validate a multiplex reverse transcription-polymerase chain reaction (mRT-PCR) for simultaneous detection of avian rotavirus, turkey astrovirus-2 (TAstV-2), and avian reovirus. Primers targeting the conserved regions of NSP4 gene of avian rotavirus, polymerase gene of TAstV-2, and S4 gene of avian reovirus were used. The position of bands at 630, 802, and 1120 base pairs on agarose gel confirmed the presence of rotavirus, TAstV-2, and reovirus, respectively. This mRT-PCR was found to be specific as no amplification was observed with avian influenza virus, Newcastle disease virus, turkey coronavirus, avian metapneumovirus, and intestinal contents of uninfected turkey poults. Intestinal contents of poults from flocks suspected of exhibiting "poult enteritis syndrome" were pooled and tested. Of the 120 pooled samples tested, 70% were positive for TAstV-2, 45% for avian rotavirus, and 18% for avian reovirus. These three viruses were detected alone or in different combinations. Of the samples tested, 20% were negative for these three viruses, 38% were positive for a single virus (TAstV or rotavirus or reovirus), and 42% were positive for two or three viruses. This single-tube mRT-PCR assay has the potential to serve as a rapid diagnostic method for the simultaneous detection of the three enteric viruses in turkeys. PMID:23050480

  1. Simultaneous detection of four causal agents of tobacco bushy top disease by a multiplex one-step RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tobacco bushy top disease is a complex disease caused by mixed infection of Tobacco bushy top virus (TBTV), Tobacco vein distorting virus (TVDV), satellite RNA of TBTV (Sat-TBTV) and Tobacco vein distorting virus associate RNA (TVDVaRNA). A one-tube multiplex reverse transcription-PCR (RT-PCR) assay...

  2. Development and Characterization of a Multiplexed RT-PCR Species Specific Assay for Bovine and one for Porcine Foot-and-Mouth Disease Virus Rule-Out Supplemental Materials

    SciTech Connect

    Smith, S; Danganan, L; Tammero, L; Lenhoff, R; Naraghi-arani, P; Hindson, B

    2007-08-06

    which are of two bovine types bovine papular stomatitis virus (BPSV) and psuedocowpox (PCP). This document provides details of signature generation, evaluation, and testing, as well as the specific methods and materials used. A condensed summary of the development, testing and performance of the multiplexed assay panel was presented in a 126 page separate document, entitled 'Development and Characterization of A Multiplexed RT-PCR Species Specific Assay for Bovine and one for Porcine Foot-and-Mouth Disease Virus Rule-Out'. This supplemental document provides additional details of large amount of data collected for signature generation, evaluation, and testing, as well as the specific methods and materials used for all steps in the assay development and utilization processes. In contrast to last years effort, the development of the bovine and porcine panels is pending additional work to complete analytical characterization of FMDV, VESV, VSV, SVD, RPV and MCF. The signature screening process and final panel composition impacts this effort. The unique challenge presented this year was having strict predecessor limitations in completing characterization, where efforts at LLNL must preceed efforts at PIADC, such challenges were alleviated in the 2006 reporting by having characterization data from the interlaboratory comparison and at Plum Island under AgDDAP project. We will present an addendum at a later date with additional data on the characterization of the porcine and bovine multiplex assays when that data is available.

  3. A multiple RT-PCR assay for simultaneous detection and differentiation of latent viruses and apscarviroids in apple trees.

    PubMed

    Hao, Lu; Xie, Jipeng; Chen, Shanyi; Wang, Shaojie; Gong, Zhuoqun; Ling, Kai-Shu; Guo, Liyun; Fan, Zaifeng; Zhou, Tao

    2016-08-01

    Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), and Apple stem pitting virus (ASPV) are three latent viruses frequently occurring in apple trees worldwide. In field orchards, these viruses are frequently found in a mixed infection with viroids in the genus Apscarviroid, including Apple scar skin viroid, and Apple dimple fruit viroid. Together these viruses and viroids could cause serious damage to apple fruit production worldwide. Rapid and efficient detection methods are pivotal to identify and select the virus-free propagation material for healthy apple orchard management. In this study a multiplex Reverse Transcription-PCR (RT-PCR) was developed and optimized for simultaneous detection and differentiation of the three latent viruses and apscarviroids. With newly designed specific primers for ACLSV, ASGV, APSV, and EF-1α (as an internal control), and a pair of degenerate primers for apscarviroids, optimized parameters for multiplex RT-PCR were determined. The resulting PCR products from each target virus and viroid could be easily identified because their product sizes differ by at least a 100bp. The multiplex RT-PCR method is expected to detect different variants of the viruses as the test results showed that a variety of isolates from different regions in China gave positive results. To the best of our knowledge, this multiplex RT-PCR assay is the first to simultaneously detect multiple viruses and viroids infecting apple trees in a single reaction tube. This assay, therefore, offers a useful tool for routine certification and quarantine programs. PMID:27054889

  4. Multiplex real-time RT-PCR for the simultaneous detection and quantification of GI, GII and GIV noroviruses.

    PubMed

    Farkas, Tibor; Singh, Amy; Le Guyader, Françoise S; La Rosa, Giuseppina; Saif, Linda; McNeal, Monica

    2015-10-01

    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. PMID:26248055

  5. Real-time RT-PCR high-resolution melting curve analysis and multiplex RT-PCR to detect and differentiate grapevine leafroll-associated virus 3 variant groups I, II, III and VI

    PubMed Central

    2012-01-01

    Background Grapevine leafroll-associated virus 3 (GLRaV-3) is the main contributing agent of leafroll disease worldwide. Four of the six GLRaV-3 variant groups known have been found in South Africa, but their individual contribution to leafroll disease is unknown. In order to study the pathogenesis of leafroll disease, a sensitive and accurate diagnostic assay is required that can detect different variant groups of GLRaV-3. Methods In this study, a one-step real-time RT-PCR, followed by high-resolution melting (HRM) curve analysis for the simultaneous detection and identification of GLRaV-3 variants of groups I, II, III and VI, was developed. A melting point confidence interval for each variant group was calculated to include at least 90% of all melting points observed. A multiplex RT-PCR protocol was developed to these four variant groups in order to assess the efficacy of the real-time RT-PCR HRM assay. Results A universal primer set for GLRaV-3 targeting the heat shock protein 70 homologue (Hsp70h) gene of GLRaV-3 was designed that is able to detect GLRaV-3 variant groups I, II, III and VI and differentiate between them with high-resolution melting curve analysis. The real-time RT-PCR HRM and the multiplex RT-PCR were optimized using 121 GLRaV-3 positive samples. Due to a considerable variation in melting profile observed within each GLRaV-3 group, a confidence interval of above 90% was calculated for each variant group, based on the range and distribution of melting points. The intervals of groups I and II could not be distinguished and a 95% joint confidence interval was calculated for simultaneous detection of group I and II variants. An additional primer pair targeting GLRaV-3 ORF1a was developed that can be used in a subsequent real-time RT-PCR HRM to differentiate between variants of groups I and II. Additionally, the multiplex RT-PCR successfully validated 94.64% of the infections detected with the real-time RT-PCR HRM. Conclusion The real-time RT-PCR HRM

  6. Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus

    NASA Astrophysics Data System (ADS)

    Huang, Su-Hua; Yang, Tsuey-Ching; Tsai, Ming-Hong; Tsai, I.-Shou; Lu, Huang-Chih; Chuang, Pei-Hsin; Wan, Lei; Lin, Ying-Ju; Lai, Chih-Ho; Lin, Cheng-Wen

    2008-10-01

    Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples.

  7. DEVELOPMENT OF HOMOLOGOUS VIRAL INTERNAL CONTROLS FOR USE IN RT-PCR ASSAYS OF WATERBORNE ENTERIC VIRUSES

    EPA Science Inventory

    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 ...

  8. Multiplex nested RT-PCR for detecting avian influenza virus, infectious bronchitis virus and Newcastle disease virus.

    PubMed

    Nguyen, Thanh Trung; Kwon, Hyuk-Joon; Kim, Il-Hwan; Hong, Seung-Min; Seong, Won-Jin; Jang, Jin-Wook; Kim, Jae-Hong

    2013-03-01

    In this study, multiplex nested RT-PCR (mnRT-PCR) was applied to simultaneous detect multiplex PCR with the higher sensitivity of nested PCR that is required for avian influenza, infectious bronchitis and Newcastle disease virus using two steps of amplification. For the first PCR, primers that were specific for each virus were newly designed from the nucleoprotein gene of AIV, the nucleocapsid protein gene of IBV and the fusion protein gene of NDV to amplify products of 665, 386 and 236 nucleotides, respectively. The multiplex PCR step provides mass amplification using common primers, which increased markedly the sensitivity of the test. Non-specific reactions were not observed when other viruses and bacteria were used for evaluating the mnRT-PCR. As a field application, 172 samples were tested by RT-PCR and mnRT-PCR. Among these samples, the concordance rates for mnRT-PCR and the single conventional RT-PCR showed 98.9% (kappa=0.98) and 98.8% (kappa=0.96) similarity for IBV and AIV, respectively. As a result, it is recommended the multiplex nested PCR as an effective tool for detecting and studying the molecular epidemiology of various mixed infections of one or more of these viruses in poultry. PMID:23261801

  9. Multiplex qRT-PCR for the Detection of Western Equine Encephalomyelitis, St. Louis Encephalitis, and West Nile Viral RNA in Mosquito Pools (Diptera: Culicidae).

    PubMed

    Brault, Aaron C; Fang, Ying; Reisen, William K

    2015-05-01

    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. PMID:26334826

  10. Two RT-PCR based assays to detect human metapneumovirus in nasopharyngeal aspirates.

    PubMed

    López-Huertas, María Rosa; Casas, Inmaculada; Acosta-Herrera, Belsy; García, María Luz; Coiras, María Teresa; Pérez-Breña, Pilar

    2005-10-01

    Two sensitive and specific RT-PCR assays were standardised for testing the presence of human metapneumovirus. A total of 300 nasopharyngeal aspirates collected from infants suffering from bronchiolitis since October 2000 to June 2003 and shown previously as negative to common respiratory viruses were examined. Matrix and polymerase viral genes, which show a low rate of variation, were chosen to design amplification assays to ensure that any genotype of the human metapneumovirus could be detected. A RT-PCR followed by a reverse line blotting hybridisation was developed for viral polymerase gene. For the matrix gene, after the RT-PCR assay, a subsequent nested PCR was carried out. Both assays had similar sensitivity, equivalent to 0.1 TCID50 of human metapneumovirus strain NL/1/99 which was used as the positive control. The human metapneumovirus was present in 16.6% of the specimens studied. The approaches described below are not only a robust method for rapid diagnosis of the human metapneumovirus, but also to establish an etiological surveillance tool for epidemiological studies. Based on the results obtained, human metapneumovirus infections in Madrid followed a seasonal pattern, with most of the infections occurring between February and April. PMID:15961167

  11. Universal Single-Probe RT-PCR Assay for Diagnosis of Dengue Virus Infections

    PubMed Central

    Alm, Erik; Lesko, Birgitta; Lindegren, Gunnel; Ahlm, Clas; Söderholm, Sandra; Falk, Kerstin I.; Lagerqvist, Nina

    2014-01-01

    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

  12. A real-time RT-PCR assay for rapid detection of coxsackievirus A10.

    PubMed

    Mu, C Y; Wang, A Y; Chen, C; Zhao, L; Li, Z

    2015-01-01

    Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) have been the primary causative agents of hand, foot, and mouth disease (HFMD) outbreaks in mainland China in the past. Hence, the surveillance of HFMD has mostly focused on these viruses. However, in recent years, coxsackievirus A10 (CA10) has also been associated with the increasing sporadic HFMD cases and outbreaks. Therefore, a sensitive assay for rapid detection of the CA10 RNA is necessary for disease control. Here, we have developed a specific TaqMan real-time RT-PCR assay by analyzing VP1 gene sequences of CA10 strains from different locations. The assay has been shown to be specific, sensitive, and robust through detection of other related viruses, standard curves, and clinical samples, respectively. This is the first report on development of a VP1 gene-based TaqMan real-time RT-PCR assay for rapid diagnosis of CA10 virus. PMID:26782393

  13. Detection of feline calicivirus, feline herpesvirus 1 and Chlamydia psittaci mucosal swabs by multiplex RT-PCR/PCR.

    PubMed

    Sykes, J E; Allen, J L; Studdert, V P; Browning, G F

    2001-07-26

    A single tube, multiplex reverse transcription (RT)-polymerase chain reaction (PCR)/PCR assay was developed for detection of feline herpesvirus 1 (FHV1), Chlamydia psittaci and feline calicivirus (FCV) in cats with upper respiratory tract disease (URTD), incorporating a simple, rapid extraction procedure capable of extracting both DNA and RNA. The assay was found to be as sensitive in vitro as simplex assays that have previously been shown to be as sensitive as, or more sensitive than, culture for each pathogen in experimentally infected cats. Conjunctival alone or both conjunctival and oropharyngeal swabs were collected from cats in 104 households with URTD. FHV1 was detected in 18 (17.3%) and C. psittaci was detected in 12 (11.5%) households. The prevalence of C. psittaci was not significantly different to that determined using a duplex PCR assay for C. psittaci and FHV1. The prevalence of FCV was affected by sample storage temperature. Of samples stored at -70 degrees C, 0/31 were positive for FCV but FCV was detected in 10/73 (13.7%) samples stored at 4 degrees C (P=0.006). Of the samples stored at 4 degrees C, 3/19 (15.8%) conjunctival swabs were positive for FCV and 6/32 (18.8%) oropharyngeal/conjunctival swabs were positive for FCV (P=0.79). The potential utility of restriction endonuclease analysis of RT-PCR products resulting from amplification of the hypervariable region of the capsid protein gene of FCV in field samples, without prior cultivation, was also examined. The assay may have considerable importance for diagnosis and epidemiological surveys of feline upper respiratory tract pathogens. PMID:11376956

  14. A multiplex RT-PCR for simultaneous detection and identification of five viruses and two viroids infecting chrysanthemum.

    PubMed

    Zhao, Xiting; Liu, Xingliang; Ge, Beibei; Li, Mingjun; Hong, Bo

    2015-05-01

    Pathogens causing significant economic losses in chrysanthemum include tomato aspermy virus (TAV), chrysanthemum virus B (CVB), cucumber mosaic virus (CMV), tobacco mosaic virus (TMV), potato virus Y (PVY), chrysanthemum stunt viroid (CSVd) and chrysanthemum chlorotic mottle viroid (CChMVd). A multiplex reverse transcription polymerase chain reaction (RT-PCR) method, using specific primer sets for each virus or viroid, was developed for simultaneous detection and differentiation of TAV, CVB, CMV, TMV, PVY, CChMVd, and CSVd. The RT-PCR method was validated by testing chrysanthemum samples collected from different regions of China. In this study, CVB, TAV, TMV, PVY, CSVd, CMV, and CChMVd were detected, respectively, in 24.7 %, 17.5 %, 4.4 %, 4.4 %, 2.9 %, 2.5 %, and 1.5 % of the samples tested. These results indicate that CVB and TAV (24.7 % and 17.5 %) are common, whereas CMV, TMV, CChMVd, CSVd, and PVY (all below 5 %) are less frequently encountered. This new multiplex RT-PCR method has potential to be used routinely in large-scale virus and viroid surveys. PMID:25698104

  15. Real time RT-PCR assays for detection and typing of African horse sickness virus.

    PubMed

    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

    2014-01-01

    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

  16. Development of Multiplex RT-PCR for Simultaneous Detection of Garlic Viruses and the Incidence of Garlic Viral Disease in Garlic Genetic Resources

    PubMed Central

    Nam, Moon; Lee, Yeong-Hoon; Park, Chung Youl; Lee, Min-A; Bae, Yang-Soo; Lim, Seungmo; Lee, Joong Hwan; Moon, Jae Sun; Lee, Su-Heon

    2015-01-01

    Garlic generally becomes coinfected with several types of viruses belonging to the Potyvirus, Carlavirus, and Allexivirus genera. These viruses produce characteristically similar symptoms, they cannot be easily identified by electron microscopy (EM) or immunological detection methods, and they are currently widespread around the world, thereby affecting crop yields and crop quality adversely. For the early and reliable detection of garlic viruses, virus-specific sets of primers, including species-specific and genus-specific primers were designed. To effectively detect the twelve different types of garlic viruses, primer mixtures were tested and divided into two independent sets for multiplex polymerase chain reaction (PCR). The multiplex PCR assays were able to detect specific targets up to the similar dilution series with monoplex reverse transcription (RT)-PCR. Seventy-two field samples collected by the Gyeongbuk Agricultural Technology Administration were analyzed by multiplex RT-PCR. All seventy two samples were infected with at least one virus, and the coinfection rate was 78%. We conclude that the simultaneous detection system developed in this study can effectively detect and differentiate mixed viral infections in garlic. PMID:25774116

  17. A short target real-time RT-PCR assay for detection of pestiviruses infecting cattle.

    PubMed

    La Rocca, S A; Sandvik, T

    2009-10-01

    A rapid single step real-time duplex TaqMan RT-PCR was developed for detection of bovine viral diarrhoea virus (BVDV)-1, BVDV-2 and border disease virus (BDV). Based on alignment of available and newly generated partial 5'-UTR nucleotide sequences, one forward and two reverse primers were designed, which amplify a 104bp PCR product. Two TaqMan probes labelled with different fluorochromes were designed to detect BVDV-1/BVDV-2 and BDVs, respectively. The assay was able to detect a selection of strains and isolates that represent the genetic diversity of these three viruses, with an analytical sensitivity that corresponded to 3.6, 48 and 4.8 TCID(50) of BVDV-1, BVDV-2 and BDV, respectively. With an overall cycling time of around 70 min, the assay allows rapid diagnosis and efficient use of modern thermocycling machines. Although developed principally for the diagnosis of BVD, the assay should be equally useful for diagnosis of BD in sheep. PMID:19523981

  18. Methods for detection and differentiation of existing and new crinivirus species through multiplex and degenerate primer RT-PCR.

    PubMed

    Wintermantel, William M; Hladky, Laura L

    2010-12-01

    A method was developed for rapid identification and differentiation of both known and novel crinivirus species involving both multiplex and degenerate reverse transcription-polymerase chain reaction (RT-PCR). The multiplex method can discriminate among known criniviruses infecting vegetable and small fruit crops, and rapidly identify viruses associated with disease symptoms, as well as identification of mixed crinivirus infections. Four host groups for multiplex detection of criniviruses were selected based on the types of crops where specific criniviruses would be expected to occur. Each detection group contained three to four crop-specific primers designed to the same region of the gene encoding the highly conserved RNA-dependent RNA polymerase gene (RdRp) of criniviruses for rapid, single-reaction determination of which crinivirus(es) may be infecting a plant. Degenerate reverse primers used for RT and in PCR were designed to amplify all members of each host group, and were coupled with species-specific forward primers resulting in four separate single-reaction cocktails for detection of most criniviruses sequenced to date, whether present in single or mixed virus infections. Additional viruses can be added to multiplex detection by adjustment of primer concentration for balanced detection of target viruses. In order to identify unknown putative criniviruses or those for which sequence information is not yet available, a genus-wide, universal degenerate primer set was developed. These primers also targeted the crinivirus RdRp gene, and amplify a wide range of crinivirus sequences. Both detection systems can be used with most RNA extraction methods, and with RT-PCR reagents common in most laboratories. PMID:20833203

  19. Dopamine Receptors in Human Lymphocytes: Radioligand Binding and Quantitative RT-PCR Assays

    PubMed Central

    Kirillova, Galina P.; Hrutkay, Rebecca J.; Shurin, Michael R.; Shurin, Galina V.; Tourkova, Irina L.; Vanyukov, Michael M.

    2008-01-01

    Analysis of dopamine receptors (DR) in lymphocytes of the human peripheral blood mononuclear cell (PBMC) fraction is an attractive tool for evaluation of functional properties of dopaminergic function underlying variation in complex psychological/psychopathological traits. Receptor binding assays (RBA) with selective radioligands, which are widely used in CNS studies, have not produced consistent results when applied to isolated PBMC. We tested the assay conditions that could be essential for detection of DR in human PBMC and their membrane preparations. Using [3H]SCH23390, a dopamine D1-like receptor antagonist, we demonstrated the presence of two binding sites in PBMC-derived membrane fraction. One of them is characterized by the Kd value consistent with that reported for D5 dopamine receptors in human lymphocytes, whereas the other Kd value possibly corresponds to serotonin receptor(s). Although D5 receptor binding sites in PBMC membranes could be characterized by binding assays, the low protein expression and the large volume of blood needed for membrane preparation render the binding method impracticable for individual phenotyping. In contrast, real-time RT-PCR may be used for this purpose, contingent on the relationship between DR expression in the brain and in lymphocytes. The expression of the DRD2-DRD5 genes, as detected by this method, varied widely among samples, whereas the DRD1 expression was not detected. The expression levels were comparable with those in the brain for DRD3 and DRD4, and were significantly lower for DRD2 and DRD5. PMID:18721826

  20. Duplex Real-Time RT-PCR Assays for the Detection and Typing of Epizootic Haemorrhagic Disease Virus

    PubMed Central

    Viarouge, Cyril; Breard, Emmanuel; Zientara, Stephan; Vitour, Damien; Sailleau, Corinne

    2015-01-01

    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

  1. Real-time RT-PCR assay for detection and differentiation of Citrus tristeza virus isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  2. A one step real-time RT-PCR assay for the quantitation of Wheat yellow mosaic virus (WYMV)

    PubMed Central

    2013-01-01

    Background Wheat yellow mosaic virus (WYMV) is an important pathogen in China and other countries. It is the member of the genus Bymovirus and transmitted primarily by Polymyxa graminis. The incidence of wheat infections in endemic areas has risen in recent years. Prompt and dependable identification of WYMV is a critical component of response to suspect cases. Methods In this study, a one step real-time RT-PCR, followed by standard curve analysis for the detection and identification of WYMV, was developed. Two reference genes, 18s RNA and β-actin were selected in order to adjust the veracity of the real-time RT-PCR assay. Results We developed a one-step Taqman-based real-time quantitative RT-PCR (RT-qPCR) assay targeting the conserved region of the 879 bp long full-length WYMV coat protein gene. The accuracy of normalized data was analyzed along with appropriate internal control genes: β-actin and 18s rRNA which were included in detecting of WYMV-infected wheat leaf tissues. The detectable end point sensitivity in RT-qPCR assay was reaching the minimum limit of the quantitative assay and the measurable copy numbers were about 30 at106-fold dilution of total RNA. This value was close to 104-fold more sensitive than that of indirect enzyme-linked immunosorbent assay. More positive samples were detected by RT-qPCR assay than gel-based RT-PCR when detecting the suspected samples collected from 8 regions of China. Based on presented results, RT-qPCR will provide a valuable method for the quantitative detection of WYMV. Conclusions The Taqman-based RT-qPCR assay is a faster, simpler, more sensitive and less expensive procedure for detection and quantification of WYMV than other currently used methods. PMID:23725024

  3. Pilot Evaluation of RT-PCR/Electrospray Ionization Mass Spectrometry (PLEX-ID/Flu assay) on Influenza-Positive Specimens.

    PubMed

    Cordey, Samuel; Thomas, Yves; Suter, Patricia; Kaiser, Laurent

    2012-01-01

    The PLEX-ID/Flu assay has been recently developed to enable the detection and typing of influenza viruses based on the RT-PCR/electrospray ionization mass spectrometry technology.This novel assay was evaluated for typing performance on 201 positive influenza A or B nasopharyngeal swab specimens (NPS) detected by real-time RT-PCR during the 2010-2011 season. The PLEX-ID/Flu assay detected and characterized 91.3% and 95.3% of all influenza A and B samples, respectively. All non-typeable influenza A and B specimens by the assay showed low viral loads with threshold cycle values ≥ 33. Taken together, and although our results need to be confirmed by further prospective studies, the PLEX-ID/Flu assay detected positively and gave a typing result for 93% of all NPS detected positively by real-time RT-PCR, thus suggesting a potential role for influenza virus surveillance among other techniques. PMID:22611461

  4. Pilot Evaluation of RT-PCR/Electrospray Ionization Mass Spectrometry (PLEX-ID/Flu assay) on Influenza-Positive Specimens

    PubMed Central

    Cordey, Samuel; Thomas, Yves; Suter, Patricia; Kaiser, Laurent

    2012-01-01

    The PLEX-ID/Flu assay has been recently developed to enable the detection and typing of influenza viruses based on the RT-PCR/electrospray ionization mass spectrometry technology. This novel assay was evaluated for typing performance on 201 positive influenza A or B nasopharyngeal swab specimens (NPS) detected by real-time RT-PCR during the 2010-2011 season. The PLEX-ID/Flu assay detected and characterized 91.3% and 95.3% of all influenza A and B samples, respectively. All non-typeable influenza A and B specimens by the assay showed low viral loads with threshold cycle values ≥ 33. Taken together, and although our results need to be confirmed by further prospective studies, the PLEX-ID/Flu assay detected positively and gave a typing result for 93% of all NPS detected positively by real-time RT-PCR, thus suggesting a potential role for influenza virus surveillance among other techniques. PMID:22611461

  5. Comparison of the BD Directigen Flu A+B Kit and the Abbott TestPack RSV with a multiplex RT-PCR ELISA for rapid detection of influenza viruses and respiratory syncytial virus.

    PubMed

    Gröndahl, B; Puppe, W; Weigl, J; Schmitt, H-J

    2005-10-01

    The Directigen Flu A+B enzyme immunoassay and the Abbott TestPack RSV enzyme immunoassay were each compared with a multiplex RT-PCR ELISA by testing 635 nasopharyngeal aspirates collected from children aged < 16 years who had been hospitalised with acute respiratory tract infection during the epidemic season 2002-2003. In this study, the sensitivity of the Directigen Flu A+B assay was unacceptably low (29.3% and 10.0%, respectively) for the detection of influenza A and B viruses. The sensitivity of the Abbott TestPack RSV assay (77.4%) was acceptable and in agreement with the multiplex RT-PCR ELISA. PMID:16153263

  6. Quantitation of Taura syndrome virus by real-time RT-PCR with a TaqMan assay.

    PubMed

    Tang, Kathy F J; Wang, Jun; Lightner, Donald V

    2004-01-01

    A real-time RT-PCR assay was developed using a TaqMan probe to detect and quantify Taura syndrome virus (TSV) in penaeid shrimp. A pair of RT-PCR primers, which amplify a 72 bp DNA fragment, and a TaqMan probe were selected from open reading frame 1 (ORF1) of the TSV genome. The primers and TaqMan probe used in this assay reacted with TSV isolates from Hawaii, Texas, Colombia, Mexico, Belize, Indonesia, and Thailand, but neither with RNA of healthy shrimp nor with an isolate of yellow head virus. A plasmid (pTSV-1) that contains the target TSV sequence was constructed and used to generate positive control RNA through in vitro transcription. The positive control RNA was used to demonstrate that the real-time RT-PCR assay has a detection limit of 100 copies and a log-linear range up to 10(8) copies of TSV RNA. This quantitative method was found to be highly reproducible, with low intra- and inter-assay variation. Coefficient of variation (CVs) values were 0.04-8.9 and 0.05-3.7%, respectively, for replicates within and among assays. This assay was used to quantify TSV in both acutely and chronically infected shrimp in a laboratory experiment. The quantities of TSV in the tissues of pleopods and gills were not significantly different, and there was no difference in TSV levels between the acutely and chronically infected groups. However, in the chronically infected shrimp, the quantities of TSV were one to two orders of magnitude higher in the lymphoid organ than in either gills or pleopods. This assay proved to be specific with high sensitivity, and it can be used to detect and quantify TSV in shrimp samples. PMID:14656468

  7. RSV Growth and Quantification by Microtitration and qRT-PCR Assays.

    PubMed

    Caidi, Hayat; Harcourt, Jennifer L; Haynes, Lia M

    2016-01-01

    Defective interfering viral particles have been reported as important determinants of the course of viral infection, and they can markedly temper the virulence of the infection. Here, we describe a simple method, based on limiting dilution, for the removal of defective interfering particles from RSV. This method results in a high-titer viral preparation from both HEp-2 and Vero cell lines. We evaluated two concentrations of sucrose to stabilize the virus preparation, and demonstrate that RSV is stable when prepared and stored in 25 % sucrose at -152 °C. In addition, this chapter describes some commonly used methods of RSV titration, detection using microtitration and quantitative real-time RT-PCR, and the use of immunostaining for antigenic characterization. PMID:27464684

  8. Broad respiratory virus detection in infants hospitalized for bronchiolitis by use of a multiplex RT-PCR DNA microarray system.

    PubMed

    Huguenin, Antoine; Moutte, Lauryane; Renois, Fanny; Leveque, Nicolas; Talmud, Deborah; Abely, Michel; Nguyen, Yohan; Carrat, Fabrice; Andreoletti, Laurent

    2012-06-01

    Newly available molecular tools allow a sensitive detection of a broad panel of viruses in respiratory tract specimens. In the present study, the application of a multiplex RT-PCR DNA microarray in diagnosis and epidemiological survey of viral infections in infants hospitalized for bronchiolitis was assessed. One hundred and thirty-eight nasopharyngeal aspirates collected from October 2007 to September 2008 were tested by direct immunofluorescence and viral culture, a combination of referenced RT-PCRs and the DNA microarray. One or more viruses were detected in 96, 126 and 126 of the specimens by direct immunofluorescence and viral culture, RT-PCRs and DNA microarray, respectively (70 vs. 91 vs. 91%, P < 10(-3)). The RT-PCRs and the DNA microarray yielded concordant results for 99% of specimens and identified mixed viral infections in 85 (62%). The most common associations were: human bocavirus and respiratory syncytial virus (32%), adenovirus and respiratory syncytial virus (30%), and parainfluenza virus type 3 and respiratory syncytial virus (23%). None of the bronchiolitis severity parameters including intensive care unit admission, O(2) supply, O(2) saturation percentage, O(2) length and length of stay at the hospital appeared to be significantly increased in multiple viral infections compared to single viral infections (P > 0.1). In conclusion, the use of this DNA microarray in clinical virology practice allows rapid and accurate identification of common and uncommon viral respiratory pathogens in infants hospitalized for bronchiolitis. It should improve the clinical management, the epidemiological survey, and the prevention of the nosocomial transmission of respiratory viruses in pediatric wards. PMID:22499022

  9. Simultaneous diagnosis of Cetacean morbillivirus infection in dolphins stranded in the Spanish Mediterranean sea in 2011 using a novel Universal Probe Library (UPL) RT-PCR assay.

    PubMed

    Rubio-Guerri, Consuelo; Melero, Mar; Rivera-Arroyo, Belén; Bellière, Edwige Nina; Crespo, Jose Luis; García-Párraga, Daniel; Esperón, Fernando; Sánchez-Vizcaíno, Jose Manuel

    2013-07-26

    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. PMID:23380457

  10. Development of a One-Step Immunocapture Real-Time RT-PCR Assay for Detection of Tobacco Mosaic Virus in Soil

    PubMed Central

    Yang, Jin-Guang; Wang, Feng-Long; Chen, De-Xin; Shen, Li-Li; Qian, Yu-Mei; Liang, Zhi-Yong; Zhou, Wen-Chang; Yan, Tai-He

    2012-01-01

    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

  11. Evaluation of a broad range real-time polymerase chain reaction (RT-PCR) assay for the diagnosis of septic synovitis in horses

    PubMed Central

    Elmas, Colette R.; Koenig, Judith B.; Bienzle, Dorothee; Cribb, Nicola C.; Cernicchiaro, Natalia; Coté, Nathalie M.; Weese, J. Scott

    2013-01-01

    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. PMID:24101798

  12. Quantitative RT-PCR assay for high-throughput screening (HTS) of drugs against the growth of Cryptosporidium parvum in vitro

    PubMed Central

    Zhang, Haili; Zhu, Guan

    2015-01-01

    Our laboratory has previously developed a qRT-PCR assay to assess drug efficacy on the growth of Cryptosporidium parvum in vitro by detecting the levels of parasite 18S rRNA. This approach displayed up to four orders of magnitude of linear dynamic range and was much less labor-intensive than the traditional microscopic methods. However, conventional qRT-PCR protocol is not very amendable to high-throughput analysis when total RNA needs to be purified by lengthy, multi-step procedures. Recently, several commercial reagents are available for preparing cell lysates that could be directly used in downstream qRT-PCR analysis (e.g., Ambion Cell-to-cDNA kit and Bio-Rad iScript sample preparation reagent). Using these reagents, we are able to adapt the qRT-PCR assay into high-throughput screening of drugs in vitro (i.e., 96-well and 384-well formats for the cultivation of parasites and qRT-PCR detection, respectively). This qRT-PCR protocol is able to give a >150-fold linear dynamic range using samples isolated from cells infected with various numbers of parasites. The new assay is also validated by the NIH-recommended intra-plate, inter-plate, and inter-day uniformity tests. The robustness and effectiveness of the assay are also confirmed by evaluating the anti-cryptosporidial efficacy of paromomycin and by a small scale screening of compounds. PMID:26441920

  13. Strand-specific, real-time RT-PCR assays for quantification of genomic and positive-sense RNAs of the fish rhabdovirus, Infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Purcell, Maureen K.; Hart, S. Alexandra; Kurath, Gael; Winton, James R.

    2006-01-01

    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.

  14. Evaluation of Xpert® Norovirus Assay performance in comparison with real-time RT-PCR in hospitalized adult patients with acute gastroenteritis.

    PubMed

    Rovida, Francesca; Premoli, Marta; Campanini, Giulia; Sarasini, Antonella; Baldanti, Fausto

    2016-08-01

    Xpert® Norovirus Assay (Cepheid, Sunnyvale, CA) was compared with a laboratory-developed real-time RT-PCR assay for the detection of Norovirus GI and GII in hospitalized patients with acute gastroenteritis. The two assays showed a high level of concordance but Xpert® Norovirus Assay allowed faster detection of Norovirus and a simpler sample handling. PMID:27233425

  15. Development of a two-step SYBR Green I based real time RT-PCR assay for detecting and quantifying peste des petits ruminants virus in clinical samples.

    PubMed

    Abera, Tsegalem; Thangavelu, Ardhanary

    2014-12-01

    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. PMID:25194891

  16. Simultaneous detection of five notifiable viral diseases of cattle by single-tube multiplex real-time RT-PCR.

    PubMed

    Wernike, Kerstin; Hoffmann, Bernd; Beer, Martin

    2015-06-01

    Multiplexed real-time PCR (qPCR) assays enable the detection of several target genes in a single reaction, which is applicable for simultaneous testing for the most important viral diseases in samples obtained from ruminants with unspecific clinical symptoms. Here, reverse transcription qPCR (RT-qPCR) systems for the detection of bovine viral diarrhoea virus (BVDV) and bluetongue virus (BTV) were combined with an internal control system based on the beta-actin gene. Additionally, a background screening for three further major pathogens of cloven-hoofed animals reportable to the World Organisation for Animal Health, namely foot-and-mouth disease virus, epizootic haemorrhagic disease virus, and Rift Valley fever virus, was integrated using the identical fluorophore for the respective RT-qPCR assays. Every pathogen-specific assay had an analytical sensitivity of at least 100 genome copies per reaction within the multiplex approach, and a series of reference samples and clinical specimens obtained from cattle, but also from small ruminants, were detected reliably. The qPCR systems integrated in the background screening were even not influenced by the simultaneous amplification of very high BVDV and BTV genome copy numbers. The newly developed multiplex qPCR allows the specific and sensitive detection of five of the most important diseases of ruminants and could be used in the context of monitoring programs or for differential diagnostics. PMID:25746154

  17. Performance of Simplexa Dengue Molecular Assay Compared to Conventional and SYBR Green RT-PCR for Detection of Dengue Infection in Indonesia

    PubMed Central

    Wardhani, Puspa; Yohan, Benediktus; Trimarsanto, Hidayat; Fahri, Sukmal; Setianingsih, Tri Y.; Meutiawati, Febrina

    2014-01-01

    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. PMID:25102066

  18. Development of a novel hepatitis B virus encapsidation detection assay by viral nucleocapsid-captured quantitative RT-PCR.

    PubMed

    Ryu, Dong-Kyun; Ahn, Yeji; Ryu, Wang-Shick; Windisch, Marc P

    2015-11-01

    After encapsidation, where pregenomic RNA (pgRNA) is packaged into viral nucleocapsids, hepatitis B virus (HBV) uses the pgRNA as a template to replicate its DNA genome by reverse transcription. To date, there are only two encapsidation detection methods for evaluating the amount of pgRNA packaged into nucleocapsids: (i) the RNase protection assay and (ii) the native agarose gel electrophoresis assay. However, these methods are complex and laborious because they require multiple pgRNA purification steps followed by detection via an isotope-labeled probe. Moreover, both assays are unsuitable for evaluating a large number of antiviral agents in a dose-dependent manner. To overcome these limitations, we devised a novel HBV encapsidation assay in a 96-well plate format using nucleocapsid capture plates coated with an anti-HBV core (HBc) antibody, usually employed in enzyme-linked immunosorbent assays, to immobilize viral nucleocapsids. Viral pgRNA is then detected by quantitative RT-PCR (RT-qPCR). This strategy allows fast, convenient, and quantitative analysis of multiple viral RNA samples to evaluate encapsidation inhibitors. Furthermore, our protocol is potentially suitable for high-throughput screening (HTS) of compounds targeting HBV pgRNA encapsidation. PMID:26554506

  19. Detection of Australasian Flavivirus encephalitic viruses using rapid fluorogenic TaqMan RT-PCR assays.

    PubMed

    Pyke, Alyssa T; Smith, Ina L; van den Hurk, Andrew F; Northill, Judith A; Chuan, Teck F; Westacott, Alan J; Smith, Greg A

    2004-05-01

    The development of single, sensitive, fluorogenic reverse transcriptase-polymerase chain reaction (TaqMan) assays were required for the rapid and specific detection of three encephalitic viruses found in the Australasian region, namely; Japanese encephalitis virus (JEV), Murray Valley encephalitis virus (MVEV), and Kunjin virus (KUNV). Primers and a fluorogenic probe were individually designed to be complementary to a nucleotide region encompassing the 3' terminus of the nonstructural (NS) 5 gene and a portion of the 3' untranslated region (NS5-3'UTR) of each of the viral genomes respectively. Synthetically produced primer and probe controls were developed to minimize the likelihood of contamination and generation of false positives. Viral RNA from singly infected mosquitoes could be detected in pools of 1000 mosquitoes and positive mosquito pools collected from the field have been identified using each assay, indicating a high level of sensitivity and suitability for use in mosquito surveillance programs. In addition, the JEV TaqMan assay has been used to detect successfully viral RNA in sentinel pig serum samples. These assays potentially offer superior and timely detection of encephalitic viruses from surveillance samples, which is essential for the rapid implementation of vector control measures and continued monitoring of virus activity in the Australasian region. PMID:15041213

  20. Multiplex titration RT-PCR: rapid determination of gene expression patterns for a large number of genes

    NASA Technical Reports Server (NTRS)

    Nebenfuhr, A.; Lomax, T. L.

    1998-01-01

    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.

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

    PubMed

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

    2016-03-01

    We collected 599 Canadian retail pork chops and 283 pork livers routinely (usually weekly) from April 2011 to March 2012 using the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) retail sampling platform. Samples were assayed using validated real-time (q) reverse transcriptase polymerase chain reaction (RT-PCR) and nested classical RT-PCR for the detection of hepatitis E virus (HEV), porcine enteric calicivirus (PEC) and rotavirus (RV). The presence of Escherichia coli, Salmonella spp. and Campylobacter spp. was measured on a subset of our samples. Exact logistic regression models were fitted for predictors for HEV detection, for each assay. For both assays, sample type (pork chop versus liver) was a significant predictor for HEV RNA detection. For nested classical RT-PCR but not qRT-PCR, region of sample collection was a significant predictor (P = 0.008) of HEV detection. Odds of HEV detection were greatest in spring relative to other seasons. E. coli was a significant predictor for HEV RNA detection using the qRT-PCR (P = 0.03). Overall, the prevalence of E. coli, Salmonella spp. and Campylobacter spp. was significantly greater than HEV, PEC or RV on our retail pork samples. Our sparse data set for the detection of PEC and RV precluded modelling of risk factors for the detection of these viruses. PMID:26192650

  2. A rapid real-time qRT-PCR assay for ovine beta-actin mRNA.

    PubMed

    Bjarnadottir, Helga; Jonsson, Jon J

    2005-05-01

    Beta-Actin mRNA is often used for normalization in gene expression experiments. We describe a sensitive, rapid and specific quantitative assay for the cytoplasmic ovine beta-actin mRNA. The assay was based on the polymerase chain reaction (PCR) with real-time fluorescence resonance energy transfer (FRET) measurements to amplify cDNA products reverse transcribed from mRNA. A part of the ovine beta-actin sequence was amplified from cDNA from fetal ovine synovial (FOS) cells with mRNA-specific primers and cloned into a plasmid clone. The assay standard curve was constructed with dilutions of this plasmid. The assay was linear over five orders of magnitude and detected down to 600 copies per reaction of target DNA. Intraassay coefficient of variation was 12%. Detection of the beta-actin gene was eliminated by designing FRET probes at splice junctions and detection of putative processed pseudogenes was minimized by using FRET assay design with four oligonucleotides. We measured 0.2 copies per cell in RNA preparations without reverse transcription and DNase digestion. This might represent processed pseudogenes. In constrast, we measured 1400 beta-actin mRNA copies per cell in RNA preparations after the RT and DNase steps. The assay should, therefore, be sensitive enough to measure beta-actin from a single individual cell. Dilution of target DNA in murine RNA or ovine cDNA preparations did not effect efficiency of PCR or linearity of the assay. The quantitative assay described in this work can be used to correct for variations in various real-time qRT-PCR experiments in ovine cells with diverse goals, including gene expression studies, quantitation of viral load in infected cells and in various gene therapy experiments measuring vector load and expression in transduced cells. PMID:15823406

  3. Comparison of Luminex xTAG® RVP fast assay and real time RT-PCR for the detection of respiratory viruses in adults with community-acquired pneumonia.

    PubMed

    Luchsinger, Vivian; Prades, Yara; Ruiz, Mauricio; Pizarro, Rolando; Rossi, Patricio; Lizama, Luis; Garmendia, María Luisa; Meza, Angela; Larrañaga, Carmen; Avendaño, Luis F

    2016-07-01

    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. J. Med. Virol. 88:1173-1179, 2016. © 2016 Wiley Periodicals, Inc. PMID:27061405

  4. Development of RT-LAMP and real-time RT-PCR assays for the rapid detection of the new duck Tembusu-like BYD virus.

    PubMed

    Jiang, Tao; Liu, Juan; Deng, Yong-Qiang; Su, Jing-Liang; Xu, Li-Juan; Liu, Zhi-Hui; Li, Xiao-Feng; Yu, Xue-Dong; Zhu, Shun-Ya; Gao, George Fu; Qin, E-De; Qin, Cheng-Feng

    2012-12-01

    A new duck Tembusu virus (TMUV), also known as BYD virus, has been identified as the causative agent for the emerging duck egg-drop syndrome in mainland China. The rapid spread and wide distribution of the new TMUV in mainland China result in heavy loss to the poultry industry and pose great threats to public health. Rapid and sensitive detection methods are critical for prevention and control of TMUV infections. In this study, a reverse-transcription loop-mediated isothermal amplification assay (RT-LAMP) and an SYBR Green-I-based real-time RT-PCR assay specific for the duck TMUV were developed and validated with laboratory and field samples, respectively. The detection limits were 1 × 10(-4) and 1 × 10(-3) PFU per reaction for the RT-LAMP assay and real-time RT-PCR assay, respectively. The specificities were analyzed with other related members of the genus Flavivirus, and no cross-reaction was observed. Furthermore, both assays were evaluated with field samples, and they exhibited high sensitivity and specificity. In addition, the real-time RT-PCR assay worked well in viral load analysis, which revealed that the spleen may be the primary target for the replication of new duck TMUV in ducks. The TMUV-specific RT-LAMP and real-time RT-PCR assays will provide useful tools for the diagnosis and epidemiological surveillance of TMUV infection. PMID:22865206

  5. One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees.

    PubMed

    Malandraki, Ioanna; Varveri, Christina; Olmos, Antonio; Vassilakos, Nikon

    2015-03-01

    A one-step multiplex real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan chemistry was developed for the simultaneous detection of Pear blister canker viroid and Apple scar skin viroid along with universal detection of phytoplasmas, in pome trees. Total nucleic acids (TNAs) extraction was performed according to a modified CTAB protocol. Primers and TaqMan MGB probes for specific detection of the two viroids were designed in this study, whereas for phytoplasma detection published universal primers and probe were used, with the difference that the later was modified to carry a MGB quencher. The pathogens were detected simultaneously in 10-fold serial dilutions of TNAs from infected plant material into TNAs of healthy plant up to dilutions 10(-5) for viroids and 10(-4) for phytoplasmas. The multiplex real-time assay was at least 10 times more sensitive than conventional protocols for viroid and phytoplasma detection. Simultaneous detection of the three targets was achieved in composite samples at least up to a ratio of 1:100 triple-infected to healthy tissue, demonstrating that the developed assay has the potential to be used for rapid and massive screening of viroids and phytoplasmas of pome fruit trees in the frame of certification schemes and surveys. PMID:25479356

  6. Development of a reliable dual-gene amplification RT-PCR assay for the detection of Turkey Meningoencephalitis virus in Turkey brain tissues.

    PubMed

    Davidson, Irit; Raibstein, Israel; Al-Tori, Amira; Khinich, Yevgeny; Simanov, Michael; Yuval, Chanoch; Perk, Shimon; Lublin, Avishai

    2012-11-01

    The Turkey Meningoencephalitis virus (TMEV) causes neuroparalytic signs, paresis, in-coordination, morbidity and mortality in turkeys. In parallel to the increased worldwide scientific interest in veterinary avian flaviviruses, including the Bagaza, Tembusu and Tembusu-related BYD virus, TMEV-caused disease also reemergence in commercial turkeys during late summer of 2010. While initially TMEV was detected by NS5-gene RT-PCR, subsequently, the env-gene RT-PCR was employed. As lately several inconsistencies were observed between the clinical, serological and molecular detection of the TMEV env gene, this study evaluated whether genetic changes occurred in the recently isolated viruses, and sought to optimize and improve the direct TMEV amplification from brain tissues of affected turkeys. The main findings indicated that no changes occurred during the years in the TMEV genome, but the PCR detection sensitivities of the env and NS5 genes differed. The RT-PCR and RNA purification were optimized for direct amplification from brain tissues without pre-replication of clinical samples in tissue cultures or in embryonated eggs. The amplification sensitivity of the NS5-gene was 10-100 times more than the env-gene when separate. The new dual-gene amplification RT-PCR was similar to that of the NS5 gene, therefore the assay can be considered as a reliable diagnostic assay. Cases where one of the two amplicons would be RT-PCR negative would alert and warn on the virus identity, and possible genetic changes. In addition, the biochemical environment of the dual-gene amplification reaction seemed to contribute in deleting non-specific byproducts that occasionally appeared in the singular RT-PCR assays on RNA purified from brain tissues. PMID:22705084

  7. Establishment of a nanoparticle-assisted RT-PCR assay to distinguish field strains and attenuated strains of porcine epidemic diarrhea virus.

    PubMed

    Zhu, Yu; Wang, Gui-Hua; Cui, Yu-Dong; Cui, Shang-Jin

    2016-09-01

    Porcine epidemic diarrhea virus (PEDV) can cause serious disease and even death in neonatal piglets, resulting in serious damage to the swine industry worldwide. Open reading frame 3 (ORF3) is the only accessory gene in the PEDV genome. Previous studies have indicated that PEDV vaccine strains have a partial deletion in ORF3. In this study, a nanoparticle-assisted polymerase chain reaction (nanoparticle-assisted RT-PCR) assay targeting the ORF3 of PEDV was developed to distinguish PEDV field strains from attenuated strains by using a specific pair of primers. The PCR products of field strains and attenuated strains were 264 bp and 215 bp in length, respectively. The sensitivity and specificity of this assay were also assessed. The nanoparticle-assisted RT-PCR assay was 10-100 times more sensitive than the conventional RT-PCR assay, with no cross-reactions when amplifying porcine pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine rotavirus (RV), and porcine transmissible gastroenteritis virus (TGEV). The nanoparticle-assisted RT-PCR assay we describe here can be used to distinguish field strains from vaccine strains of PEDV, and it shows promise for reducing economic loss due to PEDV infection. PMID:27287433

  8. Development of a SYBR green I based RT-PCR assay for yellow fever virus: application in assessment of YFV infection in Aedes aegypti

    PubMed Central

    2012-01-01

    Background Yellow Fever virus (YFV) is an important arboviral pathogen in much of sub-Saharan Africa and the tropical Americas. It is the prototype member of the genus Flavivirus and is transmitted primarily by Aedes (Stegomyia) mosquitoes. The incidence of human infections in endemic areas has risen in recent years. Prompt and dependable identification of YFV is a critical component of response to suspect cases. Results We developed a one-step SYBR Green I-based real-time quantitative RT-PCR (qRT-PCR) assay targeting the 5'NTR and capsid-gene junction--for rapid detection and quantification of YFV. The detection limit was 1 PFU/mL, 10-fold more sensitive than conventional RT-PCR, and there was no cross-reactivity with closely related flaviviruses or with alphaviruses. Viral load in samples was determined by standard curve plotted from cycle threshold (Ct) values and virus concentration. The efficacy of the assay in mosquitoes was assessed with spiked samples. The utility of the assay for screening of pooled mosquitoes was also confirmed. Replication of a Cameroon isolate of YFV in Ae. aegypti revealed a marked variation in susceptibility among different colonies at different days post infection (pi). Conclusions The SYBR Green-1 based qRT-PCR assay is a faster, simpler, more sensitive and less expensive procedure for detection and quantification of YFV than other currently used methods. PMID:22264275

  9. A SYBR Green I based real time RT-PCR assay for specific detection and quantitation of Peste des petits ruminants virus

    PubMed Central

    2014-01-01

    Background Peste des petits ruminants (PPR) is an economically important disease of small ruminants such as sheep and goats. The disease is characterized by severe pyrexia, oculo-nasal discharge, pneumonia, necrosis and ulceration of the mucous membrane and inflammation of the gastro-intestinal tract leading to severe diarrhea. A SYBR Green I based real time RT-PCR targeting the N gene of PPRV has not been established for PPRV detection. Thus, the objective of present study was to develop highly sensitive N gene target SYBR Green I real time RT-PCR for specific detection and quantification of PPRV in clinical samples. A set of primers was designed to detect the nucleocapsid (N) gene of PPRV. Results The assay exhibited high specificity as all the viruses which have clinical and structural similarities to PPRV including Canine distemper virus (CDV), Measles virus (MV), Bluetongue virus (BTV) and Newcastle disease virus (NDV) failed to show an amplification signal. The lower detection limit of the assay was 5.11 copies/μl (Ct value of 33.67 ± 0.5) and 0.001 TCID50/ml (Ct value of 34.7 ± 0.5) based on plasmid copy number and tissue culture infectivity titre. The assay was 3-log more sensitive than the conventional RT-PCR. The coefficient of variation (CV) values for intra- and inter-assay variability were low, ranging from 0.32% - 2.31%, and 0.71% - 5.32%, respectively. To evaluate the performance of the newly developed assay, a total of 36 clinical samples suspected of PPR were screened for the presence of PPRV in parallel with conventional RT-PCR. The real time RT-PCR assay detected PPRV in 30 (83.3%) of clinical samples compared to 16 (44.4%) by conventional RT-PCR. Conclusions The two-step SYBR Green I based real time RT-PCR assay reported here is highly sensitive, specific, reproducible and rapid for detection and quantification of PPRV nucleic acids. PMID:24423231

  10. An rRT-PCR assay to detect the matrix gene of a broad range of avian paramyxovirus serotype-1 strains.

    PubMed

    Hines, Nichole L; Killian, Mary Lea; Pedersen, Janice C; Reising, Monica M; Mosos, Nestor A; Mathieu-Benson, Christian; Miller, Cathy L

    2012-06-01

    The current U.S. Department of Agriculture (USDA)-validated real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay designed to detect the matrix gene of avian paramyxovirus serotype-1 (APMV-1) is the primary screening assay used in the United States. It has previously been shown to be unable to consistently detect all members of class I APMV-1. Diagnostic testing relies on rRT-PCR to quickly detect APMV-1 in wild birds, backyard flocks, live bird markets, commercial poultry, and for export testing. Limitations of the current USDA assay have raised concerns about the potential for some strains of APMV-1 to remain undetected by the primary screening assay. Mismatches in the probe were shown to cause a loss in template binding efficiency, resulting in lack of detection by the assay. Here, we describe the development and analytical validation of a new rRT-PCR assay designed to target a highly conserved region of the matrix gene across a wide range of APMV-1 strains. Limit of detection testing revealed a 3 log10 decrease in sensitivity for one low-virulence strain when compared to the USDA validated assay. Conversely, the assay showed increased sensitivity for a class I isolate and two virulent strains of APMV-1 that were not detected by the USDA-validated assay. The new assay also demonstrated a high degree of specificity by the lack of detection of 43 non-APMV-1 viruses. PMID:22856199

  11. Design and testing of multiplex RT-PCR primers for the rapid detection of influenza A virus genomic segments: Application to equine influenza virus.

    PubMed

    Lee, EunJung; Kim, Eun-Ju; Shin, Yeun-Kyung; Song, Jae-Young

    2016-02-01

    The avian influenza A virus causes respiratory infections in animal species. It can undergo genomic recombination with newly obtained genetic material through an interspecies transmission. However, the process is an unpredictable event, making it difficult to predict the emergence of a new pandemic virus and distinguish its origin, especially when the virus is the result of multiple infections. Therefore, identifying a novel influenza is entirely dependent on sequencing its whole genome. Occasionally, however, it can be time-consuming, costly, and labor-intensive when sequencing many influenza viruses. To compensate for the difficulty, we developed a rapid, cost-effective, and simple multiplex RT-PCR to identify the viral genomic segments. As an example to evaluate its performance, H3N8 equine influenza virus (EIV) was studied for the purpose. In developing this protocol to amplify the EIV eight-segments, a series of processes, including phylogenetic analysis based on different influenza hosts, in silico analyses to estimate primer specificity, coverage, and variation scores, and investigation of host-specific amino acids, were progressively conducted to reduce or eliminate the negative factors that might affect PCR amplification. Selectively, EIV specific primers were synthesized with dual priming oligonucleotides (DPO) system to increase primer specificity. As a result, 16 primer pairs were selected to screen the dominantly circulating H3N8 EIV 8 genome segments: PA (3), PB2 (1), PA (3), NP (3), NA8 (2), HA3 (1), NS (1), and M (2). The diagnostic performance of the primers was evaluated with eight sets composing of four segment combinations using viral samples from various influenza hosts. The PCR results suggest that the multiplex RT-PCR has a wide range of applications in detection and diagnosis of newly emerging EIVs. Further, the proposed procedures of designing multiplex primers are expected to be used for detecting other animal influenza A viruses. PMID

  12. Establishment and validation of two duplex one-step real-time RT-PCR assays for diagnosis of foot-and-mouth disease.

    PubMed

    Gorna, K; Relmy, A; Romey, A; Zientara, S; Blaise-Boisseau, S; Bakkali-Kassimi, L

    2016-09-01

    Two duplex one-step TaqMan-based RT-PCR protocols for detection of foot-and-mouth disease virus (FMDV) were established and validated. Each RT-PCR test consists of a ready-to-use master mix for simultaneous detection of the well established 3D or IRES FMDV targets and incorporates the host β-actin mRNA as an internal control target, in a single-tube assay. The two real-time RT-PCR 3D/β-actin and IRES/β-actin tests are highly sensitive and able to detect up to 7TCID50/ml of FMDV and 10 copies/1μl of viral RNA. In field epithelium samples, the diagnostic sensitivity was 100% (95% CI; 91-100%) for the 3D/β-actin test and 97% (95% CI; 87-100%) for the IRES/β-actin test. The diagnostic specificity was 100% (95% CI; 95-100%) for both RT-PCRs. In addition, the two protocols proved to be robust, showing inter-assay coefficients of variation ranging from 1.94% to 6.73% for the IRES target and from 2.33% to 5.42% for the 3D target for different RNA extractions and different RT-PCR conditions. The internally controlled one-step real-time RT-PCR protocols described in this study provide a rapid, effective and reliable method for the detection of FMDV and thus may improve the routine diagnosis for foot-and-mouth disease. PMID:27317973

  13. Development of sensitive, high-throughput one-tube RT-PCR-enzyme hybridisation assay to detect selected bacterial fish pathogens.

    PubMed

    Wilson, T; Carson, J

    2003-03-31

    Bacterial monitoring and surveillance is critical for the early detection of pathogens to avoid the spread of disease. To facilitate this, an efficient, high-performance and high-throughput method to detect the presence of femotgram amounts of ribosomal RNA from 4 bacterial fish pathogens: Aeromonas salmonicida; Tenacibaculum maritimum (formerly Flexibacter maritimus); Lactococcus garvieae; and Yersinia ruckeri was developed. The system uses NucleoLink strips for liquid- and solid-phase PCR in 1 tube, to perform RT-PCR-enzyme hybridisation assays (RT-PCR-EHA) detecting 4 fg or less of rRNA from pure cultures and between 1 and 9 CFU per 200 microl sample volume from selective-enrichment culture media. The liquid-phase amplicons were visualised by gel electrophoresis and the solid-phase amplicons detected using internal probes and visualised using colorimetric detection and p-nitrophenylphosphate. PMID:12747638

  14. Novel Molecular Beacon Probe-Based Real-Time RT-PCR Assay for Diagnosis of Crimean-Congo Hemorrhagic Fever Encountered in India

    PubMed Central

    Kamboj, Aman; Pateriya, Atul Kumar; Mishra, Anamika; Ranaware, Pradip; Kulkarni, Diwakar D.; Raut, Ashwin Ashok

    2014-01-01

    Crimean-Congo hemorrhagic fever (CCHF) is an emerging zoonotic disease in India and requires immediate detection of infection both for preventing further transmission and for controlling the infection. The present study describes development, optimization, and evaluation of a novel molecular beacon-based real-time RT-PCR assay for rapid, sensitive, and specific diagnosis of Crimean-Congo hemorrhagic fever virus (CCHFV). The developed assay was found to be a better alternative to the reported TaqMan assay for routine diagnosis of CCHF. PMID:24877102

  15. Microfluidic based multiplex qRT-PCR identifies diagnostic and prognostic microRNA signatures in sera of prostate cancer patients

    PubMed Central

    Moltzahn, Felix; Olshen, Adam B.; Baehner, Lauren; Peek, Andrew; Fong, Lawrence; Stöppler, Hubert; Simko, Jeffry; Hilton, Joan F.; Carroll, Peter; Blelloch, Robert

    2010-01-01

    Recent prostate specific antigen (PSA) based screening trials indicate an urgent need for novel and non-invasive biomarker identification strategies to improve the prediction of prostate cancer behavior. Non-coding microRNAs (miRNAs) in the serum and plasma have been shown to have potential as non-invasive markers for physiological and pathological conditions. To identify serum miRNAs that diagnose and correlate with prognosis of prostate cancer, we developed a multiplex quantitative reverse transcription PCR (qRT-PCR) method involving purification of multiplex PCR products followed by uniplex analysis on a microfluidics chip to evaluate 384 human miRNAs. Using Dgcr8 and Dicer knockout (small RNA - deficient) mouse ES cells (mESC) as the benchmark, we confirmed the validity of our technique, while uncovering a significant lack of accuracy in previously published methods. Profiling 48 sera from healthy men and untreated prostate cancer patients with differing CAPRA (Cancer of the Prostate Risk Assessment) scores, we identified miRNA signatures that allow to diagnose cancer patients and correlate with prognosis. These serum signatures include oncogenic and tumor suppressive miRNAs suggesting functional roles in prostate cancer progression. PMID:21098088

  16. High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

    PubMed Central

    Renn, Lynnsey A.; Theisen, Terence C.; Navarro, Maria B.; Mane, Viraj P.; Schramm, Lynnsie M.; Kirschman, Kevin D.; Fabozzi, Giulia; Hillyer, Philippa; Puig, Montserrat; Verthelyi, Daniela; Rabin, Ronald L.

    2015-01-01

    Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-α subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-λ2 and -λ3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts. PMID:25867042

  17. Modification of Rotavirus Multiplex RT-PCR for the Detection of G12 Strains Based on Characterization of Emerging G12 Rotavirus Strains From South India

    PubMed Central

    Banerjee, Indrani; Ramani, Sasirekha; Primrose, Beryl; Iturriza-Gomara, Miren; Gray, James J.; Brown, David W.; Kang, Gagandeep

    2008-01-01

    Rotaviruses are the major etiological agents of diarrhea in children less than 5 years of age. The commonest G types in humans are G1-4 and G9. G12 is a rare human rotavirus (HRV) strain first reported in the Philippines. In this study, 13 G12 strains obtained from a community-based cohort and a hospital-based surveillance system in 2005 were characterized by phylogenetic analysis of partial nucleotide sequences of VP7, VP6, and NSP4 genes. Sequence and phylogenetic analysis of VP7 gene sequences showed that these southern Indian strains had the greatest homology with G12 strains recently reported from eastern India (97-99% identity both at the nucleotide level and deduced amino acid level) and less homology with the prototype G12 strain, L26 (89-90% identity at the nucleotide level and 90-94% at the deduced amino acid level). Phylogenetic analysis of the VP6 and the NSP4 genes revealed that the Vellore G12 strains belonged to VP6 subgroup II and NSP4 genotype B. The P types associated with these strains were P[6] and P[8]. A G12 type-specific primer was designed for inclusion in an established VP7 G-typing multiplex RT PCR, and tested against a panel of known G types and untyped samples and was found to detect G12 strains in the multiplex-PCR. Close homology of the South Indian G12 strains to those from Kolkata suggests that G12 HRV strains are emerging in India. Methods for characterization of rotaviruses in epidemiological studies need to be updated frequently, particularly in developing countries. PMID:17607780

  18. A one-step duplex rRT-PCR assay for the simultaneous detection of duck hepatitis A virus genotypes 1 and 3.

    PubMed

    Hu, Qin; Zhu, Dekang; Ma, Guangpeng; Cheng, Anchun; Wang, Mingshu; Chen, Shun; Jia, Renyong; Liu, Mafeng; Sun, Kunfeng; Yang, Qiao; Wu, Ying; Chen, Xiaoyue

    2016-10-01

    Duck hepatitis A virus (DHAV) is a highly infectious pathogen that causes significant bleeding lesions in the viscera of ducklings less than 3 weeks old. There are three serotypes of DHAV: serotype 1 (DHAV-1), serotype 2 (DHAV-2) and serotype 3 (DHAV-3). These serotypes have no cross-antigenicity with each other. To establish an rRT-PCR assay for the rapid detection of a mixed infection of DHAV-1 and DHAV-3, two pairs of primers and a pair of matching TaqMan probes were designed based on conserved regions of DHAV-1 VP0 and DHAV-3 VP3. Finally, we established a one-step duplex rRT-PCR assay with high specificity and sensitivity for the simultaneous detection of DHAV-1 and DHAV-3. This method showed no cross-antigenicity with the other pathogens tested, including duck plague virus, Muscovy duck parvovirus, Riemerella anatipestifer, and pathogenic E. coli from ducks. Sensitivity tests identified the minimum detection limits of this method as 98 (DHAV-1) and 10 (DHAV-3) copies/reaction. To validate the method, thirty-eight clinical samples and thirty artificially infected samples collected from dead duck embryos were studied. Thirty-seven samples were positive for DHAV-1, seventeen samples were positive for DHAV-3, and fourteen samples were positive for a mixed infection using the duplex rRT-PCR method. The method established in this study is specific, sensitive, convenient and timesaving and is a powerful tool for detecting DHAV-1, DHAV-3, and their mixed infection and for conducting surveys of pandemic virus strains. PMID:27435338

  19. Evaluation of a multiplex real-time polymerase chain reaction assay for the detection of influenza and respiratory syncytial viruses.

    PubMed

    Esposito, Susanna; Scala, Alessia; Tagliabue, Claudia; Zampiero, Alberto; Bianchini, Sonia; Principi, Nicola

    2016-01-01

    Nasopharyngeal swabs from 424 children were used to compare the performances of the new multiplex real-time polymerase chain reaction (RT-PCR) RIDA®GENE Flu & RSV kit and monospecific RT-PCR assays in detecting respiratory syncytial and influenza viruses. The easy-to-use kit was highly sensitive and specific and is recommended for routine practice. PMID:26458277

  20. Radiolabeled semi-quantitative RT-PCR assay for the analysis of alternative splicing of interleukin genes.

    PubMed

    Shakola, Felitsiya; Byrne, Stephen; Javed, Kainaat; Ruggiu, Matteo

    2014-01-01

    Alternative splicing evolved as a very efficient way to generate proteome diversity from a limited number of genes, while at the same time modulating posttranscriptional events of gene expression-such as stability, turnover, subcellular localization, binding properties, and general activity of both mRNAs and proteins. Since the vast majority of human genes undergo alternative splicing, it comes to no surprise that interleukin genes also show extensive alternative splicing. In fact, there is a growing body of evidence indicating that alternative splicing plays a central role in modulating the pleiotropic functions of cytokines, and aberrant expression of alternatively spliced interleukin mRNAs has been linked to disease. However, while several interleukin splice variants have been described, their function is still poorly understood. This is particularly relevant, since alternatively spliced cytokine isoforms can act both as disease biomarkers and as candidate entry points for therapeutic intervention. In this chapter we describe a protocol that uses radiolabeled semi-quantitative RT-PCR to efficiently detect, analyze, and quantify alternative splicing patterns of cytokine genes. PMID:24908320

  1. Development of a rapid, sensitive TaqMan real-time RT-PCR assay for the detection of Rose rosette virus using multiple gene targets.

    PubMed

    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

    2016-09-01

    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. PMID:27210549

  2. Development of in situ hybridization and RT-PCR assay for the detection of a nodavirus (PvNV) that causes muscle necrosis in Penaeus vannamei.

    PubMed

    Tang, Kathy F J; Pantoja, Carlos R; Redman, Rita M; Lightner, Donald V

    2007-05-01

    A nodavirus (tentatively named PvNV, Penaeus vannamei nodavirus) that causes muscle necrosis in P. vannamei was found in Belize in 2004. From 2004 to 2006, shrimp samples collected from Belize exhibited clinical signs, white, opaque lesions in the tails and histopathology similar to those of shrimps infected by infectious myonecrosis virus (IMNV). Histological examination revealed multifocal necrosis and hemocytic fibrosis in the skeletal muscle. In addition, basophilic, cytoplasmic inclusions were found in striated muscle, lymphoid organ and connective tissues. However, IMNV was not detected in these shrimps by either RT-PCR or in situ hybridization, suggesting that these lesions may be caused by another RNA virus. Thus, a cDNA library was constructed from total RNA extracted from hemolymph collected from infected shrimp. One clone (designated PvNV-4) with a 928 bp insert was sequenced and found to be similar (69% similarity when comparing the translated amino acid sequences) to the capsid protein gene of MrNV (Macrobrachium rosenbergii nodavirus). The insert of PvNV-4 was labeled with digoxigenin-11-deoxyuridine triphosphate (dUTP) and hybridized to tissue sections of P. vannamei with muscle necrosis collected in Belize and from laboratory bioassays. The samples were positive for PvNV infection. Positively reacting tissues included skeletal muscle, connective tissues, the lymphoid organ, and hemocytes in the heart and gills. In addition, we experimentally infected both P. vannamei and P. monodon with PvNV prepared from Belize samples. A nested RT-PCR assay developed from the PvNV-4 cloned sequence showed that both species are susceptible to PvNV infection. PMID:17629112

  3. Use of novel RT-PCR assay to detect picobirnavirus in Poultry Intestinal samples collected from the field

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recent metagenomic analyses of the turkey gut ribonucleic acid (RNA) virus community in our laboratory have identified novel enteric RNA viruses that may play roles in the poultry enteric diseases and in performance problems noted in the field. This has lead to new molecular diagnostic assays for ce...

  4. A one-step RT-PCR assay to detect and discriminate porcine reproductive and respiratory syndrome viruses in clinical specimens.

    PubMed

    Yang, Keli; Li, Yanhe; Duan, Zhengying; Guo, Rui; Liu, Zewen; Zhou, Danna; Yuan, Fangyan; Tian, Yongxiang

    2013-12-01

    Outbreaks of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) have led to large economic losses and, subsequently, have drawn great attention to its diagnosis and prevention. To facilitate rapid discrimination of HP-PRRSV from classical PRRSV (C-PRRSV), we developed a one-step RT-PCR assay. Primer specificities were evaluated with RNA extracted from 8 viral strains and our results revealed that the primers had a high specificity for PRRSV. The assay sensitivity was 25 copies/μL for both HP-PRRSV and C-PRRSV. A total of 929 serum samples were identified, of which 20.45% were HP-PRRSV-positive and 1.51% were C-PRRSV-positive, which was completely consistent with that of immunochromatochemistry and sequencing method. The proposed assay can detect the virus 2 days prior the onset of symptoms and it can be performed in 2h, thereby providing a rapid method to discriminate HP-PRRSV from C-PRRSV for the identification and prevention of PRRSV infections. PMID:24035936

  5. Measurement of the Infection and Dissemination of Bluetongue Virus in Culicoides Biting Midges Using a Semi-Quantitative RT-PCR Assay and Isolation of Infectious Virus

    PubMed Central

    Veronesi, Eva; Antony, Frank; Gubbins, Simon; Golding, Nick; Blackwell, Alison; Mertens, Peter PC.; Brownlie, Joe; Darpel, Karin E.; Mellor, Philip S.; Carpenter, Simon

    2013-01-01

    Background Culicoides biting midges (Diptera: Ceratopogonidae) are the biological vectors of globally significant arboviruses of livestock including bluetongue virus (BTV), African horse sickness virus (AHSV) and the recently emerging Schmallenberg virus (SBV). From 2006–2009 outbreaks of BTV in northern Europe inflicted major disruption and economic losses to farmers and several attempts were made to implicate Palaearctic Culicoides species as vectors. Results from these studies were difficult to interpret as they used semi-quantitative RT-PCR (sqPCR) assays as the major diagnostic tool, a technique that had not been validated for use in this role. In this study we validate the use of these assays by carrying out time-series detection of BTV RNA in two colony species of Culicoides and compare the results with the more traditional isolation of infectious BTV on cell culture. Methodology/Principal Findings A BTV serotype 1 strain mixed with horse blood was fed to several hundred individuals of Culicoides sonorensis (Wirth & Jones) and C. nubeculosus (Mg.) using a membrane-based assay and replete individuals were then incubated at 25°C. At daily intervals 25 Culicoides of each species were removed from incubation, homogenised and BTV quantified in each individual using sqPCR (Cq values) and virus isolation on a KC-C. sonorensis embryonic cell line, followed by antigen enzyme-linked immunosorbent assay (ELISA). In addition, comparisons were also drawn between the results obtained with whole C. sonorensis and with individually dissected individuals to determine the level of BTV dissemination. Conclusions/Significance Cq values generated from time-series infection experiments in both C. sonorensis and C. nubeculosus confirmed previous studies that relied upon the isolation and detection of infectious BTV. Implications on the testing of field-collected Culicoides as potential virus vectors by PCR assays and the use of such assays as front-line tools for use in

  6. A quantitative real-time reverse transcription PCR (qRT-PCR) assay to detect genome segment 9 of all 26 bluetongue virus serotypes.

    PubMed

    Maan, Narender S; Maan, Sushila; Belaganahalli, Manjunatha; Pullinger, Gillian; Montes, Antonio J Arenas; Gasparini, Marcela R; Guimera, Marc; Nomikou, Kyriaki; Mertens, Peter P C

    2015-03-01

    Bluetongue (BT) is an arboviral disease, which can often be fatal in naïve sheep and white tailed deer, but is usually less severe, or unapparent in other ruminants. Twenty-six bluetongue virus (BTV) serotypes have been recognised so far, two of which (BTV-25 and BTV-26) were recently identified by phylogenetic comparisons of genome-segment/outer-capsid protein VP2 (subsequently confirmed by serological 'virus-neutralisation' assays). Rapid, sensitive, reliable and quantitative diagnostic-assays for detection and identification of BTV represent important components of effective surveillance and control strategies. The BTV genome comprises 10 linear segments of dsRNA. We describe a 'TaqMan' fluorescence-probe based quantitative real-time RT-PCR assay, targeting the highly conserved genome-segment-9 (encoding the viral-helicase 'VP6' and NS4). The assay detected Seg-9 from isolates of all 26 BTV types, as well as from clinical samples derived from BTV-6w and BTV-8w outbreaks (in Europe), BTV-25 from Switzerland, BTV-26 from Kuwait, BTV-1w, BTV-4w and BTV-8w from Spain, BTV-4w, BTV-8, BTV-10 and BTV-16 from Brazil. Assay efficiency was evaluated with RNA derived from the reference strain of BTV-1w [RSArrrr/01] and was 99.6%, detecting down to 4 copies per reaction. Samples from uninfected insect or mammalian cell-cultures, hosts-species (uninfected sheep blood) or vector-insects, all gave negative results. The assay failed to detect RNA from heterologous but related Orbivirus species (including the nine African horse sickness virus [AHSV] and seven epizootic haemorrhagic disease virus [EHDV] serotypes). PMID:25486080

  7. One-step multiplex real time RT-PCR for the detection of bovine respiratory syncytial virus, bovine herpesvirus 1 and bovine parainfluenza virus 3

    PubMed Central

    2012-01-01

    Background Detection of respiratory viruses in veterinary species has traditionally relied on virus detection by isolation or immunofluorescence and/or detection of circulating antibody using ELISA or serum neutralising antibody tests. Multiplex real time PCR is increasingly used to diagnose respiratory viruses in humans and has proved to be superior to traditional methods. Bovine respiratory disease (BRD) is one of the most common causes of morbidity and mortality in housed cattle and virus infections can play a major role. We describe here a one step multiplex reverse transcriptase quantitative polymerase chain reaction (mRT-qPCR) to detect the viruses commonly implicated in BRD. Results A mRT-qPCR assay was developed and optimised for the simultaneous detection of bovine respiratory syncytial virus (BRSV), bovine herpes virus type 1 (BoHV-1) and bovine parainfluenza virus type 3 (BPI3 i & ii) nucleic acids in clinical samples from cattle. The assay targets the highly conserved glycoprotein B gene of BoHV-1, nucleocapsid gene of BRSV and nucleoprotein gene of BPI3. This mRT-qPCR assay was assessed for sensitivity, specificity and repeatability using in vitro transcribed RNA and recent field isolates. For clinical validation, 541 samples from clinically affected animals were tested and mRT-qPCR result compared to those obtained by conventional testing using virus isolation (VI) and/or indirect fluorescent antibody test (IFAT). Conclusions The mRT-qPCR assay was rapid, highly repeatable, specific and had a sensitivity of 97% in detecting 102 copies of BRSV, BoHV-1 and BPI3 i & ii. This is the first mRT-qPCR developed to detect the three primary viral agents of BRD and the first multiplex designed using locked nucleic acid (LNA), minor groove binding (MGB) and TaqMan probes in one reaction mix. This test was more sensitive than both VI and IFAT and can replace the aforesaid methods for virus detection during outbreaks of BRD. PMID:22455597

  8. Real-time RT-PCR assays to differentiate wild-type group A rotavirus strains from Rotarix® and RotaTeq® vaccine strains in stool samples

    PubMed Central

    Gautam, Rashi; Esona, Mathew D; Mijatovic-Rustempasic, Slavica; Ian Tam, Ka; Gentsch, Jon R; Bowen, Michael D

    2014-01-01

    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. PMID:24342877

  9. Real-time RT-PCR assays to differentiate wild-type group A rotavirus strains from Rotarix(®) and RotaTeq(®) vaccine strains in stool samples.

    PubMed

    Gautam, Rashi; Esona, Mathew D; Mijatovic-Rustempasic, Slavica; Ian Tam, Ka; Gentsch, Jon R; Bowen, Michael D

    2014-01-01

    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. PMID:24342877

  10. Development, application and validation of a Taqman real-time RT-PCR assay for the detection of infectious salmon anaemia virus (ISAV) in Atlantic salmon (Salmo salar).

    PubMed

    Snow, M; McKay, P; McBeath, A J A; Black, J; Doig, F; Kerr, R; Cunningham, C O; Nylund, A; Devold, M

    2006-01-01

    Infectious salmon anaemia (ISA) is a disease of cultured Atlantic salmon (Salmo salar) which was successfully eradicated from Scotland following its emergence in 1998. The rapid deployment of sensitive diagnostic methods for the detection of ISA virus (ISAV) was fundamental to the swift eradication of ISA disease in Scotland and continues to be of crucial importance to surveillance of the aquaculture industry. This study reports the development, validation, application and interpretation of two independent, highly sensitive and specific semi-quantitative Taqman real-time RT-PCR (qRT-PCR) methods for the detection of ISAV. Such technology offers considerable advantages over conventional RT-PCR methods in current routine use for ISAV surveillance. These include an increased sensitivity, enhanced specificity, semi-quantification using endogenous controls, a lack of subjectivity in results interpretation, speed of processing and improved contamination control. PMID:17058489

  11. Development of a One-Step Immunocapture Real-Time TaqMan RT-PCR Assay for the Broad Spectrum Detection of Pepino Mosaic Virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A Real-time reverse transcription-polymerase chain reaction (RT-PCR) was developed for efficient detection of genetically diverse PepMV isolates. The novel detection system was designed to use a duo-primer system targeting the conserved region in the triple gene block 2 (TGB2) gene with a single co...

  12. Performance of a commercial assay for the diagnosis of influenza A (H1N1) infection in comparison to the Centers for Disease Control and Prevention protocol of real time RT-PCR.

    PubMed

    Barbás, María G; Gallego, Sandra V; Castro, Gonzalo M; Baumeister, Elsa; Kademian, Silvia; De Leon, Juan; Cudolá, Analía

    2012-01-01

    At the time of influenza A (H1N1) emergency, the WHO responded with remarkable speed by releasing guidelines and a protocol for a real-time RT-PCR assay (rRT-PCR). The aim of the present study was to evalúate the performance of the "Real Time Ready Influenza A/H1N1 Detection Set" (June 2009)-Roche kit in comparison to the CDC reference rRT-PCR protocol. The overall sensitivity of the Roche assay for detection of the Inf A gene in the presence or absence of the H1 gene was 74.5 %. The sensitivity for detecting samples that were only positive for the Inf A gene (absence of the H1 gene) was 53.3 % whereas the sensitivity for H1N1-positive samples (presence of the Inf A gene and any other swine gene) was 76.4 %. The specificity of the assay was 97.1 %. A new version of the kit (November 2009) is now available, and a recent evaluation of its performance showed good sensitivity to detect pandemic H1N1 compared to other molecular assays. PMID:22610294

  13. Assessment of Preparation of Samples Under the Field Conditions and a Portable Real-Time RT-PCR Assay for the Rapid On-Site Detection of Newcastle Disease Virus.

    PubMed

    Liu, L; Benyeda, Z; Zohari, S; Yacoub, A; Isaksson, M; Leijon, M; LeBlanc, N; Benyeda, J; Belák, S

    2016-04-01

    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. PMID:25209697

  14. Determination of cut-off cycle threshold values in routine RT-PCR assays to assist differential diagnosis of norovirus in children hospitalized for acute gastroenteritis.

    PubMed

    Trang, N V; Choisy, M; Nakagomi, T; Chinh, N T M; Doan, Y H; Yamashiro, T; Bryant, J E; Nakagomi, O; Anh, D D

    2015-11-01

    Norovirus (NV) is an important cause of acute gastroenteritis in children, but is also frequently detected in asymptomatic children, which complicates the interpretation of NV detection results in both the clinical setting and population prevalence studies. A total of 807 faecal samples from children aged <5 years hospitalized for acute gastroenteritis were collected in Thai Binh, Vietnam, from January 2011 to September 2012. Real-time RT-PCR was used to detect and quantify NV-RNA in clinical samples. A bimodal distribution of cycle threshold (Ct) values was observed in which the lower peak was assumed to represent cases for which NV was the causal agent of diarrhoea, whereas the higher peak was assumed to represent cases involving an alternative pathogen other than NV. Under these assumptions, we applied finite-mixture modelling to estimate a threshold of Ct <21·36 (95% confidence interval 20·29-22·46) to distinguish NV-positive patients for which NV was the likely cause of diarrhoea. We evaluated the validity of the threshold through comparisons with NV antigen ELISA results, and comparisons of Ct values in patients co-infected with rotavirus. We conclude that the use of an appropriate cut-off value in the interpretation of NV real-time RT-PCR results may improve differential diagnosis of enteric infections, and could contribute to improved estimates of the burden of NV disease. PMID:26418350

  15. 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.

    PubMed

    Chai, Zheng; Ma, Wenjun; Fu, Fang; Lang, Yuekun; Wang, Wei; Tong, Guangzhi; Liu, Qinfang; Cai, Xuehui; Li, Xi

    2013-02-01

    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. PMID:23070137

  16. Padlock probe-mediated qRT-PCR for DNA computing answer determination

    PubMed Central

    Xiong, Fusheng; Frasch, Wayne D.

    2011-01-01

    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

  17. Development and evaluation of a one-step SYBR-Green I-based real-time RT-PCR assay for the detection and quantification of Chikungunya virus in human, monkey and mosquito samples.

    PubMed

    Ummul Haninah, A; Vasan, S S; Ravindran, T; Chandru, A; Lee, H L; Shamala Devi, S

    2010-12-01

    This paper reports the development of a one-step SYBR-Green I-based realtime RT-PCR assay for the detection and quantification of Chikungunya virus (CHIKV) in human, monkey and mosquito samples by targeting the E1 structural gene. A preliminary evaluation of this assay has been successfully completed using 71 samples, consisting of a panel of negative control sera, sera from healthy individuals, sera from patients with acute disease from which CHIKV had been isolated, as well as monkey sera and adult mosquito samples obtained during the chikungunya fever outbreak in Malaysia in 2008. The assay was found to be 100-fold more sensitive than the conventional RT-PCR with a detection limit of 4.12x10(0) RNA copies/μl. The specificity of the assay was tested against other related viruses such as Dengue (serotypes 1-4), Japanese encephalitis, Herpes Simplex, Parainfluenza, Sindbis, Ross River, Yellow fever and West Nile viruses. The sensitivity, specificity and efficiency of this assay were 100%, 100% and 96.8% respectively. This study on early diagnostics is of importance to all endemic countries, especially Malaysia, which has been facing increasingly frequent and bigger outbreaks due to this virus since 1999. PMID:21399603

  18. Evaluation of the Xpert Flu test and comparison with in-house real-time RT-PCR assays for detection of influenza virus from 2008 to 2011 in Marseille, France.

    PubMed

    Salez, N; Ninove, L; Thirion, L; Gazin, C; Zandotti, C; de Lamballerie, X; Charrel, R N

    2012-04-01

    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. PMID:22360446

  19. Design of a multiplex real time RT-PCR assay to detect Newcastle disease viruses from classes I and II

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Newcastle disease (ND) is a major concern for poultry producers around the world and the rapid diagnosis of an outbreak is crucial to any control program. Prompt detection of the causative agent of ND, virulent strains of Newcastle disease virus (vNDV), and differentiation of these viruses from tho...

  20. Detection sensitivity and quantitation of Plasmodium falciparum var gene transcripts by real-time RT-PCR in comparison with conventional RT-PCR.

    PubMed

    Gatton, Michelle L; Peters, Jennifer M; Gresty, Karryn; Fowler, Elizabeth V; Chen, Nanhua; Cheng, Qin

    2006-08-01

    Antigenic variation in Plasmodium falciparum erythrocyte membrane protein 1, caused by a switch in transcription of the encoding var gene, is an important feature of malaria. In this study, we quantified the relative abundance of var gene transcripts present in P. falciparum parasite clones using real-time reverse transcription-polymerase chain reaction (RT-PCR) and conventional RT-PCR combined with cloning and sequencing, with the aim of directly comparing the results obtained. When there was sufficient abundance of RNA for the real-time RT-PCR assay to be operating within the region of good reproducibility, RT-PCR and real-time RT-PCR tended to identify the same dominant transcript, although some transcript-specific issues were identified. When there were differences in the estimated relative amounts of minor transcripts, the RT-PCR assay tended to produce higher estimates than real-time RT-PCR. These results provide valuable information comparing RT-PCR and real-time RT-PCR analysis of samples with small quantities of RNA as might be expected in the analysis of field or clinical samples. PMID:16896121

  1. PALATAL DYSMORPHOGENESIS: QUANTITATIVE RT-PCR

    EPA Science Inventory

    ABSTRACT

    Palatal Dysmorphogenesis : Quantitative RT-PCR

    Gary A. Held and Barbara D. Abbott

    Reverse transcription PCR (RT-PCR) is a very sensitive method for detecting mRNA in tissue samples. However, as it is usually performed it is does not yield quantitativ...

  2. Development of an In-House TaqMan Real Time RT-PCR Assay to Quantify Hepatitis C Virus RNA in Serum and Peripheral Blood Mononuclear Cells in Patients With Chronic Hepatitis C Virus Infection

    PubMed Central

    Khalvati Fahlyani, Bahman; Behzad-Behbahani, Abbas; Taghavi, Seiied Alireza; Farhadi, Ali; Salehi, Saeede; Adibzadeh, Setare; Aboualizadeh, Farzaneh; Alavi, Parniyan; Nikouyan, Negin; Okhovat, Mohammad Ali; Ranjbaran, Reza; Rafiei Dehbidi, Gholam Reza; Shakibzadeh, Arash

    2015-01-01

    Background: Viral load measurements are commonly used to monitor HCV infection in patients with chronic diseases or determining the number of HCV-genomes in serum samples of patients after sustained virological response. However, in some patients, HCV viral load in serum samples is too low to be detected by PCR, especially after treatment. Objectives: The aim of this study was to develop a highly specific, sensitive, and reproducible in-house quantitative PCR using specific primers and probe cited in highly conservative region of HCV genome that allows simultaneous detection of HCV genotypes 1 - 4. Materials and Methods: In this study, three sets of primer pairs and a TaqMan probe for amplification and detection of selected region within 5’-non-coding (5’NCR) of four HCV genotypes were used. Using plasmid containing 5’NCR region of HCV, standard curve, threshold, and threshold cycle (CT) values were determined. Real-time and nested PCR were performed on HCV genotypes 1 - 4 extracted from plasma and peripheral blood mononuclear cells (PBMCs) samples collected from patients with chronic HCV infection. Results: The lower limit detection of this in-house HCV real-time RT-PCR was determined as 100 RNA copies/mL. Inter- and intra-assay coefficient of variation (CV) of this in-house HCV real-time RT-PCR ranged from 0.9% to 1.8% and 1.76% to 3.94%, respectively. The viral load of the genotyped samples ranged from 2.0 × 106 ± 0.31 to 2.7 × 105 ± 0.46 copies/mL in serum samples and 5 × 102 ± 0.36 to 4.0 × 103 ± 0.51 copies/106 cells/mL of PBMCs. Conclusions: The quite sensitive in-house TaqMan real time RT-PCR assay was able to detect and quantify all four main HCV genotypes prevailing around all geographical regions of Iran. PMID:26425128

  3. Development of generic Taqman PCR and RT-PCR assays for the detection of DNA and mRNA of β-actin-encoding sequences in a wide range of animal species.

    PubMed

    Piorkowski, Géraldine; Baronti, Cécile; de Lamballerie, Xavier; de Fabritus, Lauriane; Bichaud, Laurence; Pastorino, Boris A; Bessaud, Maël

    2014-06-01

    As a member of the European Virus Archive (EVA) consortium, our laboratory is developing and maintaining a large collection of viruses. This collection implies the use of a panel of cell lines originating from various animal species. In order to make easier the handling of such a large panel of cell lines, wide spectrum real-time PCR and RT-PCR assays were developed to allow the detection and the quantification of DNA and mRNA of β-actin, one of the most commonly used eukaryotic housekeeping genes. By using two degenerated primers and a unique probe, these two assays were shown to detect nucleic acids of a panel of vertebrate and invertebrate cell lines commonly used in animal virology. This panel included human, monkey, rodent, dog, pig, fish, batrachian, mosquito and tick cell lines. Additionally, the two assays amplified successfully β-actin-encoding sequences of sandflies. Sensitivity evaluation performed on synthetic DNA and RNA sequences showed that the two assays were very sensitive and suitable for accurate quantification. The two assays constitute together a convenient method suitable for multiple purposes. They can be used for instance to estimate the amount of contaminating cellular genetic material prior to sequence-independent amplification of viral genomes achieved before high-throughput sequencing, to evaluate the efficiency of DNase and/or RNase treatments performed on cellular extract and to check nucleic acid extraction by using β-actin-encoding sequences as endogenous control. This assay will constitute a precious tool for virologists working with multiple cell lines or animal models. PMID:24642236

  4. A highly specific q-RT-PCR assay to address the relevance of the JAK2WT and JAK2V617F expression levels and control genes in Ph-negative myeloproliferative neoplasms.

    PubMed

    Fantasia, Francesca; Di Capua, Emma Nora; Cenfra, Natalia; Pessina, Gloria; Mecarocci, Sergio; Rago, Angela; Cotroneo, Ettore; Busanello, Anna; Equitani, Francesco; Lo-Coco, Francesco; Nervi, Clara; Cimino, Giuseppe

    2014-04-01

    In Ph- myeloproliferative neoplasms, the quantification of the JAK2V617F transcripts may provide some advantages over the DNA allele burden determination. We developed a q-RT-PCR to assess the JAK2WT and JAK2V617F mRNA expression in 105 cases (23 donors, 13 secondary polycythemia, 22 polycythemia vera (PV), 38 essential thrombocythemia (ET), and 9 primary myelofibrosis (PMF)). Compared with the standard allele-specific oligonucleotide (ASO)-PCR technique, our assay showed a 100 % concordance rate detecting the JAK2V617F mutation in 22/22 PV (100 %), 29/38 (76.3 %) ET, and 5/9 (55.5 %) PMF cases, respectively. The sensitivity of the assay was 0.01 %. Comparing DNA and RNA samples, we found that the JAK2V617F mutational ratios were significantly higher at the RNA level both in PV (p = 0.005) and ET (p = 0.001) samples. In PV patients, JAK2WT expression levels positively correlated with the platelets (PLTs) (p = 0.003) whereas a trend to negative correlation was observed with the Hb levels (p = 0.051). JAK2V617F-positive cases showed the lowest JAK2WT and ABL1 mRNA expression levels. In all the samples, the expression pattern of beta-glucoronidase (GUSB) was more homogeneous than that of ABL1 or β2 microglobulin (B2M). Using GUSB as normalizator gene, a significant increase of the JAK2V617F mRNA levels was seen in two ET patients at time of progression to PV. In conclusion, the proposed q-RT-PCR is a sensitive and accurate method to quantify the JAK2 mutational status that can also show clinical correlations suggesting the impact of the residual amount of the JAK2WT allele on the Ph- MPN disease phenotype. Our observations also preclude the use of ABL1 as a housekeeping gene for these neoplasms. PMID:24173087

  5. Evaluation of a real-time two-step RT-PCR assay for quantitation of Chronic bee paralysis virus (CBPV) genome in experimentally-infected bee tissues and in life stages of a symptomatic colony.

    PubMed

    Blanchard, Philippe; Ribière, Magali; Celle, Olivier; Lallemand, Perrine; Schurr, Frank; Olivier, Violaine; Iscache, Anne Laure; Faucon, Jean Paul

    2007-04-01

    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. PMID:17166598

  6. Rapid Differential Diagnosis between Extrapulmonary Tuberculosis and Focal Complications of Brucellosis Using a Multiplex Real-Time PCR Assay

    PubMed Central

    Queipo-Ortuño, María Isabel; Colmenero, Juan D.; Bermudez, Pilar; Bravo, María José; Morata, Pilar

    2009-01-01

    Background Arduous to differ clinically, extrapulmonary tuberculosis and focal complications of brucellosis remain important causes of morbidity and mortality in many countries. We developed and applied a multiplex real-time PCR assay (M RT-PCR) for the simultaneous detection of Mycobacterium tuberculosis complex and Brucella spp. Methodology Conventional microbiological techniques and M RT-PCR for M. tuberculosis complex and Brucella spp were performed on 45 clinical specimens from patients with focal complications of brucellosis or extrapulmonary tuberculosis and 26 control samples. Fragments of 207 bp and 164 bp from the conserved region of the genes coding for an immunogenic membrane protein of 31 kDa of B. abortus (BCSP31) and the intergenic region SenX3-RegX3 were used for the identification of Brucella and M. tuberculosis complex, respectively. Conclusions The detection limit of the M RT-PCR was 2 genomes per reaction for both pathogens and the intra- and inter-assay coefficients of variation were 0.44% and 0.93% for Brucella and 0.58% and 1.12% for Mycobacterium. M RT-PCR correctly identified 42 of the 45 samples from patients with tuberculosis or brucellosis and was negative in all the controls. Thus, the overall sensitivity, specificity, PPV and NPV values of the M RT PCR assay were 93.3%, 100%, 100% and 89.7%, respectively, with an accuracy of 95.8% (95% CI, 91.1%–100%). Since M RT-PCR is highly reproducible and more rapid and sensitive than conventional microbiological tests, this technique could be a promising and practical approach for the differential diagnosis between extrapulmonary tuberculosis and focal complications of brucellosis. PMID:19225565

  7. Sensitivity and reproducibility of standardized-competitive RT-PCR for transcript quantification and its comparison with real time RT-PCR

    PubMed Central

    Pagliarulo, Vincenzo; George, Ben; Beil, Stephen J; Groshen, Susan; Laird, Peter W; Cai, Jie; Willey, James; Cote, Richard J; Datar, Ram H

    2004-01-01

    Background Probe based detection assays form the mainstay of transcript quantification. Problems with these assays include varying hybridization efficiencies of the probes used for transcript quantification and the expense involved. We examined the ability of a standardized competitive RT-PCR (StaRT PCR) assay to quantify transcripts of 4 cell cycle associated genes (RB, E2F1, CDKN2A and PCNA) in two cell lines (T24 & LD419) and compared its efficacy with the established Taqman real time quantitative RT-PCR assay. We also assessed the sensitivity, reproducibility and consistency of StaRT PCR. StaRT PCR assay is based on the incorporation of competitive templates (CT) in precisely standardized quantities along with the native template (NT) in a PCR reaction. This enables transcript quantification by comparing the NT and CT band intensities at the end of the PCR amplification. The CT serves as an ideal internal control. The transcript numbers are expressed as copies per million transcripts of a control gene such as β-actin (ACTB). Results The NT and CT were amplified at remarkably similar rates throughout the StaRT PCR amplification cycles, and the coefficient of variation was least (<3.8%) when the NT/CT ratio was kept as close to 1:1 as possible. The variability between the rates of amplification in different tubes subjected to the same StaRT PCR reaction was very low and within the range of experimental noise. Further, StaRT PCR was sensitive enough to detect variations as low as 10% in endogenous actin transcript quantity (p < 0.01 by the paired student's t-test). StaRT PCR correlated well with Taqman real time RT-PCR assay in terms of transcript quantification efficacy (p < 0.01 for all 4 genes by the Spearman Rank correlation method) and the ability to discriminate between cell types and confluence patterns. Conclusion StaRT PCR is thus a reliable and sensitive technique that can be applied to medium-high throughput quantitative transcript measurement. Further

  8. Prevalence study of Bovine viral diarrhea virus by evaluation of antigen capture ELISA and RT-PCR assay in Bovine, Ovine, Caprine, Buffalo and Camel aborted fetuses in Iran

    PubMed Central

    2011-01-01

    Bovine viral diarrhea virus is a pestivirus in the family Flaviviridae that cause abortions and stillbirths in livestock and its traditional diagnosis is based on cell culture and virus neutralization test. In this study, for more sensitive, specific detection and determined the prevalence of virus in aborted Bovine, Ovine, Caprine, Buffalo and Camel fetuses the antigen capture ELISA and RT-PCR were recommended. From the total of 2173 aborted fetuses, 347 (15.96%) and 402 (18.49%) were positive for presence of Bovine viral diarrhea virus by antigen capture ELISA and RT-PCR respectively. Statistical analysis of data showed significant differences between ELISA and RT-PCR for detection of virus in aborted fetuses. These results indicate a high presence of this pathogen in Iran and that RT- PCR is considerably faster and more accurate than ELISA for identification of Bovine viral diarrhea virus. To our knowledge the Camels and Bovine are the most resistant and sensitive to Bovine viral diarrhea's abortions respectively and the prevalence of virus in Caprine is more than Ovine aborted fetuses. This study is the first prevalence report of Bovine viral diarrhea virus in aborted Bovine, Ovine, Caprine, Buffalo and Camel fetuses by evaluation of ELISA and RT-PCR in Iran. PMID:22018096

  9. Type A influenza virus detection from horses by real-time RT-PCR and insulated isothermal RT-PCR.

    PubMed

    Balasuriya, Udeni B R

    2014-01-01

    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. PMID:24899448

  10. Development of a multiplex PCR assay to detect gastroenteric pathogens in the feces of Mexican children.

    PubMed

    Tolentino-Ruiz, R; Montoya-Varela, D; García-Espitia, M; Salas-Benito, M; Gutiérrez-Escolano, A; Gómez-García, C; Figueroa-Arredondo, P; Salas-Benito, J; De Nova-Ocampo, M

    2012-10-01

    Acute gastroenteritis (AGE) is a major cause of childhood morbidity and mortality worldwide; the etiology of AGE includes viruses, bacteria, and parasites. A multiplex PCR assay to simultaneously identify human Astrovirus (HAstV), Calicivirus (HuCVs), Entamoeba histolytica (E. histolytica), and enteroinvasive Escherichia coli (EIEC) in stool samples is described. A total of 103 samples were individually analyzed by ELISA (enzyme-linked immunosorbent assays) and RT-PCR/PCR. HAstV and HuCVs were detected in four out of 103 samples (3.8 %) by RT-PCR, but ELISAs found only one sample as positive for HuCVs (2.5 %). E. histolytica was identified in two out of 19 samples (10.5 %) and EIEC in 13 out of 20 samples (70 %) by PCR, and all PCR products were sequenced to verify their identities. Our multiplex PCR results demonstrate the simultaneous amplification of different pathogens such as HAstV, EIEC, and E. histolytica in the same reaction, though the HuCVs signal was weak in every replicate. Regardless, this multiplex PCR protocol represents a novel tool for the identification of distinct pathogens and may provide support for the diagnosis of AGE in children. PMID:22711331

  11. Design of Multiplexed Detection Assays for Identification of Avian Influenza A Virus Subtypes Pathogenic to Humans by SmartCycler Real-Time Reverse Transcription-PCR ▿

    PubMed Central

    Wang, Wei; Ren, Peijun; Mardi, Sek; Hou, Lili; Tsai, Cheguo; Chan, Kwok Hung; Cheng, Peter; Sheng, Jun; Buchy, Philippe; Sun, Bing; Toyoda, Tetsuya; Lim, Wilina; Peiris, J. S. Malik; Zhou, Paul; Deubel, Vincent

    2009-01-01

    Influenza A virus (IAV) epidemics are the result of human-to-human or poultry-to-human transmission. Tracking seasonal outbreaks of IAV and other avian influenza virus (AIV) subtypes that can infect humans, aquatic and migratory birds, poultry, and pigs is essential for epidemiological surveillance and outbreak alerts. In this study, we performed four real-time reverse transcription-PCR (rRT-PCR) assays for identification of the IAV M and hemagglutinin (HA) genes from six known AIVs infecting pigs, birds, and humans. IAV M1 gene-positive samples tested by single-step rRT-PCR and a fluorogenic Sybr green I detection system were further processed for H5 subtype identification by using two-primer-set multiplex and Sybr green I rRT-PCR assays. H5 subtype-negative samples were then tested with either a TaqMan assay for subtypes H1 and H3 or a TaqMan assay for subtypes H2, H7, and H9 and a beacon multiplex rRT-PCR identification assay. The four-tube strategy was able to detect 10 RNA copies of the HA genes of subtypes H1, H2, H3, H5, and H7 and 100 RNA copies of the HA gene of subtype H9. At least six H5 clades of H5N1 viruses isolated in Southeast Asia and China were detected by that test. Using rRT-PCR assays for the M1 and HA genes in 202 nasopharyngeal swab specimens from children with acute respiratory infections, we identified a total of 39 samples positive for the IAV M1 gene and subtypes H1 and H3. When performed with a portable SmartCycler instrument, the assays offer an efficient, flexible, and reliable platform for investigations of IAV and AIV in remote hospitals and in the field. PMID:18971359

  12. Development of a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction assay for the differential diagnosis of Feline leukemia virus vaccine and wild strains.

    PubMed

    Ho, Chia-Fang; Chan, Kun-Wei; Yang, Wei-Cheng; Chiang, Yu-Chung; Chung, Yang-Tsung; Kuo, James; Wang, Chi-Young

    2014-05-19

    A multiplex amplification refractory mutation system reverse transcription polymerase chain reaction (ARMS RT-PCR) was developed for the differential diagnosis of Feline leukemia virus (FeLV) vaccine and wild-type strains based on a point mutation between the vaccine strain (S) and the wild-type strain (T) located in the p27 gene. This system was further upgraded to obtain a real-time ARMS RT-PCR (ARMS qRT-PCR) with a high-resolution melt analysis (HRMA) platform. The genotyping of various strains of FeLV was determined by comparing the HRMA curves with the defined wild-type FeLV (strain TW1), and the results were expressed as a percentage confidence. The detection limits of ARMS RT-PCR and ARMS qRT-PCR combined with HRMA were 100 and 1 copies of transcribed FeLV RNA per 0.5 ml of sample, respectively. No false-positive results were obtained with 6 unrelated pathogens and 1 feline cell line. Twelve FeLV Taiwan strains were correctly identified using ARMS qRT-PCR combined with HRMA. The genotypes of the strains matched the defined FeLV wild-type strain genotype with at least 91.17% confidence. A higher degree of sequence polymorphism was found throughout the p27 gene compared with the long terminal repeat region. In conclusion, the current study describes the phylogenetic relationship of the FeLV Taiwan strains and demonstrates that the developed ARMS RT-PCR assay is able to be used to detect the replication of a vaccine strain that has not been properly inactivated, thus acting as a safety check for the quality of FeLV vaccines. PMID:24842287

  13. DETECTION OF HUMAN ENTERIC VIRUSES IN STREAM WATER WITH RT-PCR AND CELL CULTURE

    EPA Science Inventory

    A multiplex RT-PCR method was used to measure virus occurrence at five stream water sites that span a range of hydroclimatic, water-quality, and land-use characteristics. The performance of the molecular method was evaluated in comparison to traditional cell culture and Escherich...

  14. Simultaneous detection of papaya ringspot virus, papaya leaf distortion mosaic virus, and papaya mosaic virus by multiplex real-time reverse transcription PCR.

    PubMed

    Huo, P; Shen, W T; Yan, P; Tuo, D C; Li, X Y; Zhou, P

    2015-12-01

    Both the single infection of papaya ringspot virus (PRSV), papaya leaf distortion mosaic virus (PLDMV) or papaya mosaic virus (PapMV) and double infection of PRSV and PLDMV or PapMV which cause indistinguishable symptoms, threaten the papaya industry in Hainan Island, China. In this study, a multiplex real-time reverse transcription PCR (RT-PCR) was developed to detect simultaneously the three viruses based on their distinctive melting temperatures (Tms): 81.0±0.8°C for PRSV, 84.7±0.6°C for PLDMV, and 88.7±0.4°C for PapMV. The multiplex real-time RT-PCR method was specific and sensitive in detecting the three viruses, with a detection limit of 1.0×10(1), 1.0×10(2), and 1.0×10(2) copies for PRSV, PLDMV, and PapMV, respectively. Indeed, the reaction was 100 times more sensitive than the multiplex RT-PCR for PRSV, and 10 times more sensitive than multiplex RT-PCR for PLDMV. Field application of the multiplex real-time RT-PCR demonstrated that some non-symptomatic samples were positive for PLDMV by multiplex real-time RT-PCR but negative by multiplex RT-PCR, whereas some samples were positive for both PRSV and PLDMV by multiplex real-time RT-PCR assay but only positive for PLDMV by multiplex RT-PCR. Therefore, this multiplex real-time RT-PCR assay provides a more rapid, sensitive and reliable method for simultaneous detection of PRSV, PLDMV, PapMV and their mixed infections in papaya. PMID:26666186

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

    PubMed

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

    2016-09-01

    There is no gold standard for detection of Rotavirus Group A (RVA), one of the main causes of diarrhea in neonatal animals. Sensitive and specific real-time RT-PCR (rtRT-PCR) assays are available for RVA but require submission of the clinical samples to diagnostic laboratories. Patient-side immunoassays for RVA protein detection have shown variable results, particularly with samples from unintended species. A sensitive and specific test for detection of RVA on the farm would facilitate rapid management decisions. The insulated isothermal RT-PCR (RT-iiPCR) assay works in a portable machine to allow sensitive and specific on-site testing. The aim of this investigation was to evaluate a commercially available RT-iiPCR assay for RVA detection in feces from different animal species. This assay was compared to an in-house rtRT-PCR assay and a commercially available rtRT-PCR kit, as well as an ELISA and EM for RVA detection. All three PCR assays targeted the well-conserved NSP5 gene. Clinical fecal samples from 108 diarrheic animals (mainly cattle and horses) were tested. The percentage of positive samples by ELISA, EM, in-house rtRT-PCR, commercial rtRT-PCR, and RT-iiPCR was 29.4%, 31%, 36.7%, 51.4%, 56.9%, respectively. The agreement between different assays was high (81.3-100%) in samples containing high viral loads. The sensitivity of the RT-iiPCR assay appeared to be higher than the commercially available rtRT-PCR assay, with a limit of detection (95% confidence index) of 3-4 copies of in vitro transcribed dsRNA. In conclusion, the user-friendly, field-deployable RT-iiPCR system holds substantial promise for on-site detection of RVA. PMID:27180038

  16. The effects of reference genes in qRT-PCR assays for determining the immune response of bovine cells (MDBK) infected with the Bovine Viral Diarrhea Virus 1 (BVDV-1).

    PubMed

    Fredericksen, Fernanda; Delgado, Fredy; Cabrera, Cristian; Yáñez, Alejandro; Gonzalo, Carrasco; Villalba, Melina; Olavarría, Víctor H

    2015-09-10

    The bovine viral diarrhea virus (BVDV) causes significant economic losses to the dairy industry worldwide, and understanding its infection mechanisms would be extremely useful in designing new and efficient treatments. Due to the limited number of specific antibodies against bovine proteins, differential gene expression analyses are vital for researching host immune responses to viral infection. qRT-PCR provides a sensitive platform to conduct such gene expression analyses, but suitable housekeeping genes are needed for accurate transcript normalization. The present study assessed nine reference genes in bovine kidney cells under conditions of BVDV-1 infection, incubation with pathogen-associated molecular patterns, and co-incubation with BAY117085, a pharmacological inhibitor of the NF-κB signaling pathway. Analyses of Ct values using the BestKeeper and Normfinder programs ranked CD81, RPL4, and GAPDH as the most reliable reference genes. This determination of a stable set of reference genes in this culture system will facilitate analyses of expression levels for genes of interest. PMID:26004977

  17. Detection of human enteric viruses in stream water with RT-PCR and cell culture.

    USGS Publications Warehouse

    Denis-Mize, K.; Fout, G.S.; Dahling, D.R.; Francy, D.S.

    2004-01-01

    A multiplex RT-PCR method was used to measure virus occurrence at five stream water sites that span a range of hydroclimatic, water-quality, and land-use characteristics. The performance of the molecular method was evaluated in comparison with traditional cell culture and Escherichia coli membrane filtration assays. The study incorporated multiple quality controls and included a control for virus recovery during the sampling procedure as well as controls to detect potentially false-negative and false-positive data. Poliovirus recovery ranged from 16 to 65% and was variable, even in samples collected within the same stream. All five sites were positive for viruses by both molecular and cell culture-based virus assays. Enteroviruses, reoviruses, rotaviruses, and hepatitis A viruses were detected, but the use of the quality controls proved critical for interpretation of the molecular data. All sites showed evidence of faecal contamination, and culturable viruses were detected in four samples that would have met the US Environmental Protection Agency's recommended E. coli guideline for safe recreational water.

  18. Multiplexed Molecular Assays for Rapid Rule-Out of Foot-and-Mouth Disease

    SciTech Connect

    Lenhoff, R; Naraghi-Arani, P; Thissen, J; Olivas, J; Carillo, C; Chinn, C; Rasmussen, M; Messenger, S; Suer, L; Smith, S M; Tammero, L; Vitalis, E; Slezak, T R; Hullinger, P J; Hindson, B J; Hietala, S; Crossley, B; Mcbride, M

    2007-06-26

    A nucleic acid-based multiplexed assay was developed that combines detection of foot-and-mouth disease virus (FMDV) with rule-out assays for two other foreign animal diseases and four domestic animal diseases that cause vesicular or ulcerative lesions indistinguishable from FMDV infection in cattle, sheep and swine. The FMDV 'look-alike' diagnostic assay panel contains five PCR and twelve reverse transcriptase PCR (RT-PCR) signatures for a total of seventeen simultaneous PCR amplifications for seven diseases plus incorporating four internal assay controls. It was developed and optimized to amplify both DNA and RNA viruses simultaneously in a single tube and employs Luminex{trademark} liquid array technology. Assay development including selection of appropriate controls, a comparison of signature performance in single and multiplex testing against target nucleic acids, as well of limits of detection for each of the individual signatures is presented. While this assay is a prototype and by no means a comprehensive test for FMDV 'look-alike' viruses, an assay of this type is envisioned to have benefit to a laboratory network in routine surveillance and possibly for post-outbreak proof of freedom from foot-and-mouth disease.

  19. Detection of Zika virus by SYBR green one-step real-time RT-PCR.

    PubMed

    Xu, Ming-Yue; Liu, Si-Qing; Deng, Cheng-Lin; Zhang, Qiu-Yan; Zhang, Bo

    2016-10-01

    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. PMID:27444120

  20. Detection and identification of infectious bronchitis virus by RT-PCR in Iran.

    PubMed

    Homayounimehr, Alireza; Pakbin, Ahmad; Momayyez, Reza; Fatemi, Seyyedeh Mahsa Rastegar

    2016-06-01

    Infectious bronchitis virus (IBV) causes severe diseases in poultry with significant economic consequences to the poultry industry in Iran. The aim of this study was the detection and identification of IBV by reverse transcription(RT)-PCR in Iran. Ten IB virus strains were detected by testing trachea, cecal tonsil, and kidney tissues collected from broiler and layer farms in Iran. In order to detect infectious bronchitis virus, an optimized RT-PCR was used. Primers targeting the conserved region of known IBV serotypes were used in the RT-PCR assay. Primers selectively detecting Massachusetts and 793/B type IB viruses were designed to amplify the S1 gene of the virus and used in the nested PCR test. Our findings indicate the circulation of at least three genotypes of IB viruses (Massachusetts, 793/B, and variant 2) among poultry flocks. PMID:27010714

  1. Application of a Master Equation for Quantitative mRNA Analysis Using qRT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The qRT-PCR has been widely accepted as the assay of choice for mRNA quantification. Gene expression as measured by mRNA dynamics varies in response to different conditions and environmental stimuli. For conventional practice, housekeeping genes have been applied as internal reference for data nor...

  2. Predicting Gene Structures from Multiple RT-PCR Tests

    NASA Astrophysics Data System (ADS)

    Kováč, Jakub; Vinař, Tomáš; Brejová, Broňa

    It has been demonstrated that the use of additional information such as ESTs and protein homology can significantly improve accuracy of gene prediction. However, many sources of external information are still being omitted from consideration. Here, we investigate the use of product lengths from RT-PCR experiments in gene finding. We present hardness results and practical algorithms for several variants of the problem and apply our methods to a real RT-PCR data set in the Drosophila genome. We conclude that the use of RT-PCR data can improve the sensitivity of gene prediction and locate novel splicing variants.

  3. Detection and typing of human-infecting influenza viruses in China by using a multiplex DNA biochip assay.

    PubMed

    Wang, Yongqiang; Qu, Jiuxin; Ba, Qi; Dong, Jiuhong; Zhang, Liang; Zhang, Hong; Wu, Aiping; Wang, Dayan; Xia, Zanxian; Peng, Daxin; Shu, Yuelong; Cao, Bin; Jiang, Taijiao

    2016-08-01

    Rapid identification of the infections of specific subtypes of influenza viruses is critical for patient treatment and pandemic control. Here we report the application of multiplex reverse transcription polymerase chain reaction (RT-PCR) coupled with membrane-based DNA biochip to the detection and discrimination of the type (A and B) and subtype (human H1N1, human H3N2, avian H5N1 and avian H7N9) of influenza viruses in circulation in China. A multiplex one-step RT-PCR assay was designed to simultaneously amplify the HA and NA genes of the four subtypes of influenza A viruses and NS genes to discriminate type A and B viruses. PCR products were analyzed by a membrane-based biochip. The analytical sensitivity of the assay was determined at a range of 2-100 copies/reactions for each of the gene transcripts. Eighty one clinical samples, containing 66 positive samples with evident seasonal influenza virus infections, were tested, which gives the clinical sensitivity and specificity of 95.5% and 100% respectively. For the avian influenza samples, 3 out of 4 H5N1 samples and 2 out of 2 H7N9 avian samples were correctly identified. We argue this method could allow a rapid, reliable and inexpensive detection and differentiation of human-infecting influenza viruses. PMID:27150046

  4. Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses.

    PubMed

    Simmons, Monika; Myers, Todd; Guevara, Carolina; Jungkind, Donald; Williams, Maya; Houng, Huo-Shu

    2016-07-01

    Dengue virus (DENV) and chikungunya virus (CHIKV) are important human pathogens with common transmission vectors and similar clinical presentations. Patient care may be impacted by the misdiagnosis of DENV and CHIKV in areas where both viruses cocirculate. In this study, we have developed and validated a one-step multiplex reverse transcriptase PCR (RT-PCR) to simultaneously detect, quantify, and differentiate between four DENV serotypes (pan-DENV) and chikungunya virus. The assay uses TaqMan technology, employing two forward primers, three reverse primers, and four fluorophore-labeled probes in a single-reaction format. Coextracted and coamplified RNA was used as an internal control (IC), and in vitro-transcribed DENV and CHIKV RNAs were used to generate standard curves for absolute quantification. The diagnostic 95% limits of detection (LOD) within the linear range were 50 and 60 RNA copies/reaction for DENV (serotypes 1 to 4) and CHIKV, respectively. Our assay was able to detect 53 different strains of DENV, representing four serotypes, and six strains of CHIKV. No cross-reactivity was observed with related flaviviruses and alphaviruses, To evaluate diagnostic sensitivity and specificity, 89 clinical samples positive or negative for DENV (serotypes 1 to 4) and CHIKV by the standard virus isolation method were tested in our assay. The multiplex RT-PCR assay showed 95% sensitivity and 100% specificity for DENV and 100% sensitivity and specificity for CHIKV. With an assay turnaround time of less than 2 h, including extraction of RNA, the multiplex quantitative RT-PCR assay provides rapid diagnosis for the differential detection of two clinically indistinguishable diseases, whose geographical occurrence is increasingly overlapping. PMID:27098955

  5. Detection of beet yellows virus by RT-PCR and immunocapture RT-PCR in Tetragonia expansa and Beta vulgaris.

    PubMed

    Kundu, K; Rysánek, P

    2004-01-01

    Two sensitive methods, RT-PCR with phenol-extracted RNA or Triton X-100-released RNA and immunocapture RT-PCR (IR-RT-PCR) were used for the detection of Beet yellows virus (BYV) in young and old leaves of Tetragonia expansa and sugar beet (Beta vulgaris) and in sugar beet roots. Four oligonucleotide primer pairs proved suitable for the detection of BYV. The release of BYV RNA with Triton X-100 was shown to be a very effective and easy as compared to isolation of total RNA by phenol extraction with the same or higher sensitivity of subsequent PCR. Using the Triton X-100 release of RNA and IC-RT-PCR the sensitivity of detection was so high that pg amounts of BYV RNA occurring in dilutions up to 10(-6) of saps from young Tetragonia and sugar beet leaves could be detected. PMID:15595212

  6. Detection of West Nile virus in mosquitoes by RT-PCR.

    PubMed

    Hadfield, T L; Turell, M; Dempsey, M P; David, J; Park, E J

    2001-06-01

    A reverse transcriptase-polymerase chain reaction (RT-PCR) assay employing detection technology was developed to identify West Nile virus in experimentally infected mosquitoes. The specificity of the assay was evaluated with the following viruses: eastern equine encephalitis, Ilheus, West Nile and yellow fever viruses. The limits of detection were determined using West Nile viral RNA extracted from serial dilutions of virus culture in infected mosquitoes. Limit of detection was 5 PFU from extracted mosquitoes. We were able to detect the presence of one infected mosquito in a pool of 50 repeatedly. When the RT-PCR was used with coded samples of intrathoracically-infected and uninfected mosquitoes, the assay detected the virus in all infected mosquitoes. Analytic sensitivity and specificity were 100%. This assay offers an efficient and rapid method of identifying West Nile virus in infected mosquitoes or cell culture. PMID:11352595

  7. Probe-free and sensitive detection of diarrhea-causing pathogens using RT-PCR combined high resolution melting analysis.

    PubMed

    Wang, Hai-Bo; Mo, Qiu-Hua; Wang, Qi; Wu, Bi-Mei; Feng, Zi-Li; Lin, Ji-Can; Yang, Ze

    2016-09-01

    Rapid and sensitive diagnostic methods are needed to help physicians make faster and better treatment decision for patients suffered from diarrhea. In the present study, a probe-free and sensitive RT-PCR combined high resolution melting analysis (HRMA) assay was established successfully for the detection of four major diarrhea-causing pathogens. The lower limit of detection of the assay were 10(0), 10(2), 10(0) and 10(3) copies/reaction for rotaviruses group A, astroviruses serotype 1, noroviruses genogroup II, and sapoviruses genegroup I, respectively, which were 1000-fold, 10-fold, 1000-fold and 10-fold more sensitive than conventional RT-PCR assay developed in parallel and comparable to or higher than commercially available real-time RT-PCR assay. Blinded sample evaluation showed that the assay was 100% concordant to both conventional RT-PCR and commercial real-time RT-PCR, indicating high reliability of the new assay. Therefore, the assay could provide a valuable platform for the probe-free and sensitive diagnosis of these pathogens. PMID:27461241

  8. Development of SYBR green I based one-step real-time RT-PCR assay for the detection and differentiation of very virulent and classical strains of infectious bursal disease virus.

    PubMed

    Kong, Lih Ling; Omar, Abdul Rahman; Hair Bejo, Mohd; Ideris, Aini; Tan, Sheau Wei

    2009-11-01

    A SYBR Green I based one-step real-time reverse transcriptase polymerase chain reaction was developed for the detection and differentiation of very virulent (vv) and classical strains of infectious bursal disease virus (IBDV). The assay showed high PCR efficiency >93% and high reproducibility with coefficient of variation less than 0.5%. When tested on characterized IBDV strains, the very virulent and classical-specific primers detected accurately only vvIBDV and classical IBDV strains, respectively. The diagnostic efficacy of the assay was also tested on 140 bursal samples from experimental infection and 37 bursal samples from cases suspected of IBD. The assay was able to detect IBDV from bursal samples collected at days 3 and 5 post-infection with the vvIBDV strain UPM94/273 and the classical IBDV strain D78. The assay was also able to detect bursal samples infected dually with D78 and UPM94/273. The melting temperature values of the amplification products from the classical and very virulent viral infection were statistically significant (P<0.05). The specificity of the assay for detecting IBDV from suspected cases was confirmed by sequence analysis of the VP2 gene. The assay showed high sensitivity since bursal samples which were negative for IBDV were confirmed by virus isolation and PCR amplification. Hence, the new assay offers an attractive method for rapid detection of strains of IBDV. PMID:19591873

  9. VARIATION OF THE EXPRESSION OF ENDOGENOUS "HOUSEKEEPING" GENES IN B[A]P TREATED MOUSE LUNGS MEASURED BY qRT-PCR

    EPA Science Inventory

    Quantitative RT-PCR is frequently used to analyze gene expression in different experimental systems. In this assay, housekeeping genes are frequently used to normalize for the variability between samples (relative quantification). We have examined the utility of using qRT-PCR and...

  10. Development and evaluation of a real-time RT-PCR assay for the detection of Ebola virus (Zaire) during an Ebola outbreak in Guinea in 2014-2015.

    PubMed

    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

    2016-02-01

    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. PMID:26597659

  11. Development of a RT-PCR ELISA for simultaneous detection of BVDV-1, BVDV-2 and BDV in ruminants and its evaluation on clinical samples.

    PubMed

    Dubey, Pooja; Mishra, N; Rajukumar, K; Behera, S P; Kalaiyarasu, S; Nema, R K; Prakash, A

    2015-03-01

    The aim of this study was to develop a reverse transcription polymerase chain reaction ELISA (RT-PCR ELISA) for detection of ruminant pestiviruses and to evaluate its diagnostic performance on clinical samples obtained from cattle, sheep and goats. Optimization was carried out by serial dilution of home-made digoxygenin-labelled RT-PCR product standards obtained from pestivirus isolates and pestivirus infected animals. The detection limit of the assay was 10TCID50/ml, similar to virus isolation and real-time RT-PCR but 10-fold higher than RT-PCR. The assay had high analytical specificity along with a good reproducibility. When the assay was evaluated on the samples obtained from animals infected experimentally with BVDV and from the field using virus isolation as standard, it showed a high diagnostic sensitivity (95.9%) and specificity (98.6%) and there was strong agreement (97.5% concordance) between the two tests. However, it displayed an increased diagnostic specificity and sensitivity over RT-PCR. Additionally, when a few samples (n=26) were tested by RT-PCR ELISA and real-time RT-PCR, 100% concordance was obtained between them. Our results showed that RT-PCR ELISA can be a rapid, cost effective and alternative molecular diagnostic test for simultaneous detection of BVDV-1, BVDV-2 and BDV in ruminants in ordinary laboratory settings. PMID:25486086

  12. Simultaneous Measurement of Multiple Mouse Ear Proteins with Multiplex ELISA Assays

    PubMed Central

    Trune, Dennis R.; Larrain, Barbara E.; Hausman, Frances A.; Kempton, J. Beth; MacArthur, Carol J.

    2011-01-01

    RT-PCR. The Quansys array showed a limit of detection for ear IL-6 down to 2–4 pg/ml, indicating it is sufficiently sensitive to detect ear proteins present in low concentrations. Thus, the multiplex ELISA procedures appear suitable and reliable for the study of hearing related proteins, providing accurate, quantitative, reproducible results with considerable improvement in sensitivity and economy. PMID:21144888

  13. Sensitive, semi-nested RT-PCR amplification of fusion gene sequences for the rapid detection and differentiation of Newcastle disease virus.

    PubMed

    Zhang, Lei; Pan, Zhiming; Geng, Shizhong; Chen, Xiang; Hu, Shunlin; Liu, Huimo; Wu, Yantao; Jiao, Xinan; Liu, Xiufan

    2010-10-01

    A rapid, sensitive and specific semi-nested RT-PCR was developed to detect and differentiate virulent and avirulent strains of Newcastle disease virus (NDV). For a total of 67 NDV strains, the results obtained from the semi-nested RT-PCR were consistent with those from nucleotide sequence analysis, plaque forming assays, mean death time (MDT) measurements and intracerebral pathogenicity index (ICPI). Furthermore, 13 class I NDV strains can be characterized by the semi-nested RT-PCR approach, which was feasible by the conventional methods. The detection limit for the semi-nested RT-PCR was two plaque forming units (PFU) both for NDV strain F48E9 in allantoic fluid and for isolate APMV1/ch/ChinaND4031 in oral or cloacal swabs. In conclusion, this semi-nested RT-PCR method offers a new assay for the rapid detection and differentiation of NDVs. PMID:20219221

  14. A multiplexed reverse transcriptase PCR assay for identification of viral respiratory pathogens at point-of-care

    SciTech Connect

    Letant, S E; .Ortiz, J I; Tammero, L; Birch, J M; Derlet, R W; Cohen, S; Manning, D; McBride, M T

    2007-04-11

    We have developed a nucleic acid-based assay that is rapid, sensitive, specific, and can be used for the simultaneous detection of 5 common human respiratory pathogens including influenza A, influenza B, parainfluenza type 1 and 3, respiratory syncytial virus, and adenovirus group B, C, and E. Typically, diagnosis on an un-extracted clinical sample can be provided in less than 3 hours, including sample collection, preparation, and processing, as well as data analysis. Such a multiplexed panel would enable rapid broad-spectrum pathogen testing on nasal swabs, and therefore allow implementation of infection control measures, and timely administration of antiviral therapies. This article presents a summary of the assay performance in terms of sensitivity and specificity. Limits of detection are provided for each targeted respiratory pathogen, and result comparisons are performed on clinical samples, our goal being to compare the sensitivity and specificity of the multiplexed assay to the combination of immunofluorescence and shell vial culture currently implemented at the UCDMC hospital. Overall, the use of the multiplexed RT-PCR assay reduced the rate of false negatives by 4% and reduced the rate of false positives by up to 10%. The assay correctly identified 99.3% of the clinical negatives, 97% of adenovirus, 95% of RSV, 92% of influenza B, and 77% of influenza A without any extraction performed on the clinical samples. The data also showed that extraction will be needed for parainfluenza virus, which was only identified correctly 24% of the time on un-extracted samples.

  15. Combination of conventional immunohistochemistry and qRT-PCR to detect ALK rearrangement

    PubMed Central

    2014-01-01

    Background Compared with FISH and qRT-PCR analyses, immunohistochemistry (IHC) is the preferred screening test in most pathology practices for ALK-rearrangement detection. With 100% sensitivity and 98% specificity, the VENTANA ALK (D5F3) IHC assay has been approved in the EU and some Asian countries for ALK-rearrangement detection. However, an automated Ventana IHC platform is not available in most pathology labs. In this study, we evaluated the applicability of conventional IHC with D5F3 antibody in routine pathological practice and proposed detection methods and procedures that ensure that patients with ALK+ are not missed. Methods FISH and IHC analyses were performed on 297 lung adenocarcinoma cases. VENTANA IHC and qRT-PCR assay were applied to evaluate ALK-fusion status in the discordant cases of FISH and IHC. The association of ALK+ with clinicopathological characteristics was statistically analyzed. Results IHC had 100% sensitivity and 81.8% specificity for detecting ALK+. Eight ALK-expressed cases were ALK-, five of which had ALK fusion detected by qRT-PCR analysis. Three of these five cases showed ALK expression using VENTANA IHC assay. ALK+ was associated with younger age and lymph node metastasis in this Chinese lung adenocarcinoma patient cohort. Conclusions The advantages of low cost and 100% sensitivity allow conventional IHC to serve as a robust diagnostic tool for screening patients with ALK+, especially in pathology labs without a VENTANA IHC platform. For cases in which ALK is weakly expressed, qRT-PCR is necessary as a diagnostic test for ALK-fusion detection. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2269448351088278. PMID:24422905

  16. Detection of Banana mild mosaic virus and Banana virus X by polyvalent degenerate oligonucleotide RT-PCR (PDO-RT-PCR).

    PubMed

    Teycheney, Pierre-Yves; Acina, Isabelle; Lockhart, Benham E L; Candresse, Thierry

    2007-06-01

    Viruses are important constraints to the movement and propagation of plant germplasm, especially for vegetatively propagated crops such as banana and plantain. Their control relies primarily on the use of virus-free plant material, whose production and certification requires sensitive and reliable detection methods. An existing polyvalent degenerate oligonucleotide RT-PCR (PDO-RT-PCR) assay was adapted to the detection of Banana mild mosaic virus (BanMMV) and Banana virus X, two Flexiviridae infecting Musa spp. PDO inosine-containing primers were found to be well suited to the detection of BanMMV, despite its high molecular diversity, but not to that of the highly conserved BVX, for which species-specific primers were designed. Sampling and sample processing steps were optimized in order to avoid nucleic acid purification prior to the reverse transcription step. A polyclonal anti-BanMMV antiserum was raised and successfully used for the immunocapture (IC) of BanMMV viral particles from leaf extracts, leading to the development of a PDO-IC-RT-nested PCR assay. Although the anti-BanMMV antiserum could to some extent recognize BVX viral particles, direct binding (DB) was shown to be a more efficient method for processing BVX-infected samples and a PDO-DB-RT-nested PCR assay was developed for the detection of BVX from leaf extracts. PMID:17280722

  17. A multiplex real-time PCR panel assay for simultaneous detection and differentiation of 12 common swine viruses.

    PubMed

    Shi, Xiju; Liu, Xuming; Wang, Qin; Das, Amaresh; Ma, Guiping; Xu, Lu; Sun, Qing; Peddireddi, Lalitha; Jia, Wei; Liu, Yanhua; Anderson, Gary; Bai, Jianfa; Shi, Jishu

    2016-10-01

    Mixed infection with different pathogens is common in swine production systems especially under intensive production conditions. Quick and accurate detection and differentiation of different pathogens are necessary for epidemiological surveillance, disease management and import and export controls. In this study, we developed and validated a panel of multiplex real-time PCR/RT-PCR assays composed of four subpanels, each detects three common swine pathogens. The panel detects 12 viruses or viral serotypes, namely, VSV-IN, VSV-NJ, SVDV, CSFV, ASFV, FMDV, PCV2, PPV, PRV, PRRSV-NA, PRRSV-EU and SIV. Correlation coefficients (R(2)) and PCR amplification efficiencies of all singular and triplex real-time PCR reactions are within the acceptable range. Comparison between singular and triplex real-time PCR assays of each subpanel indicates that there is no significant interference on assay sensitivities caused by multiplexing. Specificity tests on 226 target clinical samples or 4 viral strains and 91 non-target clinical samples revealed that the real-time PCR panel is 100% specific, and there is no cross amplification observed. The limit of detection of each triplex real-time PCR is less than 10 copies per reaction for DNA, and less than 16 copies per reaction for RNA viruses. The newly developed multiplex real-time PCR panel also detected different combinations of co-infections as confirmed by other means of detections. PMID:27506582

  18. Real-Time RT-PCR for the Detection of Lyssavirus Species

    PubMed Central

    Deubelbeiss, A.; Zahno, M.-L.; Zanoni, M.; Bruegger, D.; Zanoni, R.

    2014-01-01

    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

  19. Real-Time RT-PCR for the Detection of Lyssavirus Species.

    PubMed

    Deubelbeiss, A; Zahno, M-L; Zanoni, M; Bruegger, D; Zanoni, R

    2014-01-01

    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

  20. Misidentification of Bordetella bronchiseptica as Bordetella pertussis using a Newly Described RT-PCR Targeting the Pertactin Gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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....

  1. Diagnostic real-time RT-PCR for the simultaneous detection of Citrus exocortis viroid and Hop stunt viroid.

    PubMed

    Papayiannis, Lambros C

    2014-02-01

    Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) are two important viroids known to infect several plant species worldwide. In this study, a real-time reverse transcription (RT) TaqMan polymerase chain reaction (PCR) assay was developed and optimized for the simultaneous detection of CEVd and HSVd. The assay's analytical and diagnostic sensitivity and specificity were evaluated using reference isolates. Two different RNA extraction methods and one rapid crude template preparation procedure were compared in terms of extraction purity and efficiency for PCR applications. Extraction method Q included a commercially available kit, whereas method C was a modified chloroform-phase extraction in house protocol. Procedure S involved blotting the sap extract on a positively charged nylon membrane and elution. The multiplex RT-TaqMan PCR assay successfully discriminated the two viroid species from all reference samples and its recorded diagnostic sensitivity (Dse) and specificity (Dsp) was 100%. On the contrary, in conventional RT-PCR tests, the overall Dse and Dsp were lower and estimated at 94 and 95% for CEVd, and 97 and 98% for HSVd, respectively. In a direct comparison, the developed assay presented 1000-fold more analytical sensitivity. Spectrophotometric results showed that RNA extraction methods Q and C, yielded the purest RNA, and gave the lowest mean Ct values. Alternative template preparation method S resulted in Ct values statistically similar to those obtained with methods Q to C when tested by RT-TaqMan PCR. The developed assay, using crude template preparation S, allows the simple, accurate and cost-effective testing of a large number of plant samples, and can be applied in surveys and certification schemes. PMID:24252553

  2. Novel Multitarget Real-Time PCR Assay for Rapid Detection of Bordetella Species in Clinical Specimens ▿

    PubMed Central

    Tatti, Kathleen M.; Sparks, Kansas N.; Boney, Kathryn O.; Tondella, Maria Lucia

    2011-01-01

    A novel multitarget real-time PCR (RT-PCR) assay for the rapid identification of Bordetella pertussis, B. parapertussis, and B. holmesii was developed using multicopy insertion sequences (ISs) in combination with the pertussis toxin subunit S1 (ptxS1) singleplex assay. The RT-PCR targets for the multiplex assay include IS481, commonly found in B. pertussis and B. holmesii; IS1001 of B. parapertussis; and the IS1001-like sequence of B. holmesii. Overall, 402 Bordetella species and 66 non-Bordetella species isolates were tested in the multitarget assay. Cross-reactivity was found only with 5 B. bronchiseptica isolates, which were positive with IS1001 of B. parapertussis. The lower limit of detection (LLOD) of the multiplex assay was similar to the LLOD of each target in an individual assay format, which was approximately 1 genomic equivalent per reaction for all targets. A total of 197 human clinical specimens obtained during cough-illness outbreak investigations were used to evaluate the multitarget RT-PCR assay. The multiplex assay results from 87 clinical specimens were compared to the individual RT-PCR assay and culture results. The multitarget assay is useful as a diagnostic tool to confirm B. pertussis infections and to rapidly identify other Bordetella species. In conclusion, the use of this multitarget RT-PCR approach increases specificity, while it decreases the amount of time, reagents, and specimen necessary for RT-PCRs used for accurate diagnosis of pertussis-like illness. PMID:21940464

  3. Inactivation conditions for human Norovirus measured by an in situ capture-qRT-PCR Method

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Human noroviruses (HuNoVs) are the major cause of epidemic non-bacterial gastroenteritis. Due to the inability to cultivate HuNoVs, it has been a challenge to determine their infectivity. Quantitative real-time RT-PCR (qRT-PCR) is widely used in detecting HuNoVs. However, qRT-PCR only detects the...

  4. Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs

    PubMed Central

    Varkonyi-Gasic, Erika; Wu, Rongmei; Wood, Marion; Walton, Eric F; Hellens, Roger P

    2007-01-01

    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

  5. Rapid detection of lineage IV peste des petits ruminants virus by real-time RT-PCR.

    PubMed

    Li, Lin; Wu, Xiaodong; Liu, Fuxiao; Wang, Zhiliang; Liu, Chunju; Wang, Qinghua; Bao, Jingyue

    2016-09-01

    Peste des petits ruminants virus (PPRV) is the cause agent of peste des petitis ruminants (PPR). A novel lineage IV PPRV has reemerged in China in 2013 and 2014. Mass vaccination was implemented in most provinces in China. In order to detect lineage IV PPRV in clinical samples and to distinguish rapidly it from the other lineages PPRVs, a real-time RT-PCR assay was developed. This assay showed high sensitivity, specificity and efficiency in differentiating the lineage IV PPRV from others. The performance of this assay was evaluated by positive clinical samples of lineage IV viruses. This new real-time RT-PCR assay will facilitate epidemiological investigations and rapid differentiatial diagnosis in areas where lineage IV viruses are circulating. PMID:27260657

  6. Diffuse large B-cell lymphoma: sub-classification by massive parallel quantitative RT-PCR.

    PubMed

    Xue, Xuemin; Zeng, Naiyan; Gao, Zifen; Du, Ming-Qing

    2015-01-01

    Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous entity with remarkably variable clinical outcome. Gene expression profiling (GEP) classifies DLBCL into activated B-cell like (ABC), germinal center B-cell like (GCB), and Type-III subtypes, with ABC-DLBCL characterized by a poor prognosis and constitutive NF-κB activation. A major challenge for the application of this cell of origin (COO) classification in routine clinical practice is to establish a robust clinical assay amenable to routine formalin-fixed paraffin-embedded (FFPE) diagnostic biopsies. In this study, we investigated the possibility of COO-classification using FFPE tissue RNA samples by massive parallel quantitative reverse transcription PCR (qRT-PCR). We established a protocol for parallel qRT-PCR using FFPE RNA samples with the Fluidigm BioMark HD system, and quantified the expression of the COO classifier genes and the NF-κB targeted-genes that characterize ABC-DLBCL in 143 cases of DLBCL. We also trained and validated a series of basic machine-learning classifiers and their derived meta classifiers, and identified SimpleLogistic as the top classifier that gave excellent performance across various GEP data sets derived from fresh-frozen or FFPE tissues by different microarray platforms. Finally, we applied SimpleLogistic to our data set generated by qRT-PCR, and the ABC and GCB-DLBCL assigned showed the respective characteristics in their clinical outcome and NF-κB target gene expression. The methodology established in this study provides a robust approach for DLBCL sub-classification using routine FFPE diagnostic biopsies in a routine clinical setting. PMID:25418578

  7. Lung Cancer Lymph Node Micrometastasis Detection Using RT-PCR – Correlation with Vascular Endothelial Growth Factor (VEGF) expression

    PubMed Central

    Nwogu, Chukwumere E.; Yendamuri, Sai; Tan, Wei; Kannisto, Eric; Bogner, Paul; Morrison, Carl; Cheney, Richard; Dexter, Elisabeth; Picone, Anthony; Hennon, Mark; Hutson, Alan; Reid, Mary; Adjei, Alex; Demmy, Todd L.

    2013-01-01

    Objectives Lymph node (LN) staging provides critical information in non-small cell lung cancer (NSCLC) patients. Lymphangiogenesis may be an important contributor to the pathophysiology of lymphatic metastases. We hypothesized that the presence of lymph node micrometastases positively correlates with VEGF-A/C/D and VEGF-receptor-3 (lymphangiogenic factors) expression in lymph nodes. Methods Forty NSCLC patients had pre-operative PET-CT and mediastinoscopy. RT-PCR assays for mRNA expression of epithelial markers (CK-7, CEACAM-5 and PLUNC) were performed in selected fluorodeoxyglucose (FDG)-avid lymph nodes. VEGF-A/C/D and VEGF-receptor-3 expression levels were measured in primary tumors and lymph nodes. Wilcoxon rank sum test was run for the association between the RT-PCR epithelial marker levels and VEGF expression levels in the LNs. Results RT-PCR for CK-7, CEACAM5 or PLUNC indicated lymph node micrometastatic disease in 19 of 35 patients (54%). There was a high correlation between detection of micrometastases and VEGF-A/C/D or VEGF-receptor-3 expression levels in lymph nodes. Median follow-up was 12.6 months. Conclusions RT-PCR analysis of FDG-avid lymph nodes results in up-staging of patients. Micrometastases correlate with the expression of VEGF in lymph nodes in NSCLC patients. This may reflect the role of lymphangiogenesis in promoting metastases. PMID:23414988

  8. Detection and quantitation of Citrus leaf blotch virus by TaqMan real-time RT-PCR.

    PubMed

    Ruiz-Ruiz, Susana; Ambrós, Silvia; Vives, María del Carmen; Navarro, Luis; Moreno, Pedro; Guerri, José

    2009-09-01

    A real-time RT-PCR assay based on the TaqMan chemistry was developed for reliable detection and quantitation of Citrus leaf blotch virus (CLBV) in citrus plants. Detection by this method was highly specific and about one thousand times more sensitive than detection by conventional RT-PCR. An external standard curve using in vitro synthesized RNA transcripts of the selected target allowed a reproducible quantitative assay, with a wide dynamic range (seven logarithmic units of concentration) and very low variation coefficient values. This protocol enabled detection of as little as 100 copies of CLBV RNA in various tissues and citrus varieties infected with CLBV sources from different geographical origins. The new assay greatly improves current detection methods for CLBV and it has been most helpful for the Spanish citrus sanitation, quarantine and certification programs, and fitness evaluation of infectious cDNA clones of CLBV, useful potentially as viral vectors for citrus. PMID:19406167

  9. Quantitative real-time RT-PCR and chromogenic in situ hybridization: precise methods to detect HER-2 status in breast carcinoma

    PubMed Central

    2009-01-01

    Background HER-2 gene testing has become an integral part of breast cancer patient diagnosis. The most commonly used assay in the clinical setting for evaluating HER-2 status is immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). These procedures permit correlation between HER-2 expression and morphological features. However, FISH signals are labile and fade over time, making post-revision of the tumor difficult. CISH (chromogenic in situ hybridization) is an alternative procedure, with certain advantages, although still limited as a diagnostic tool in breast carcinomas. Methods To elucidate the molecular profile of HER-2 status, mRNA and protein expression in 75 invasive breast carcinomas were analyzed by real time quantitative RT-PCR (qRT-PCR) and IHC, respectively. Amplifications were evaluated in 43 of these cases by CISH and in 11 by FISH. Results The concordance rate between IHC and qRT-PCR results was 78.9%, and 94.6% for qRT-PCR and CISH. Intratumoral heterogeneity of HER-2 status was identified in three cases by CISH. The results of the three procedures were compared and showed a concordance rate of 83.8%; higher discordances were observed in 0 or 1+ immunostaining cases, which showed high-level amplification (15.4%) and HER-2 transcript overexpression (20%). Moreover, 2+ immunostaining cases presented nonamplified status (50%) by CISH and HER-2 downexpression (38.5%) by qRT-PCR. In general, concordance occurred between qRT-PCR and CISH results. A high concordance was observed between CISH/qRT-PCR and FISH. Comparisons with clinicopathological data revealed a significant association between HER-2 downexpression and the involvement of less than four lymph nodes (P = 0.0350). Conclusion Based on these findings, qRT-PCR was more precise and reproducible than IHC. Furthermore, CISH was revealed as an alternative and useful procedure for investigating amplifications involving the HER-2 gene. PMID:19309522

  10. Quantitative RT-PCR analysis of the MOZ-CBP fusion transcript in therapy-related acute myeloid leukemia with t(8;16)(p11;p13).

    PubMed

    Fujiki, Atsushi; Imamura, Toshihiko; Furutani, Akiyo; Hatano, Waka; Asai, Daisuke; Hirashima, Yoshifumi; Miyachi, Mitsuru; Tamura, Shinichi; Tsuchiya, Kunihiko; Iehara, Tomoko; Ishida, Hiroyuki; Yoshihara, Takao; Hosoi, Hajime

    2012-07-01

    We developed a real time reverse transcriptase polymerase chain reaction (RT-PCR) assay system for detecting the MOZ-CBP fusion transcript and used it to monitor minimal residual disease (MRD) status in a patient with therapy related acute myeloid leukemia (t-AML) harboring t(8;16)(p11;p13). Expression of the MOZ-CBP fusion transcript was determined by RT-PCR analysis of the patient's bone marrow at the time of diagnosis. Thereafter, real time RT-PCR was used to evaluate MRD levels throughout the entire course of treatment. The sensitivity of quantitative RT-PCR for the MOZ-CBP fusion transcript was 10(-5). Below this level, MRD was classified as negative. Real time RT-PCR of the bone marrow after induction therapy showed the reduction of MOZ-CBP transcript to approximately 10(-3) level when compared to the diagnostic sample. MRD was classified as negative (< 10(-5) compared with that in the bone marrow at diagnosis) after 5 courses of chemotherapy, a level that was maintained post-allo-hematopoietic stem cell transplantation. Real time RT-PCR of the MOZ-CBP transcript is a useful tool for assessing MRD status for a patient with therapy related acute myeloid leukemia who was initially predicted to have a poor prognosis. PMID:22278196

  11. Simultaneous detection of West Nile and Japanese encephalitis virus RNA by duplex TaqMan RT-PCR.

    PubMed

    Barros, Silvia C; Ramos, Fernanda; Zé-Zé, Líbia; Alves, Maria J; Fagulha, Teresa; Duarte, Margarida; Henriques, Margarida; Luís, Tiago; Fevereiro, Miguel

    2013-11-01

    West Nile virus (WNV) and Japanese encephalitis virus (JEV) are important mosquito-borne viruses of the Flaviviridae family, associated with encephalitis, mainly in humans and horses. WNV is also pathogen for many bird species. The incidence of human and animal WNV infections in Europe has risen, mostly in recent years, and JEV was detected in 2011 in mosquitoes collected in Italy and may emerge in Europe in the same way as other flaviviruses had emerged recently (USUTU and Bagaza virus) and should be regarded as a potential threat to public health. Prompt identification and discrimination between WNV and JEV provides critical epidemiological data for prevalence studies and public and animal health management policies. Here we describe a quantitative one-step duplex TaqMan RT-PCR, targeting non-structural protein 2A gene (NS2A-qRT-PCR), based on only one primer pair and two probes for differential diagnosis of WNV and JEV. Also this assay enables the detection of both WNV lineages (WNV-1 and WNV-2). To access the specificity of NS2A-qRT-PCR a panel of different arboviruses were used. The assay was shown to be specific for both WNV lineages (WNV-1 and WNV-2), WNV related Kunjin virus and JEV, since no cross-reactions were observed with other tested arboviruses. Sensitivity of the assay was determined using serial dilutions of in vitro-transcribed RNA from WNV and JEV. The duplex NS2A-qRT-PCR assay was shown to be very sensitive, being able to detect 10 copies of WNV and JEV RNA. This assay is a suitable tool for the diagnosis of WNV and JEV, and provides a valuable addition to the methods currently available for routine diagnosis of these zoonoses and for surveillance studies. PMID:23892127

  12. Evaluation of viral extraction methods on a broad range of Ready-To-Eat foods with conventional and real-time RT-PCR for Norovirus GII detection.

    PubMed

    Baert, Leen; Uyttendaele, Mieke; Debevere, Johan

    2008-03-31

    Noroviruses (NoV) are a common cause of foodborne outbreaks. In spite of that, no standard viral detection method is available for food products. Therefore, three viral elution-concentration methods and one direct RNA isolation method were evaluated on a broad range of Ready-To-Eat (RTE) food products (mixed lettuce, fruit salad, raspberries and two RTE dishes) artificially seeded with a diluted stool sample contaminated with NoV genogroup II. These seeding experiments revealed two categories of RTE products, fruits and vegetables grouped together and RTE dishes (penne and tagliatelle salads) which are rich in proteins and fat formed another category. The RNA extracts were amplified and detected with two conventional RT-PCR systems (Booster and Semi-nested GII) and one real-time RT-PCR (Real-time GII) assay. A fast direct RNA isolation method detected 10(2) RT-PCRU on 10 g penne and tagliatelle salads with the conventional RT-PCR assays. However real-time RT-PCR was less sensitive for penne salad. A viral elution-concentration method, including a buffer solution for the elution step and one polyethylene glycol (PEG) precipitation step, was able to detect 10(2) RT-PCRU on 50 g frozen raspberries with conventional and real-time RT-PCR assays. Moreover the latter extraction method used no environmental hazardous chemical reagents and was easy to perform. PMID:18258325

  13. Influenza A virus drift variants reduced the detection sensitivity of a commercial multiplex nucleic acid amplification assay in the season 2014/15.

    PubMed

    Huzly, Daniela; Korn, Klaus; Bierbaum, Sibylle; Eberle, Björn; Falcone, Valeria; Knöll, Antje; Steininger, Philipp; Panning, Marcus

    2016-09-01

    The influenza season 2014/15 was dominated by drift variants of influenza A(H3N2), which resulted in a reduced vaccine effectiveness. It was not clear if the performance of commercial nucleic-acid-based amplification (NAT) assays for the detection of influenza was affected. The purpose of this study was to perform a real-life evaluation of two commercial NAT assays. During January-April 2015, we tested a total of 665 samples from patients with influenza-like illness using the Fast Track Diagnostics Respiratory pathogens 21, a commercial multiplex kit, (cohorts 1 and 2, n = 563 patients) and the Xpert Flu/RSV XC assay (cohort 3, n = 102 patients), a single-use cartridge system. An in-house influenza real-time RT-PCR (cohort 1) and the RealStar Influenza RT-PCR 1.0 Kit (cohort 2 and 3) served as reference tests. Compared to the reference assay, an overall agreement of 95.9 % (cohort 1), 95 % (cohort 2), and 98 % (cohort 3) was achieved. A total of 24 false-negative results were observed using the Fast Track Diagnostics Respiratory pathogens 21 kit. No false-negative results occurred using the Xpert Flu/RSV XC assay. The Fast Track Diagnostics Respiratory pathogens 21 kit and the Xpert Flu/RSV XC assay had sensitivities of 90.7 % and 100 % and specificities of 100 % and 94.1 %, respectively, compared to the RealStar 1.0 kit. Upon modification of the Fast Track Diagnostics Respiratory pathogens 21 kit, the sensitivity increased to 97.3 %. Influenza virus strains circulating during the 2014/15 season reduced the detection sensitivity of a commercial NAT assay, and continuous monitoring of test performance is therefore necessary. PMID:27316440

  14. Data transformation methods for multiplexed assays

    DOEpatents

    Tammero, Lance F. Bentley; Dzenitis, John M; Hindson, Benjamin J

    2013-07-23

    Methods to improve the performance of an array assay are described. A correlation between fluorescence intensity-related parameters and negative control values of the assay is determined. The parameters are then adjusted as a function of the correlation. As a result, sensitivity of the assay is improved without changes in its specificity.

  15. Detection of Langat virus by TaqMan real-time one-step qRT-PCR method

    PubMed Central

    Muhd Radzi, Siti Fatimah; Rückert, Claudia; Sam, Sing-Sin; Teoh, Boon-Teong; Jee, Pui-Fong; Phoon, Wai-Hong; Abubakar, Sazaly; Zandi, Keivan

    2015-01-01

    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

  16. Multiplexed Recombinase Polymerase Amplification Assay To Detect Intestinal Protozoa.

    PubMed

    Crannell, Zachary; Castellanos-Gonzalez, Alejandro; Nair, Gayatri; Mejia, Rojelio; White, A Clinton; Richards-Kortum, Rebecca

    2016-02-01

    This work describes a proof-of-concept multiplex recombinase polymerase amplification (RPA) assay with lateral flow readout that is capable of simultaneously detecting and differentiating DNA from any of the diarrhea-causing protozoa Giardia, Cryptosporidium, and Entamoeba. Together, these parasites contribute significantly to the global burden of diarrheal illness. Differential diagnosis of these parasites is traditionally accomplished via stool microscopy. However, microscopy is insensitive and can miss up to half of all cases. DNA-based diagnostics such as polymerase chain reaction (PCR) are far more sensitive; however, they rely on expensive thermal cycling equipment, limiting their availability to centralized reference laboratories. Isothermal DNA amplification platforms, such as the RPA platform used in this study, alleviate the need for thermal cycling equipment and have the potential to broaden access to more sensitive diagnostics. Until now, multiplex RPA assays have not been developed that are capable of simultaneously detecting and differentiating infections caused by different pathogens. We developed a multiplex RPA assay to detect the presence of DNA from Giardia, Cryptosporidium, and Entamoeba. The multiplex assay was characterized using synthetic DNA, where the limits-of-detection were calculated to be 403, 425, and 368 gene copies per reaction of the synthetic Giardia, Cryptosporidium, and Entamoeba targets, respectively (roughly 1.5 orders of magnitude higher than for the same targets in a singleplex RPA assay). The multiplex assay was also characterized using DNA extracted from live parasites spiked into stool samples where the limits-of-detection were calculated to be 444, 6, and 9 parasites per reaction for Giardia, Cryptosporidium, and Entamoeba parasites, respectively. This proof-of-concept assay may be reconfigured to detect a wide variety of targets by re-designing the primer and probe sequences. PMID:26669715

  17. Detection and genogrouping of noroviruses from children's stools by Taqman One-step RT-PCR.

    PubMed

    Apaza, Sonia; Espetia, Susan; Gilman, Robert H; Montenegro, Sonia; Pineda, Susana; Herhold, Fanny; Pomari, Romeo; Kosek, Margaret; Vu, Nancy; Saito, Mayuko

    2012-01-01

    consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups. PMID:22898754

  18. Evaluation of reference genes for quantitative RT-PCR in Lolium perenne

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quantitative real-time RT-PCR provides an important tool for analyzing gene expression if proper internal standards are used. The aim of this study was to identify and evaluate reference genes for use in real-time quantitative RT-PCR in perennial ryegrass (Lolium perenne L.) during plant developmen...

  19. Methods for threshold determination in multiplexed assays

    DOEpatents

    Tammero, Lance F. Bentley; Dzenitis, John M; Hindson, Benjamin J

    2014-06-24

    Methods for determination of threshold values of signatures comprised in an assay are described. Each signature enables detection of a target. The methods determine a probability density function of negative samples and a corresponding false positive rate curve. A false positive criterion is established and a threshold for that signature is determined as a point at which the false positive rate curve intersects the false positive criterion. A method for quantitative analysis and interpretation of assay results together with a method for determination of a desired limit of detection of a signature in an assay are also described.

  20. Identification and Validation of Reference Genes for qRT-PCR Studies of Gene Expression in Dioscorea opposita

    PubMed Central

    Zhao, Xiting; Zhang, Xiaoli; Guo, Xiaobo; Li, Shujie; Han, Linlin; Song, Zhihui; Wang, Yunying; Li, Junhua; Li, Mingjun

    2016-01-01

    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

  1. Identification and Validation of Reference Genes for qRT-PCR Studies of Gene Expression in Dioscorea opposita.

    PubMed

    Zhao, Xiting; Zhang, Xiaoli; Guo, Xiaobo; Li, Shujie; Han, Linlin; Song, Zhihui; Wang, Yunying; Li, Junhua; Li, Mingjun

    2016-01-01

    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

  2. Whole genome alignment based one-step real-time RT-PCR for universal detection of avian orthoreoviruses of chicken, pheasant and turkey origins.

    PubMed

    Tang, Yi; Lu, Huaguang

    2016-04-01

    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. PMID:26812128

  3. Quantitative RT-PCR assessment of melanoma cells in peripheral blood during immunotherapy for metastatic melanoma.

    PubMed

    Schmidt, H; Sørensen, B S; von der Maase, H; Bang, C; Agger, R; Hokland, M; Nexo, E

    2002-12-01

    Circulating malignant cells in peripheral blood are thought to be precursors and surrogate markers of distant metastases and hence markers of a poor clinical outcome. In this study, we used the detection of MART-1 and tyrosinase (TYR) mRNA with a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay to identify circulating melanoma cells. Blood samples were obtained from 35 patients with metastatic melanoma before, during and after treatment with interleukin-2, interferon-alpha and cisplatin. In addition, MART-1 and TYR protein was identified by immunohistochemistry in consecutive biopsies from 15 of the patients. Analysis of three daily blood samples for 3 days demonstrated that four out of 11 patients examined were negative for both markers on all occasions, and two patients were positive for both markers on all occasions but one. The remaining five patients showed sporadic low positive results for one or the other of the two markers. By comparing the immunohistochemistry results from consecutive biopsies with the RT-PCR results, we demonstrated that patients with MART-1 and TYR protein in their tumour cells had circulating MART-1 and TYR mRNA in 77% and 54% of the cases, respectively. During treatment, the majority of patients who were positive for MART-1 and TYR mRNA converted to being negative. However, these conversions did not significantly correlate with objective response. The presence of TYR mRNA in one of the first two samples showed a trend towards being an independent prognostic factor for poor survival. PMID:12459648

  4. One-Step RT-PCR protocols improve the rate of dengue diagnosis compared to Two-Step RT-PCR approaches.

    PubMed

    De Paula, Sérgio Oliveira; de Melo Lima, Cristiane; Torres, Maria Paula; Pereira, Márcia Rodrigues Garbin; Lopes da Fonseca, Benedito Antônio

    2004-08-01

    Dengue is the most important arboviral disease transmitted to humans. In our laboratory, we have been working on the standardization of the polymerase chain reaction (PCR) diagnosis of this disease. In this work, we compared five commercial kits regularly used on reverse-transcription polymerase chain reaction (RT-PCR) protocols: two Two-Step kits (SuperScript II RT/Super Mix kit and reverse transcription system/Taq DNA polymerase) and three One-Step kits (ready-to-go RT-PCR Beads kit, QIAGEN One-Step RT-PCR kit, and AcessQuick RT-PCR system). Thirty-one serum samples of patients with clinical diagnosis of dengue fever (DF) were analyzed by RT-PCR and serology. RNA extraction was done with the QIAamp Viral RNA kit, and cDNA synthesis and PCR done according to the manufacturer's protocol for the five kits. Out of the 31 serum samples collected from patients suspected of having dengue, 27 were IgM-positive, confirming the dengue diagnosis. Out of those, 24 were positive by the ready-to-go RT-PCR Beads kit, 25 were positive by AcessQuick RT-PCR system and 27 were positive by QIAGEN One-Step RT-PCR kit. On the other hand, only six samples were positive by the SuperScript II RT/Super Mix kits and 10 were positive by reverse transcription system/Taq DNA polymerase kit. The best performance observed with the One-Step kits was confirmed in spiked samples with known quantities of dengue-1 virus since they detected up 1 x 10(2) PFU/ml, while the most sensitive Two-Step kit detected up 1 x 10(4) PFU/ml. These data show that One-Step RT-PCR kits yielded a higher rate of dengue virus detection than the Two-Step kits and correlated well with the serological diagnosis. PMID:15163417

  5. RT-PCR analysis of dystrophin mRNA in DND/BMD patients

    SciTech Connect

    Ciafaloni, E.; Silva, H.A.R. de; Roses, A.D.

    1994-09-01

    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.

  6. Computational tradeoffs in multiplex PCR assay design for SNP genotyping

    PubMed Central

    Rachlin, John; Ding, Chunming; Cantor, Charles; Kasif, Simon

    2005-01-01

    Background Multiplex PCR is a key technology for detecting infectious microorganisms, whole-genome sequencing, forensic analysis, and for enabling flexible yet low-cost genotyping. However, the design of a multiplex PCR assays requires the consideration of multiple competing objectives and physical constraints, and extensive computational analysis must be performed in order to identify the possible formation of primer-dimers that can negatively impact product yield. Results This paper examines the computational design limits of multiplex PCR in the context of SNP genotyping and examines tradeoffs associated with several key design factors including multiplexing level (the number of primer pairs per tube), coverage (the % of SNP whose associated primers are actually assigned to one of several available tube), and tube-size uniformity. We also examine how design performance depends on the total number of available SNPs from which to choose, and primer stringency criterial. We show that finding high-multiplexing/high-coverage designs is subject to a computational phase transition, becoming dramatically more difficult when the probability of primer pair interaction exceeds a critical threshold. The precise location of this critical transition point depends on the number of available SNPs and the level of multiplexing required. We also demonstrate how coverage performance is impacted by the number of available snps, primer selection criteria, and target multiplexing levels. Conclusion The presence of a phase transition suggests limits to scaling Multiplex PCR performance for high-throughput genomics applications. Achieving broad SNP coverage rapidly transitions from being very easy to very hard as the target multiplexing level (# of primer pairs per tube) increases. The onset of a phase transition can be "delayed" by having a larger pool of SNPs, or loosening primer selection constraints so as to increase the number of candidate primer pairs per SNP, though the latter

  7. Molecular simultaneous detection of Cherry necrotic rusty mottle virus and Cherry green ring mottle virus by real-time RT-PCR and high resolution melting analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  8. Change your conventional practice of qRT-PCR: A simple robust quality control standard for yeast mRNA quantification analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real time quantitative reverse transcription polymerase chain reaction (qRT-PCR) has been realized to be the assay of choice among available techniques for mRNA quantification analysis. For conventional practice, housekeeping genes have been applied as internal reference for data normalization and a...

  9. Real-time RT-PCR for detection of Raspberry bushy dwarf virus, Raspberry leaf mottle virus and characterizing synergistic interactions in mixed infections

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  10. A Multiplex Assay for the Diagnosis of Mucopolysaccharidoses and Mucolipidoses

    PubMed Central

    Langereis, Eveline J.; Wagemans, Tom; Kulik, Wim; Lefeber, Dirk J.; van Lenthe, Henk; Oussoren, Esmee; van der Ploeg, Ans T.; Ruijter, George J.; Wevers, Ron A.; Wijburg, Frits A.; van Vlies, Naomi

    2015-01-01

    Introduction Diagnosis of the mucopolysaccharidoses (MPSs) generally relies on an initial analysis of total glycosaminoglycan (GAG) excretion in urine. Often the dimethylmethylene blue dye-binding (DMB) assay is used, although false-negative results have been reported. We report a multiplexed diagnostic test with a high sensitivity for all MPSs and with the potential to identify patients with I-cell disease (ML II) and mucolipidosis III (ML III). Methods Urine samples of 100 treatment naive MPS patients were collected and analyzed by the conventional DMB assay and a multiplex assay based on enzymatic digestion of heparan sulfate (HS), dermatan sulfate (DS) and keratan sulfate (KS) followed by quantification by LC-MS/MS. Specificity was calculated by analyzing urine samples from a cohort of 39 patients suspected for an inborn error of metabolism, including MPSs. Results The MPS cohort consisted of 18 MPS I, 16 MPS II, 34 MPS III, 10 MPS IVA, 3 MPS IVB, 17 MPS VI and 2 MPS VII patients. All 100 patients were identified by the LC-MS/MS assay with typical patterns of elevation of HS, DS and KS, respectively (sensitivity 100%). DMB analysis of the urine was found to be in the normal range in 10 of the 100 patients (sensitivity 90%). Three out of the 39 patients were identified as false-positive, resulting in a specificity of the LS-MS/MS assay of 92%. For the DMB this was 97%. All three patients with MLII/MLIII had elevated GAGs in the LC-MS/MS assay while the DMB test was normal in 2 of them. Conclusion The multiplex LC-MS/MS assay provides a robust and very sensitive assay for the diagnosis of the complete spectrum of MPSs and has the potential to identify MPS related disorders such as MLII/MLIII. Its performance is superior to that of the conventional DMB assay. PMID:26406883

  11. Quantification of Bacterial Transcripts during Infection Using Competitive Reverse Transcription-PCR (RT-PCR) and LightCycler RT-PCR

    PubMed Central

    Goerke, Christiane; Bayer, Manfred G.; Wolz, Christiane

    2001-01-01

    Bacteria have evolved sophisticated regulatory circuits to modulate their gene expression in response to disparate environments. In order to monitor bacterial gene expression and regulation in the host, methods for direct transcript analysis from clinical specimens are needed. For most bacterial infections, amplification of the mRNAs of interest is necessary due to the low numbers of cells present and the low levels of specific transcripts. Here we compare two methods of quantitative reverse transcription-PCR (RT-PCR)—competitive RT-PCR using a one-tube system followed by standard gel analysis and the real-time detection of PCR product formation by fluorescence resonance energy transfer technology using the LightCycler unit. We isolated Staphylococcus aureus RNA directly from clinical specimens obtained from cystic fibrosis patients with chronic S. aureus lung infection and from an animal model of foreign-body infection with no further cultivation of the bacteria. Competitive RT-PCR and LightCycler RT-PCR were tested for their ability to quantify the transcription of a constitutively expressed gyrase gene (gyr) and a highly regulated α-toxin gene (hla) of S. aureus. Reproducible results were obtained with both methods. A sensitivity of 104 (gyr) and 103 (hla) copies, respectively, was reached, which was sufficient for the quantification of transcripts during bacterial infection. Overall, the competitive RT-PCR is a robust technique which does not need special RNA purification. On the negative side, it is labor intensive and time consuming, thus limiting the numbers of samples which can be analyzed at a given time. LightCycler RT-PCR is very susceptible to even traces of inhibitors, but it allows high-throughput processing of samples. PMID:11238208

  12. Gastroenteritis outbreaks associated with Norwalk-like viruses and their investigation by nested RT-PCR

    PubMed Central

    O'Neill, Hugh J; McCaughey, Conall; Wyatt, Dorothy E; Mitchell, Frederick; Coyle, Peter V

    2001-01-01

    Background Norwalk-like viruses are the most common cause of gastroenteritis outbreaks and sporadic cases of vomiting and diarrhoea. In healthy individuals infection is often mild and short-lived but in debilitated patients infection can be severe. It is essential that the virus laboratory can offer a sensitive and specific test, delivered in a timely manner. Methods We have developed a nested reverse transcriptase PCR based on published primers against the RNA polymerase gene and after comparison with electronmicroscopy used the assay to investigate 31 outbreaks of gastroenteritis. These were in diverse situations including nursing homes, small district hospitals, large general hospitals, a ferry ship, hotels, restaurants and staff canteens. Results A positive diagnosis was made in 30/31 outbreaks investigated giving an overall outbreak positive detection rate of 97%. At an individual patient level there was a positive diagnostic rate of 11.5% in a large hospital environment to 100% in smaller outbreak situations. The average patient positive rate was 34%. In addition we investigated 532 control faecal specimens from adults. Of these 530 were negative and 2 were repeatedly positive. Conclusions It is essential that insensitive electronmicroscopy is replaced with the more sensitive reverse transcription PCR assays. These tests should be made available "on call" at weekends and public holidays. It is also important that outbreaks of NLV infection are monitored using sensitive RT-PCR assays so that the laboratory information can be used in ascertaining the spread and duration of the outbreak PMID:11511325

  13. 384-Well Multiplexed Luminex Cytokine Assays for Lead Optimization.

    PubMed

    Tang, Huaping; Panemangalore, Reshma; Yarde, Melissa; Zhang, Litao; Cvijic, Mary Ellen

    2016-07-01

    Cytokines serve as a major mechanism of communication between immune cells and are the functional molecules at the end of immune pathways. Abnormalities in cytokines are involved in a wide variety of diseases, including chronic inflammation, autoimmune diseases, and cancer. Cytokines are not only direct targets of therapeutics but also important biomarkers for assessing drug efficacy and safety. Traditionally, enzyme-linked immunosorbent assays (ELISA) were most popular for identifying and quantifying cytokines. However, ELISA is expensive, labor intensive, and low throughput. Here, we report the development of a miniaturized Luminex (Austin, TX) assay platform to establish a panel of high-throughput, multiplexed assays for measuring cytokines in human whole blood. The miniaturized 384-well Luminex assay uses <25% of the assay reagents compared with the 96-well assay. The development and validation of the 384-well Luminex cytokine assays enabled high-throughput screening of compounds in primary cells using cytokines as physiologically relevant readouts. Furthermore, this miniaturized multiplexed technology platform allows for high-throughput biomarker profiling of biofluids from animal studies and patient samples for translational research. PMID:27095819

  14. Detection of West Nile viral RNA from field-collected mosquitoes in tropical regions by conventional and real-time RT-PCR.

    PubMed

    González-Reiche, Ana Silvia; Monzón-Pineda, María de Lourdes; Johnson, Barbara W; Morales-Betoulle, María Eugenia

    2010-01-01

    West Nile virus (WNV) is an emerging mosquito-borne flavivirus, which has rapidly spread and is currently widely distributed. Therefore, efforts for WNV early detection and ecological surveillance of this disease agent have been increased around the world. Although virus isolation is known to be the standard method for detection and identification of viruses, the use of RT-PCR assays as routine laboratory tests provides a rapid alterative suitable for the detection of viral RNA on field-collected samples. A method for WNV RNA genome detection in field-collected mosquitoes is presented in this chapter. This method has been designed for virus surveillance in tropical regions endemic for other flaviviruses. Reverse Transcriptase-PCR (RT-PCR) assays, both standard and real time, to detect WNV and other flaviviruses are described. A first screening for flavivirus RNA detection is performed using a conventional RT-PCR with two different sets of flavivirus consensus primers. Mosquito samples are then tested for WNV RNA by a real-time (TaqMan) RT-PCR assay. Sample preparation and RNA extraction procedures are also described. PMID:20300994

  15. Evaluation of Four Different Systems for Extraction of RNA from Stool Suspensions Using MS-2 Coliphage as an Exogenous Control for RT-PCR Inhibition

    PubMed Central

    Shulman, Lester M.; Hindiyeh, Musa; Muhsen, Khitam; Cohen, Dani; Mendelson, Ella; Sofer, Danit

    2012-01-01

    Knowing when, and to what extent co-extracted inhibitors interfere with molecular RNA diagnostic assays is of utmost importance. The QIAamp Viral RNA Mini Kit (A); MagNA Pure LC2.0 Automatic extractor (B); KingFisher (C); and NucliSENS EasyMag (D) RNA extraction systems were evaluated for extraction efficiency and co-purification of inhibitors from stool suspensions. Real-Time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR) of MS-2 coliphage spiked into each system’s lysis buffer served as an external control for both. Cycle thresholds (Cts) of the MS2 were determined for RNA extracted from stool suspensions containing unknown (n = 93) or varying amounts of inhibitors (n = 92). Stool suspensions from the latter group were also used to determine whether MS-2 and enterovirus rRT-PCR inhibitions were correlated. Specifically 23 RNA extracts from stool suspensions were spiked with enterovirus RNA after extraction and 13 of these stool suspension were spiked with intact enterovirus before extraction. MS2 rRT-PCR inhibition varied for RNAs extracted by the different systems. Inhibition was noted in 12 (13.0%), 26 (28.3%), 7 (7.6%), and 7 (7.6%) of the first 93 RNA extracts, and 58 (63.0%), 55 (59.8%), 37 (40.2%) and 30 (32.6%) of the second 92 extracts for A, B, C, and D, respectively. Furthermore, enterovirus rRT-PCR inhibition correlated with MS2 rRT-PCR inhibition for added enterovirus RNA or virus particles. In conclusion, rRT-PCR for MS-2 RNA is a good predictor of inhibition of enterovirus RNA extracted from stool suspensions. EasyMag performed the best, however all four extraction methods were suitable provided that external controls identified problematic samples. PMID:22815706

  16. Hepatitis C Virus RNA Real-Time Quantitative RT-PCR Method Based on a New Primer Design Strategy.

    PubMed

    Chen, Lida; Li, Wenli; Zhang, Kuo; Zhang, Rui; Lu, Tian; Hao, Mingju; Jia, Tingting; Sun, Yu; Lin, Guigao; Wang, Lunan; Li, Jinming

    2016-01-01

    Viral nucleic acids are unstable when improperly collected, handled, and stored, resulting in decreased sensitivity of currently available commercial quantitative nucleic acid testing kits. Using known unstable hepatitis C virus RNA, we developed a quantitative RT-PCR method based on a new primer design strategy to reduce the impact of nucleic acid instability on nucleic acid testing. The performance of the method was evaluated for linearity, limit of detection, precision, specificity, and agreement with commercial hepatitis C virus assays. Its clinical application was compared to that of two commercial kits--Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) and Kehua. The quantitative RT-PCR method delivered a good performance, with a linearity of R(2) = 0.99, a total limit of detection (genotypes 1 to 6) of 42.6 IU/mL (95% CI, 32.84 to 67.76 IU/mL), a CV of 1.06% to 3.34%, a specificity of 100%, and a high concordance with the CAP/CTM assay (R(2) = 0.97), with a means ± SD value of -0.06 ± 1.96 log IU/mL (range, -0.38 to 0.25 log IU/mL). The method was superior to commercial assays in detecting unstable hepatitis C virus RNA (P < 0.05). This quantitative RT-PCR method can effectively eliminate the influence of RNA instability on nucleic acid testing. The principle of primer design strategy may be applied to the detection of other RNA or DNA viruses. PMID:26612712

  17. A universal TaqMan-based RT-PCR protocol for cost-efficient detection of small noncoding RNA

    PubMed Central

    Jung, Ulrike; Jiang, Xiaoou; Kaufmann, Stefan H.E.; Patzel, Volker

    2013-01-01

    Several methods for the detection of RNA have been developed over time. For small RNA detection, a stem–loop reverse primer-based protocol relying on TaqMan RT-PCR has been described. This protocol requires an individual specific TaqMan probe for each target RNA and, hence, is highly cost-intensive for experiments with small sample sizes or large numbers of different samples. We describe a universal TaqMan-based probe protocol which can be used to detect any target sequence and demonstrate its applicability for the detection of endogenous as well as artificial eukaryotic and bacterial small RNAs. While the specific and the universal probe-based protocol showed the same sensitivity, the absolute sensitivity of detection was found to be more than 100-fold lower for both than previously reported. In subsequent experiments, we found previously unknown limitations intrinsic to the method affecting its feasibility in determination of mature template RISC incorporation as well as in multiplexing. Both protocols were equally specific in discriminating between correct and incorrect small RNA targets or between mature miRNA and its unprocessed RNA precursor, indicating the stem–loop RT-primer, but not the TaqMan probe, triggers target specificity. The presented universal TaqMan-based RT-PCR protocol represents a cost-efficient method for the detection of small RNAs. PMID:24149841

  18. Multiplex primer-extension assay for identification of Yersinia species.

    PubMed

    Dalmasso, Alessandra; Civera, Tiziana; Filipello, Virginia; Bottero, Maria Teresa

    2014-10-01

    A multiplex primer-extension reaction (PER) assay, was specifically designed for the identification of ten Yersinia species. The assay, directed towards the tufA (elongation factor Tu) gene, was tested on a total of 42 samples representing Yersinia species and non-Yersinia species. The primers used in the preliminary PCR, designed in highly conserved regions upstream and downstream of the diagnosis sites, successfully amplified a 587 bp fragment. The diagnosis sites were simultaneously interrogated using a multiplex PER and the results were confirmed by fragment sequencing. The proposed test provides an appropriate tool to monitor the presence of Yersinia spp. in food samples and to evaluate the potential hazard for consumers. PMID:24985982

  19. Real-time RT-PCR detection of betanodavirus in naturally and experimentally infected fish from Spain.

    PubMed

    Hodneland, K; García, R; Balbuena, J A; Zarza, C; Fouz, B

    2011-03-01

    Infections with betanodavirus affect a wide range of wild and farmed fish species throughout the world, mostly from the marine environment. The aim of this work was to develop and validate real-time RT-PCR assays for sensitive and specific detection of nodavirus in diseased or carrier fish. The new detection assay was used to study the transmission and development of nodavirus infection in juvenile sea bass, Dicentrarchus labrax (L.), challenged by different routes, and also to screen for nodavirus in various farmed fish species. On average, the sensitivity was 10-100 times higher than a standard RT-PCR, and the assay was able to detect asymptomatic carrier fish that otherwise could have been classified as free of infection. Clinical signs of nodavirus infection were reproduced in fish infected following bath exposure or intramuscular injection, demonstrating horizontal transmission of the disease. Nodavirus was always detected in the brain of diseased fish but also in many recovered fish. The new assay enables us to confirm the presence of the virus at an early phase in the production cycle and may represent a useful tool to prevent or slow down the spread of nodavirus to new locations. PMID:21306586

  20. Detecting Newcastle disease virus in combination of RT-PCR with red blood cell absorption.

    PubMed

    Yi, Jianzhong; Liu, Chengqian

    2011-01-01

    Reverse transcription-polymerase chain reaction (RT-PCR) has limited sensitivity when treating complicated samples, such as feces, waste-water in farms, and nucleic acids, protein rich tissue samples, all the factors may interfere with the sensitivity of PCR test or generate false results. In this study, we developed a sensitive RT-PCR, combination of red blood cell adsorption, for detecting Newcastle disease virus (NDV). One pair of primers which was highly homologous to three NDV pathotypes was designed according to the consensus nucleocapsid protein (NP) gene sequence. To eliminate the interfere of microbes and toxic substances, we concentrated and purified NDV from varied samples utilizing the ability of NDV binding red blood cells (RBCs). The RT-PCR coupled with red blood cell adsorption was much more sensitive in comparison with regular RT-PCR. The approach could also be used to detect other viruses with the property of hemagglutination, such as influenza viruses. PMID:21535888

  1. Multiple RT-PCR markers for the detection of circulating tumour cells of metastatic canine mammary tumours.

    PubMed

    da Costa, A; Kohn, B; Gruber, A D; Klopfleisch, R

    2013-04-01

    In humans, detection of circulating tumour cells (CTCs) using nucleic acid-based methods such as reverse transcription polymerase chain reaction (RT-PCR) has proven to be of prognostic relevance. However, similar procedures are still lacking in veterinary oncology. To assess the correlation of CTC markers with the metastatic potential of canine mammary tumours, 120 peripheral blood samples from bitches with mammary carcinomas with (group 1) and without (group 2) histological evidence of vascular invasion and/or presence of lymph node metastases and mammary adenomas (group 3) were analyzed. Blood samples were collected in EDTA tubes and RNA was extracted within 48 h. Subsequently, the samples were tested by RT-PCR for a panel of seven CTC mRNA markers. CRYAB was the most sensitive single marker with a sensitivity of 35% and also the most specific marker with a specificity of 100% to detect group 1 blood samples. A multimarker assay combining four genes enhanced the sensitivity up to 77.5%, but decreased the specificity to 80%. CRYAB appeared to be highly specific but only moderately sensitive at detecting blood samples from dogs with metastatic tumours and detection significantly correlated with vascular invasion of primary mammary tumours. However, a multimarker assay of four genes significantly enhanced the sensitivity of the assay and is therefore preferable for CTC detection. PMID:23036177

  2. Application of real time RT-PCR for the genetic homogeneity and stability tests of the seed candidates for live attenuated influenza vaccine production

    PubMed Central

    Shcherbik, Svetlana; Sergent, Sheila B.; Davis, William G.; Shu, Bo; Barnes, John; Kiseleva, Irina; Larionova, Natalie; Klimov, Alexander; Bousse, Tatiana

    2015-01-01

    Development and improvement of quality control tests for live attenuated vaccines are a high priority because of safety concerns. Live attenuated influenza vaccine (LAIV) viruses are 6:2 reassortants containing the hemagglutinin (HA) and neuraminidase (NA) gene segments from circulating influenza viruses to induce protective immune responses, and the six internal gene segments from a cold-adapted Master Donor Virus (MDV). LAIV candidate viruses for the 2012–2013 seasons, A/Victoria/361/2011-CDC-LV1 (LV1) and B/Texas/06/2011-CDC-LV2B (LV2B), were created by classical reassortment of A/Victoria/361/2011 and MDV-A A/Leningrad/134/17/57 (H2N2) or B/Texas/06/2011 and MDV-B B/USSR/60/69. In an attempt to provide better identity and stability testing for quality control of LV1 and LV2B, sensitive real-time RT-PCR assays (rRT-PCR) were developed to detect the presence of undesired gene segments (HA and NA from MDV and the six internal genes from the seasonal influenza viruses). The sensitivity of rRT-PCR assays designed for each gene segment ranged from 0.08 to 0.8 EID50 (50% of Egg Infectious Dose) per reaction for the detection of undesired genes in LV1 and from 0.1 to 1 EID50 per reaction for the detection of undesired genes in LV2B. No undesired genes were detected either before or after five passages of LV1 or LV2B in eggs. The complete genome sequencing of LV1 and LV2B confirmed the results of rRT-PCR, demonstrating the utility of the new rRT-PCR assays to provide the evidence for the homogeneity of the prepared vaccine candidate. PMID:24056261

  3. Simultaneous detection of eight swine reproductive and respiratory pathogens using a novel GeXP analyser-based multiplex PCR assay.

    PubMed

    Zhang, Minxiu; Xie, Zhixun; Xie, Liji; Deng, Xianwen; Xie, Zhiqin; Luo, Sisi; Liu, Jiabo; Pang, Yaoshan; Khan, Mazhar I

    2015-11-01

    A new high-throughput GenomeLab Gene Expression Profiler (GeXP) analyser-based multiplex PCR assay was developed for the detection of eight reproductive and respiratory pathogens in swine. The reproductive and respiratory pathogens include North American porcine reproductive and respiratory syndrome virus (PRRSV-NA), classical swine fever virus (CSFV), porcine circovirus 2 (PCV-2), swine influenza virus (SIV) (including H1 and H3 subtypes), porcine parvovirus (PPV), pseudorabies virus (PRV) and Japanese encephalitis virus (JEV). Nine pairs of specific chimeric primers were designed and used to initiate PCRs, and one pair of universal primers was used for subsequent PCR cycles. The specificity of the GeXP assay was examined using positive controls for each virus. The sensitivity was evaluated using serial ten-fold dilutions of in vitro-transcribed RNA from all of the RNA viruses and plasmids from DNA viruses. The GeXP assay was further evaluated using 114 clinical specimens and was compared with real-time PCR/single RT-PCR methods. The specificity of the GeXP assay for each pathogen was examined using single cDNA/DNA template. Specific amplification peaks of the reproductive and respiratory pathogens were observed on the GeXP analyser. The minimum copies per reaction detected for each virus by the GeXP assay were as follows: 1000 copies/μl for PRV; 100 copies/μl for CSFV, JEV, PCV-2 and PPV; and 10 copies/μl for SIV-H1, SIV-H3 and PRRSV-NA. Analysis of 114 clinical samples using the GeXP assay demonstrated that the GeXP assay had comparable detection to real-time PCR/single RT-PCR. This study demonstrated that the GeXP assay is a new method with high sensitivity and specificity for the identification of these swine reproductive and respiratory pathogens. The GeXP assay may be adopted for molecular epidemiological surveys of these reproductive and respiratory pathogens in swine populations. PMID:26259690

  4. Field-based multiplex and quantitative assay platforms for diagnostics

    NASA Astrophysics Data System (ADS)

    Venkatasubbarao, Srivatsa; Dixon, C. Edward; Chipman, Russell; Scherer, Axel; Beshay, Manal; Kempen, Lothar U.; Chandra Sekhar, Jai Ganesh; Yan, Hong; Puccio, Ava; Okonkwo, David; McClain, Stephen; Gilbert, Noah; Vyawahare, Saurabh

    2011-06-01

    The U.S. military has a continued interest in the development of handheld, field-usable sensors and test kits for a variety of diagnostic applications, such as traumatic brain injury (TBI) and infectious diseases. Field-use presents unique challenges for biosensor design, both for the readout unit and for the biological assay platform. We have developed robust biosensor devices that offer ultra-high sensitivity and also meet field-use needs. The systems under development include a multiplexed quantitative lateral flow test strip for TBI diagnostics, a field test kit for the diagnosis of pathogens endemic to the Middle East, and a microfluidic assay platform with a label-free reader for performing complex biological automated assays in the field.

  5. Molecular analysis of dolphin morbillivirus: A new sensitive detection method based on nested RT-PCR.

    PubMed

    Centelleghe, Cinzia; Beffagna, Giorgia; Zanetti, Rossella; Zappulli, Valentina; Di Guardo, Giovanni; Mazzariol, Sandro

    2016-09-01

    Cetacean Morbillivirus (CeMV) has been identified as the most pathogenic virus for cetaceans. Over the past three decades, this RNA virus has caused several outbreaks of lethal disease in odontocetes and mysticetes worldwide. Isolation and identification of CeMV RNA is very challenging in whales because of the poor preservation status frequently shown by tissues from stranded animals. Nested reverse transcription polymerase chain reaction (nested RT-PCR) is used instead of conventional RT-PCR when it is necessary to increase the sensitivity and the specificity of the reaction. This study describes a new nested RT-PCR technique useful to amplify small amounts of the cDNA copy of Cetacean morbillivirus (CeMV) when it is present in scant quantity in whales' biological specimens. This technique was used to analyze different tissues (lung, brain, spleen and other lymphoid tissues) from one under human care seal and seven cetaceans stranded along the Italian coastline between October 2011 and September 2015. A well-characterized, 200 base pair (bp) fragment of the dolphin Morbillivirus (DMV) haemagglutinin (H) gene, obtained by nested RT-PCR, was sequenced and used to confirm DMV positivity in all the eight marine mammals under study. In conclusion, this nested RT-PCR protocol can represent a sensitive detection method to identify CeMV-positive, poorly preserved tissue samples. Furthermore, this is also a rather inexpensive molecular technique, relatively easy to apply. PMID:27220282

  6. A simplified strategy for sensitive detection of Rose rosette virus compatible with three RT-PCR chemistries.

    PubMed

    Dobhal, Shefali; Olson, Jennifer D; Arif, Mohammad; Garcia Suarez, Johnny A; Ochoa-Corona, Francisco M

    2016-06-01

    Rose rosette disease is a disorder associated with infection by Rose rosette virus (RRV), a pathogen of roses that causes devastating effects on most garden cultivated varieties, and the wild invasive rose especially Rosa multiflora. Reliable and sensitive detection of this disease in early phases is needed to implement proper control measures. This study assesses a single primer-set based detection method for RRV and demonstrates its application in three different chemistries: Endpoint RT-PCR, TaqMan-quantitative RT-PCR (RT-qPCR) and SYBR Green RT-qPCR with High Resolution Melting analyses. A primer set (RRV2F/2R) was designed from consensus sequences of the nucleocapsid protein gene p3 located in the RNA 3 region of RRV. The specificity of primer set RRV2F/2R was validated in silico against published GenBank sequences and in-vitro against infected plant samples and an exclusivity panel of near-neighbor and other viruses that commonly infect Rosa spp. The developed assay is sensitive with a detection limit of 1fg from infected plant tissue. Thirty rose samples from 8 different states of the United States were tested using the developed methods. The developed methods are sensitive and reliable, and can be used by diagnostic laboratories for routine testing and disease management decisions. PMID:26850142

  7. Multiplexed Dosing Assays by Digitally Definable Hydrogel Volumes.

    PubMed

    Faralli, Adele; Melander, Fredrik; Larsen, Esben Kjaer Unmack; Chernyy, Sergey; Andresen, Thomas L; Larsen, Niels B

    2016-01-21

    Stable and low-cost multiplexed drug sensitivity assays using small volumes of cells or tissue are in demand for personalized medicine, including patient-specific combination chemotherapy. Spatially defined projected light photopolymerization of hydrogels with embedded active compounds is introduced as a flexible and cost-efficient method for producing multiplexed dosing assays. The high spatial resolution of light projector technology defines multiple compound doses by the volume of individual compound-embedded hydrogel segments. Quantitative dosing of multiple proteins with a dynamic range of 1-2 orders of magnitude is demonstrated using fluorescently labeled albumins. The hydrogel matrix results from photopolymerization of low-cost poly(ethylene glycol) diacrylates (PEGDA), and tuning of the PEGDA composition enables fast complete dosing of all tested species. Dosing of hydrophilic and hydrophobic compounds is demonstrated using two first-line chemotherapy regimens combining oxaliplatin, SN-38, 5-fluorouracil, and folinic acid, with each compound being dosed from a separate light-defined hydrogel segment. Cytotoxicity studies using a colorectal cancer cell line show equivalent effects of dissolved and released compounds. Further control of the dosing process is demonstrated by liposomal encapsulation of oxaliplatin, stable embedding of the liposomes in hydrogels for more than 3 months, and heat-triggered complete release of the loaded oxaliplatin. PMID:26619161

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

    PubMed

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

    2016-08-01

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

  9. Inactivation conditions for human norovirus measured by an in situ capture-qRT-PCR method.

    PubMed

    Wang, Dapeng; Tian, Peng

    2014-02-17

    Human norovirus (HuNoV) is a leading cause of foodborne gastroenteritis. Unfortunately, the inactivation parameters for HuNoV in clinical, food and environmental samples have not been established. Due to the inability to cultivate HuNoV in vitro, quantitative real-time RT-PCR (qRT-PCR) is widely-used for detecting HuNoVs. However, qRT-PCR does not indicate viral infectivity. Our method employs histo-blood group antigens (HBGAs) as viral receptors/co-receptors and container-affixed capture agents to concentrate HuNoVs. The captured viruses are denatured and its genome is amplified in the same module by in situ capture qRT-PCR (ISC-qRT-PCR). Greater than three log10 reduction in the receptor-captured viral genomic signal (RCVGS) was observed when HuNoV was treated by heat at 72 °C for 4 min, by chlorine at a final concentration of 16 ppm in less than 1 min, and by UV irradiation at 1J/cm². Treatment of low-titer HuNoV (<10³ copies/sample) with 70% ethanol for 20 s reduced the RCVGS of HuNoV by two log10. However, ethanol had a limited effect on high-titer samples of HuNoV (>10³ copies/sample). The results demonstrate that ISC-qRT-PCR method could be used as an alternative method to measure encapsidated viral RNA and indirectly indicate the inactivation status of HuNoV caused by physical treatment such as heat, and chemical treatment such as chlorine, that damage the ability of the virus to bind to its receptor. PMID:24361836

  10. Development of a diagnostic one-tube RT-PCR for the detection of Rift Valley fever virus.

    PubMed

    Espach, A; Romito, M; Nel, L H; Viljoen, G J

    2002-09-01

    Diagnosis of Rift Valley fever (RVF) is based on serology and virus isolation. The disadvantages of the former include poor sensitivity, high cost, risks associated with using infectious virus as antigen, the lengthy duration of ELISA as well as cross-reactivity with other Phleboviruses. We developed, optimised and evaluated a one-tube reverse-transcription-polymerase chain reaction (RT-PCR) for the detection of Rift Valley fever virus (RVFV) in ruminants. The PCR primers for this assay were designed to anneal to a region within the M segment of the virus genome, encoding glycoproteins G1 and G2. A PCR amplicon of 363 bp was obtained. The sensitivity of the assay was determined to be 0.25 TCID50. This test should allow for the early and rapid detection of RVFV in both serum and whole blood. In addition, it could facilitate the quantification of antigen for the manufacture of current vaccines. PMID:12356173

  11. Detection of influenza A(H1N1)v virus by real-time RT-PCR.

    PubMed

    Panning, M; Eickmann, M; Landt, O; Monazahian, M; Olschläger, S; Baumgarte, S; Reischl, U; Wenzel, J J; Niller, H H; Günther, S; Hollmann, B; Huzly, D; Drexler, J F; Helmer, A; Becker, S; Matz, B; Eis-Hübinger, Am; Drosten, C

    2009-09-10

    Influenza A(H1N1)v virus was first identified in April 2009. A novel real-time RT-PCR for influenza A(H1N1)v virus was set up ad hoc and validated following industry-standard criteria. The lower limit of detection of the assay was 384 copies of viral RNA per ml of viral transport medium (95% confidence interval: 273-876 RNA copies/ml). Specificity was 100% as assessed on a panel of reference samples including seasonal human influenza A virus H1N1 and H3N2, highly pathogenic avian influenza A virus H5N1 and porcine influenza A virus H1N1, H1N2 and H3N2 samples. The real-time RT-PCR assay for the influenza A matrix gene recommended in 2007 by the World Health Organization was modified to work under the same reaction conditions as the influenza A(H1N1)v virus-specific test. Both assays were equally sensitive. Clinical applicability of both assays was demonstrated by screening of almost 2,000 suspected influenza (H1N1)v specimens, which included samples from the first cases of pandemic H1N1 influenza imported to Germany. Measuring influenza A(H1N1)v virus concentrations in 144 laboratory-confirmed samples yielded a median of 4.6 log RNA copies/ml. The new methodology proved its principle and might assist public health laboratories in the upcoming influenza pandemic. PMID:19758541

  12. Detection, discrimination and quantitation of 22 bluetongue virus serotypes using real-time RT-PCR with TaqMan MGB probes.

    PubMed

    Feng, Yufei; Yang, Tao; Xu, Qingyuan; Sun, Encheng; Li, Junping; Lv, Shuang; Wang, Haixiu; Zhang, Qin; Zhang, Jikai; Wu, Donglai

    2015-09-01

    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. PMID:26115692

  13. Multiplex RT-PCR for the detection of Astroviruses and Rotaviruses in Poultry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Viral enteric diseases cause substantial economic loss to the US poultry industry because they lead to decreased weight gain, increased morbidity, increased mortality, and increased production costs from poor feed conversions and increased use of therapeutic anti-microbial treatments. Astroviruses a...

  14. One-Step Multiplex RT-PCR for Simultaneous Detection of Four Pome Tree Viroids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Apple scar skin viroid (ASSVd), Apple dimple fruit viroid (ADFVd), Apple fruit crinkle viroid (AFCVd), and Pear blister canker viroid (PBCVd) cause natural infections in pome (apple, pear, quince) fruit trees. These viroids are found worldwide and are important quarantine pathogens for the internati...

  15. Development of strand-specific real-time RT-PCR to distinguish viral RNAs during Newcastle disease virus infection.

    PubMed

    Qiu, Xusheng; Yu, Yang; Yu, Shengqing; Zhan, Yuan; Wei, Nana; Song, Cuiping; Sun, Yingjie; Tan, Lei; Ding, Chan

    2014-01-01

    Newcastle disease virus (NDV) causes large losses in the global fowl industry. To better understand NDV replication and transcription cycle, quantitative detection methods for distinguishing NDV genomic RNA (gRNA), antigenomic RNA (cRNA), and messenger RNA (mRNA) in NDV-infected cells are indispensible. Three reverse transcription primers were designed to specifically target the nucleoprotein (NP) region of gRNA, cRNA, and NP mRNA, and a corresponding real-time RT-PCR assay was developed to simultaneously quantify the three types of RNAs in NDV-infected cells. This method showed very good specificity, sensitivity, and reproducibility. The detection range of the assay was between 5.5 × 10(2) and 1.1 × 10(9) copies/μL of the target gene. These methods were applied to investigate the dynamics of the gRNA, cRNA, and mRNA synthesis in NDV La Sota infected DF-1 cells. The results showed that the copy numbers of viral gRNA, cRNA, and NP mRNA all exponentially increased in the beginning. The viral RNA copy number then plateaued at 10'h postinfection and gradually decreased from 16 h postinfection. No synthesis priority was observed between replication (gRNA and cRNA amounts) and transcription (mRNA amounts) during NDV infection. However, the cRNA accumulated more rapidly than gRNA, as the cRNA copy number was three- to tenfold higher than gRNA starting from 2 h postinfection. Conclusion. A real-time RT-PCR for absolute quantitation of specific viral RNA fragments in NDV-infected cells was developed for the first time. The development of this assay will be helpful for further studies on the pathogenesis and control strategies of NDV. PMID:25379553

  16. Evaluation of reference genes for quantitative RT-PCR in Lolium temulentum under abiotic stress

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lolium temulentum is a valuable model grass species for the study of stress in forage and turf grasses. Gene expression analysis by quantitative real time RT-PCR relies on the use of proper internal standards. The aim of this study was to identify and evaluate reference genes for use in real-time q...

  17. Generic RT-PCR tests for detection and identification of tospoviruses.

    PubMed

    Hassani-Mehraban, A; Westenberg, M; Verhoeven, J T J; van de Vossenberg, B T L H; Kormelink, R; Roenhorst, J W

    2016-07-01

    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. PMID:27036502

  18. Type-A influenza virus detection and quantitation by real-time RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real-time RT-PCR (RRT-PCR) is a relatively new technology which has been used for AIV detection since the early 2000’s for routine surveillance, during outbreaks and for research. Some of the advantages of RRT-PCR are: quantitative nature, scalability, cost, high sensitivity, high specificity, and ...

  19. Real time RT-PCR for the H5 HA subtype

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real-time RT-PCR (RRT-PCR) for type A influenza detection is widely used with subsequent tests for subtype identification. Due to the importance of the H5 HA subtype, rapid and sensitive detection of the H5 HA subtype is particularly useful due to the importance of the recent Asian H5N1 HPAI viruse...

  20. Detecting the Presence of Nora Virus in "Drosophila" Utilizing Single Fly RT-PCR

    ERIC Educational Resources Information Center

    Munn, Bethany; Ericson, Brad; Carlson, Darby J.; Carlson, Kimberly A.

    2015-01-01

    A single fly RT-PCR protocol has recently been developed to detect the presence of the persistent, horizontally transmitted Nora virus in "Drosophila." Wild-caught flies from Ohio were tested for the presence of the virus, with nearly one-fifth testing positive. The investigation presented can serve as an ideal project for biology…

  1. Validation of a real-time RT-PCR method to quantify Newcastle Disease Virus (NDV) titer and comparison with other quantifiable methods.

    PubMed

    Jang, Juno; Hong, Sung-Hwan; Kim, Ik-Hwan

    2011-01-01

    A method for the rapid detection and quantification of Newcastle disease virus (NDV) produced in an animal cell culture-based production system was developed to enhance the speed of the NDV vaccine manufacturing process. A SYBR Green I-based real-time RT-PCR was designed with a conventional, inexpensive RT-PCR kit targeting the F gene of the NDV LaSota strain. The method developed in this study was validated for specificity, accuracy, precision, linearity, limit of detection (LOD), limit of quantification (LOQ), and robustness. The validation results satisfied the predetermined acceptance criteria. The validated method was used to quantify virus samples produced in an animal cell culture-based production system. The method was able to quantify the NDV samples from mid- or late-production phases, but not effective on samples from the early-production phase. For comparison with other quantifiable methods, immunoblotting, plaque assay, and tissue culture infectious dose 50 (TCID50) assay were also performed with the NDV samples. The results demonstrated that the real-time RT-PCR method is suitable for the rapid quantification of virus particles produced in an animal cell-culture-based production system irrespective of viral infectivity. PMID:21301199

  2. Application of F⁺RNA Coliphages as Source Tracking Enteric Viruses on Parsley and Leek Using RT-PCR.

    PubMed

    Shahrampour, Dina; Yavarmanesh, Masoud; Najafi, Mohammad Bagher Habibi; Mohebbi, Mohebbat

    2015-12-01

    The objective of this study was to identify sources of fecal contamination in leek and parsley, by using four different F(+)RNA coliphage genogroups (IV, I indicate animal fecal contamination and II, III indicate human fecal contamination). Three different concentrations (10(2), 10(4), 10(6) pfu/ml) of MS2 coliphage were inoculated on the surface of parsley and leek samples for detection of phage recovery efficiency among two methods of elution concentration (PEG-precipitation and Ultracentrifugation) by performing double agar layer (DAL) assay in three replications. Highest recovery of MS2 was observed in PEG method and in 10(6) inoculation concentration. Accordingly, the PEG method was used for washing and isolation of potentially contaminated phages of 30 collected samples (15 samples from the market and 15 samples from the farm). The final solutions of PEG method were tested for the enumeration of plaques by DAL assay. Total RNA was then extracted from recovered phages, and RT-PCR was performed by using four primer sets I, II, III, and IV. Incidence of F(+)RNA coliphages was observed in 12/15 (80 %) and 10/15 (66/6 %) of samples were obtained from farm and market, respectively, using both DAL and RT-PCR test methods. Different genotypes (I, II, and IV) of F(+)RNA coliphages were found in farm samples, while only genotype I was detected in market samples by using the primer sets. Due to the higher frequency of genotype I and IV, the absence of genotype III, and also the low frequency of genotype II, it is concluded that the contamination of vegetable (parsley and leek) in Neyshabour, Iran is most likely originated from animal sources. PMID:26264153

  3. A Multiplex Assay for Detection of Staphylococcal and Streptococcal Exotoxins.

    PubMed

    Sharma, Preeti; Wang, Ningyan; Chervin, Adam S; Quinn, Cheryl L; Stone, Jennifer D; Kranz, David M

    2015-01-01

    Staphylococcal and streptococcal exotoxins, also known as superantigens, mediate a range of diseases including toxic shock syndrome, and they exacerbate skin, pulmonary and systemic infections caused by these organisms. When present in food sources they can cause enteric effects commonly known as food poisoning. A rapid, sensitive assay for the toxins would enable testing of clinical samples and improve surveillance of food sources. Here we developed a bead-based, two-color flow cytometry assay using single protein domains of the beta chain of T cell receptors engineered for high-affinity for staphylococcal (SEA, SEB and TSST-1) and streptococcal (SpeA and SpeC) toxins. Site-directed biotinylated forms of these high-affinity agents were used together with commercial, polyclonal, anti-toxin reagents to enable specific and sensitive detection with SD50 values of 400 pg/ml (SEA), 3 pg/ml (SEB), 25 pg/ml (TSST-1), 6 ng/ml (SpeA), and 100 pg/ml (SpeC). These sensitivities were in the range of 4- to 80-fold higher than achieved with standard ELISAs using the same reagents. A multiplex format of the assay showed reduced sensitivity due to higher noise associated with the use of multiple polyclonal agents, but the sensitivities were still well within the range necessary for detection in food sources or for rapid detection of toxins in culture supernatants. For example, the assay specifically detected toxins in supernatants derived from cultures of Staphylococcus aureus. Thus, these reagents can be used for simultaneous detection of the toxins in food sources or culture supernatants of potential pathogenic strains of Staphylococcus aureus and Streptococcus pyogenes. PMID:26305471

  4. Validation of Reference Genes for Robust qRT-PCR Gene Expression Analysis in the Rice Blast Fungus Magnaporthe oryzae

    PubMed Central

    Che Omar, Sarena; Bentley, Michael A.; Morieri, Giulia; Preston, Gail M.; Gurr, Sarah J.

    2016-01-01

    The rice blast fungus causes significant annual harvest losses. It also serves as a genetically-tractable model to study fungal ingress. Whilst pathogenicity determinants have been unmasked and changes in global gene expression described, we know little about Magnaporthe oryzae cell wall remodelling. Our interests, in wall remodelling genes expressed during infection, vegetative growth and under exogenous wall stress, demand robust choice of reference genes for quantitative Real Time-PCR (qRT-PCR) data normalisation. We describe the expression stability of nine candidate reference genes profiled by qRT-PCR with cDNAs derived during asexual germling development, from sexual stage perithecia and from vegetative mycelium grown under various exogenous stressors. Our Minimum Information for Publication of qRT-PCR Experiments (MIQE) compliant analysis reveals a set of robust reference genes used to track changes in the expression of the cell wall remodelling gene MGG_Crh2 (MGG_00592). We ranked nine candidate reference genes by their expression stability (M) and report the best gene combination needed for reliable gene expression normalisation, when assayed in three tissue groups (Infective, Vegetative, and Global) frequently used in M. oryzae expression studies. We found that MGG_Actin (MGG_03982) and the 40S 27a ribosomal subunit MGG_40s (MGG_02872) proved to be robust reference genes for the Infection group and MGG_40s and MGG_Ef1 (Elongation Factor1-α) for both Vegetative and Global groups. Using the above validated reference genes, M. oryzae MGG_Crh2 expression was found to be significantly (p<0.05) elevated three-fold during vegetative growth as compared with dormant spores and two fold higher under cell wall stress (Congo Red) compared to growth under optimal conditions. We recommend the combinatorial use of two reference genes, belonging to the cytoskeleton and ribosomal synthesis functional groups, MGG_Actin, MGG_40s, MGG_S8 (Ribosomal subunit 40S S8) or MGG

  5. Validation of Reference Genes for Robust qRT-PCR Gene Expression Analysis in the Rice Blast Fungus Magnaporthe oryzae.

    PubMed

    Che Omar, Sarena; Bentley, Michael A; Morieri, Giulia; Preston, Gail M; Gurr, Sarah J

    2016-01-01

    The rice blast fungus causes significant annual harvest losses. It also serves as a genetically-tractable model to study fungal ingress. Whilst pathogenicity determinants have been unmasked and changes in global gene expression described, we know little about Magnaporthe oryzae cell wall remodelling. Our interests, in wall remodelling genes expressed during infection, vegetative growth and under exogenous wall stress, demand robust choice of reference genes for quantitative Real Time-PCR (qRT-PCR) data normalisation. We describe the expression stability of nine candidate reference genes profiled by qRT-PCR with cDNAs derived during asexual germling development, from sexual stage perithecia and from vegetative mycelium grown under various exogenous stressors. Our Minimum Information for Publication of qRT-PCR Experiments (MIQE) compliant analysis reveals a set of robust reference genes used to track changes in the expression of the cell wall remodelling gene MGG_Crh2 (MGG_00592). We ranked nine candidate reference genes by their expression stability (M) and report the best gene combination needed for reliable gene expression normalisation, when assayed in three tissue groups (Infective, Vegetative, and Global) frequently used in M. oryzae expression studies. We found that MGG_Actin (MGG_03982) and the 40S 27a ribosomal subunit MGG_40s (MGG_02872) proved to be robust reference genes for the Infection group and MGG_40s and MGG_Ef1 (Elongation Factor1-α) for both Vegetative and Global groups. Using the above validated reference genes, M. oryzae MGG_Crh2 expression was found to be significantly (p<0.05) elevated three-fold during vegetative growth as compared with dormant spores and two fold higher under cell wall stress (Congo Red) compared to growth under optimal conditions. We recommend the combinatorial use of two reference genes, belonging to the cytoskeleton and ribosomal synthesis functional groups, MGG_Actin, MGG_40s, MGG_S8 (Ribosomal subunit 40S S8) or MGG

  6. 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.

    PubMed

    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

    2014-10-01

    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. PMID:25019170

  7. Selection of suitable reference genes for normalization of quantitative RT-PCR in peripheral blood samples of bottlenose dolphins (Tursiops truncatus)

    PubMed Central

    Chen, I-Hua; Chou, Lien-Siang; Chou, Shih-Jen; Wang, Jiann-Hsiung; Stott, Jeffrey; Blanchard, Myra; Jen, I-Fan; Yang, Wei-Cheng

    2015-01-01

    Quantitative RT-PCR is often used as a research tool directed at gene transcription. Selection of optimal housekeeping genes (HKGs) as reference genes is critical to establishing sensitive and reproducible qRT-PCR-based assays. The current study was designed to identify the appropriate reference genes in blood leukocytes of bottlenose dolphins (Tursiops truncatus) for gene transcription research. Seventy-five blood samples collected from 7 bottlenose dolphins were used to analyze 15 candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ, LDHA, SDHA). HKG stability in qRT-PCR was determined using geNorm, NormFinder, BestKeeper and comparative delta Ct algorithms. Utilization of RefFinder, which combined all 4 algorithms, suggested that PGK1, HPRT1 and RPL4 were the most stable HKGs in bottlenose dolphin blood. Gene transcription perturbations in blood can serve as an indication of health status in cetaceans as it occurs prior to alterations in hematology and chemistry. This study identified HKGs that could be used in gene transcript studies, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans. PMID:26486099

  8. Selection of suitable reference genes for normalization of quantitative RT-PCR in peripheral blood samples of bottlenose dolphins (Tursiops truncatus).

    PubMed

    Chen, I-Hua; Chou, Lien-Siang; Chou, Shih-Jen; Wang, Jiann-Hsiung; Stott, Jeffrey; Blanchard, Myra; Jen, I-Fan; Yang, Wei-Cheng

    2015-01-01

    Quantitative RT-PCR is often used as a research tool directed at gene transcription. Selection of optimal housekeeping genes (HKGs) as reference genes is critical to establishing sensitive and reproducible qRT-PCR-based assays. The current study was designed to identify the appropriate reference genes in blood leukocytes of bottlenose dolphins (Tursiops truncatus) for gene transcription research. Seventy-five blood samples collected from 7 bottlenose dolphins were used to analyze 15 candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ, LDHA, SDHA). HKG stability in qRT-PCR was determined using geNorm, NormFinder, BestKeeper and comparative delta Ct algorithms. Utilization of RefFinder, which combined all 4 algorithms, suggested that PGK1, HPRT1 and RPL4 were the most stable HKGs in bottlenose dolphin blood. Gene transcription perturbations in blood can serve as an indication of health status in cetaceans as it occurs prior to alterations in hematology and chemistry. This study identified HKGs that could be used in gene transcript studies, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans. PMID:26486099

  9. Electronic microarray assays for avian influenza and Newcastle disease virus.

    PubMed

    Lung, Oliver; Beeston, Anne; Ohene-Adjei, Samuel; Pasick, John; Hodko, Dalibor; Hughes, Kimberley Burton; Furukawa-Stoffer, Tara; Fisher, Mathew; Deregt, Dirk

    2012-11-01

    Microarrays are suitable for multiplexed detection and typing of pathogens. Avian influenza virus (AIV) is currently classified into 16 H (hemagglutinin) and 9 N (neuraminidase) subtypes, whereas Newcastle disease virus (NDV) strains differ in virulence and are broadly classified into high and low pathogenicity types. In this study, three assays for detection and typing of poultry viruses were developed on an automated microarray platform: a multiplex assay for simultaneous detection of AIV and detection and pathotyping of NDV, and two separate assays for differentiating all AIV H and N subtypes. The AIV-NDV multiplex assay detected all strains in a 63 virus panel, and accurately typed all high pathogenicity NDV strains tested. A limit of detection of 10(1)-10(3) TCID(50)/mL and 200-400 EID(50)/mL was obtained for NDV and AIV, respectively. The AIV typing assays accurately typed all 41 AIV strains and a limit of detection of 4-200 EID(50)/mL was obtained. Assay validation showed that the microarray assays were generally comparable to real-time RT-PCR. However, the AIV typing microarray assays detected more positive clinical samples than the AIV matrix real-time RT-PCR, and also provided information regarding the subtype. The AIV-NDV multiplex and AIV H typing microarray assays detected mixed infections and could be useful for detection and typing of AIV and NDV. PMID:22796283

  10. Highly Specific Detection of Five Exotic Quarantine Plant Viruses using RT-PCR.

    PubMed

    Choi, Hoseong; Cho, Won Kyong; Yu, Jisuk; Lee, Jong-Seung; Kim, Kook-Hyung

    2013-03-01

    To detect five plant viruses (Beet black scorch virus, Beet necrotic yellow vein virus, Eggplant mottled dwarf virus, Pelargonium zonate spot virus, and Rice yellow mottle virus) for quarantine purposes, we designed 15 RT-PCR primer sets. Primer design was based on the nucleotide sequence of the coat protein gene, which is highly conserved within species. All but one primer set successfully amplified the targets, and gradient PCRs indicated that the optimal temperature for the 14 useful primer sets was 51.9°C. Some primer sets worked well regardless of annealing temperature while others required a very specific annealing temperature. A primer specificity test using plant total RNAs and cDNAs of other plant virus-infected samples demonstrated that the designed primer sets were highly specific and generated reproducible results. The newly developed RT-PCR primer sets would be useful for quarantine inspections aimed at preventing the entry of exotic plant viruses into Korea. PMID:25288934

  11. Mass scale screening of common arboviral infections by an affordable, cost effective RT-PCR method

    PubMed Central

    Taraphdar, Debjani; Sarkar, Arindam; Chatterjee, Shyamalendu

    2012-01-01

    Objective To develop a rapid, cost effective RT-PCR method for the mass scale diagnosis of such diseases at the viremia stage to find out the actual disease burden in that area. Methods For this purpose, cases with the history of only short febrile illness were considered. Thus 157 samples with the history of dengue/chikungunya like illness and only 58 samples with a history of acute encephalitis syndrome (AES) were selected. Results Out of 157 samples, 42 and 74 were detected as dengue and chikungunya, respectively and out of 58 AES cases only 23 could be detected as Japanese encephalitis by this RT-PCR method. Conclusions This cost effective RT-PCR method can detect the total positive cases that remain undetected by ELISA method. Moreover, this method is capable to detect the viral RNA from patients' sera even after the appearance of IgM antibody at one fifth costs as compared with the other commercially available kits. PMID:23569876

  12. A Tool Set for the Genome-Wide Analysis of Neurospora crassa by RT-PCR.

    PubMed

    Hurley, Jennifer H; Dasgupta, Arko; Andrews, Peter; Crowell, Alexander M; Ringelberg, Carol; Loros, Jennifer J; Dunlap, Jay C

    2015-10-01

    Neurospora crassa is an important model organism for filamentous fungi as well as for circadian biology and photobiology. Although the community-accumulated tool set for the molecular analysis of Neurospora is extensive, two components are missing: (1) dependable reference genes whose level of expression are relatively constant across light/dark cycles and as a function of time of day and (2) a catalog of primers specifically designed for real-time PCR (RT-PCR). To address the first of these we have identified genes that are optimal for use as reference genes in RT-PCR across a wide range of expression levels; the mRNA/transcripts from these genes have potential for use as reference noncycling transcripts outside of Neurospora. In addition, we have generated a genome-wide set of RT-PCR primers, thereby streamlining the analysis of gene expression. In validation studies these primers successfully identified target mRNAs arising from 70% (34 of 49) of all tested genes and from all (28) of the moderately to highly expressed tested genes. PMID:26248984

  13. Validation of reference genes for real-time quantitative RT-PCR studies in Talaromyces marneffei.

    PubMed

    Dankai, Wiyada; Pongpom, Monsicha; Vanittanakom, Nongnuch

    2015-11-01

    Talaromyces marneffei (or Penicillium marneffei) is an opportunistic pathogen that can cause disseminated disease in human immunodeficiency virus (HIV)-infected patients, especially in Southeast Asia. T. marneffei is a thermally dimorphic fungus. Typically, T. marneffei has an adaptive morphology. It grows in a filamentous form (mould) at 25°C and can differentiate to produce asexual spores (conidia). In contrast, at 37°C, it grows as yeast cells that divide by fission. This study aimed to validate a quantitative reverse-transcription polymerase chain reaction (qRT-PCR) for gene expression analysis in T. marneffei. Analysis of relative gene expression by qRT-PCR requires normalization of data using a proper reference gene. However, suitable reference genes have not been identified in gene expression studies across different cell types or under different experimental conditions in T. marneffei. In this study, four housekeeping genes were selected for analysis: β-actin (act); glyceraldehyde-3-phosphate dehydrogenase (gapdh); β-tubulin (benA) and 18S rRNA. Two analysis programs; BestKeeper and geNorm software tools were used to validate the expression of the candidate normalized genes. The results indicated that the actin gene was the one which was most stably expressed and was recommended for use as the endogenous control for gene expression analysis of all growth forms in T. marneffei by qRT-PCR under normal and stress conditions. PMID:26327538

  14. Investigation of Reference Genes in Vibrio parahaemolyticus for Gene Expression Analysis Using Quantitative RT-PCR

    PubMed Central

    Ma, Yue-jiao; Sun, Xiao-hong; Xu, Xiao-yan; Zhao, Yong; Pan, Ying-jie; Hwang, Cheng-An; Wu, Vivian C. H.

    2015-01-01

    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

  15. RT-PCR is a more accurate diagnostic tool for detection of BCR-ABL rearrangement

    SciTech Connect

    Zehnbauer, B.A.; Allen, A.P.; McGrath, S.D.

    1994-09-01

    Detection of the Philadelphia chromosome (Ph1) or genomic Southern hybridization for clonal gene rearrangement (GSH-R) has provided very specific identification of BCR-ABL gene rearrangement. Reverse transcriptase-polymerase chain reaction (RT-PCR) is diagnostic for patterns of BCR-ABL expression which are undetected by GSH-R and/or Ph1 and provides increased sensitivity both at diagnosis and in detection of minimal residual leukemia. Fifty-three specimens (of 150 tested from 119 consecutive leukemia patients) were RT-PCR positive for BCR-ABL gene expression confirmed by hybridization of PCR products with b{sub 3}a{sub 2}, b{sub 2}a{sub 2}, or e{sub 1}a{sub 2} junction-specific oligonucleotides. In 6 cases of CML with GSH-R{sup {minus}}at diagnosis, RT-PCR provided specific BCR-ABL identification. Deletion of BCR regions, low mitotic index, or e{sub 1}a{sub 2} expression caused failure to detect GSH-R or Ph1 translocation.

  16. Ring test evaluation of the detection of influenza A virus in swine oral fluids by real-time, reverse transcription polymerase chain reaction (rRT-PCR) and virus isolation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  17. DEVELOPMENT OF AN ASTROVIRUS RT-PCR DETECTION ASSAY FOR USE WITH CONVENTIONAL, REAL-TIME, AND INTEGRATED CELL CULTURE/RT-PCR

    EPA Science Inventory

    Astrovirus is a common cause of gastroenteritis in humans that has been determined to be responsible for outbreaks of illness in several countries. Since astrovirus can be waterborne, it is important to be able to identify this virus in environmental water. We have developed an...

  18. Biosurveillance of avian influenza and Newcastle disease viruses in the Barda region of Azerbaijan using real time RT-PCR and hemagglutination inhibition

    PubMed Central

    Zeynalova, Shalala; Guliyev, Fizuli; Vatani, Mahira; Abbasov, Bahruz

    2015-01-01

    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

  19. Effective detection of human noroviruses in Hawaiian waters using enhanced RT-PCR methods.

    PubMed

    Tong, Hsin-I; Connell, Christina; Boehm, Alexandria B; Lu, Yuanan

    2011-11-15

    The current recreational water quality criteria using growth-based measurements of fecal indicator bacteria (FIB) concentration have their limitations for swimmer protection. To evaluate the possible use of enteric viruses as an improved indicator of human sewage contamination in recreational waters for enhanced health risk assessment, human norovirus (huNoV) was tested as a model in this study. To establish a highly sensitive protocol for effective huNoV detection in waters, 16 published and newly designed reverse transcription polymerase chain reaction (RT-PCR) primer pairs specific for huNoV genogroup I (GI) and genogroup II (GII) were comparatively evaluated side-by-side using single sources of huNoV RNA stock extracted from local clinical isolates. Under optimized conditions, these RT-PCR protocols shared a very different pattern of detection sensitivity for huNoV. The primer sets COG2F/COG2R and QNIF4/NV1LCR were determined to be the most sensitive ones for huNoV GII and GI, respectively, with up to 10(5)- and 10(6)-fold more sensitive as compared to other sets tested. These two sensitive protocols were validated by positive detection of huNoV in untreated and treated urban wastewater samples. In addition, these RT-PCR protocols enabled detection of the prevalence of huNoV in 5 (GI) and 10 (GII) of 16 recreational water samples collected around the island of O'ahu, which was confirmed by DNA sequencing and sequence analysis. Findings from this study support the possible use of enteric viral pathogens for environmental monitoring and argue the importance and essentiality for such monitoring activity to ensure a safe use of recreational waters. PMID:21945082

  20. Final Report Nucleic Acid System - Hybrid PCR and Multiplex Assay Project Phase 2

    SciTech Connect

    Koopman, R P; Langlois, R G; Nasarabadi, S; Benett, W J; Colston, B W; Johnson, D C; Brown, S B; Stratton, P L; Milanovich, F P

    2002-04-17

    This report covers phase 2 (year 2) of the Nucleic Acid System--Hybrid PCR and Multiplex Assay project. The objective of the project is to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction) in a multiplex mode using flow cytometry. The Hybrid instrument consists of a flow-through PCR module capable of handling a multiplexed PCR assay, a hybridizing module capable of hybridizing multiplexed PCR amplicons and beads, and a flow cytometer module for bead-based identification, all controlled by a single computer. Multiplex immunoassay using bead-based Luminex flow cytometry is available, allowing rapid screening for many agents. PCR is highly specific and complements and verifies immunoassay. It can also be multiplexed and detection provided using the bead-based Luminex flow cytometer. This approach allows full access to the speed and 100-fold multiplex capability of flow cytometry for rapid screening as well as the accuracy and specificity of PCR. This project has two principal activities: (1) Design, build and test a prototype hybrid PCR/flow cytometer with the basic capabilities for rapid, broad spectrum detection and identification, and (2) Develop and evaluate multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products. This project requires not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This involves development and evaluation of multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products.

  1. Development of a real-time RT-PCR assay for turkey cytokines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recent pathogenesis studies with turkey-origin reoviruses (TRVs) in specific pathogen free (SPF) poults have revealed evidence of immuosuppression associated with TRV infection. The ability to quantitatively measure the abundance and/or relative amounts of cytokine mRNA in poult immune system tissu...

  2. Development of a real-time RT-PCR assay for the detection of marine caliciviruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    More than forty different marine caliciviruses (family Caliciviridae, genus Vesivirus) have been identified from marine and terrestrial host since their initial isolation in 1972. Marine vesiviruses have previously infected swine along the Western coast of the United States and produce a disease cl...

  3. Protein-based multiplex assays: mock presubmissions to the US Food and Drug Administration.

    PubMed

    Regnier, Fred E; Skates, Steven J; Mesri, Mehdi; Rodriguez, Henry; Tezak, Zivana; Kondratovich, Marina V; Alterman, Michail A; Levin, Joshua D; Roscoe, Donna; Reilly, Eugene; Callaghan, James; Kelm, Kellie; Brown, David; Philip, Reena; Carr, Steven A; Liebler, Daniel C; Fisher, Susan J; Tempst, Paul; Hiltke, Tara; Kessler, Larry G; Kinsinger, Christopher R; Ransohoff, David F; Mansfield, Elizabeth; Anderson, N Leigh

    2010-02-01

    As a part of ongoing efforts of the NCI-FDA Interagency Oncology Task Force subcommittee on molecular diagnostics, members of the Clinical Proteomic Technology Assessment for Cancer program of the National Cancer Institute have submitted 2 protein-based multiplex assay descriptions to the Office of In Vitro Diagnostic Device Evaluation and Safety, US Food and Drug Administration. The objective was to evaluate the analytical measurement criteria and studies needed to validate protein-based multiplex assays. Each submission described a different protein-based platform: a multiplex immunoaffinity mass spectrometry platform for protein quantification, and an immunological array platform quantifying glycoprotein isoforms. Submissions provided a mutually beneficial way for members of the proteomics and regulatory communities to identify the analytical issues that the field should address when developing protein-based multiplex clinical assays. PMID:20007858

  4. Detection of potato mop-top virus in soils and potato tubers using bait-plant bioassay, ELISA and RT-PCR.

    PubMed

    Arif, Muhammad; Ali, Murad; Rehman, Anayatur; Fahim, Muhammad

    2014-01-01

    The hilly region of Northwest of Pakistan is leading seed potato producing areas of the country. Soil and plant samples were collected from the region and tested for PMTV using both conventional and molecular techniques. The bait plants exhibited PMTV-characteristic v-shaped yellow leaf markings in Nicotiana debneyi plants grown in putative viruliferious soils from 20/26 locations. The results were confirmed by back inoculation of sap from both roots and leaves of bait plant on indicator hosts (N. debneyi, Nicotiana benthamiana). The root samples of bait plants grown in soils of 25 locations and leaves of 24 locations reproduced systemic infection on indicator hosts upon back inoculation. The virus was identified in bait plants grown in soils from 25/26 locations using double antibody sandwich-enzyme linked immunosorbent assay (DAS)-ELISA and reverse transcription and polymerase chain reaction (RT-PCR) methods. The products of the 566bp were amplified from coat protein region of PMTV RNA 3 in both root and leaf samples of baited plants. The virus was detected in 10 potato cultivars commercially grown in the region using DAS-ELISA and RT-PCR. The virus was also detected in zoospores of Spongospora subterranea derived from the peels of selected scabby tubers using triple antibody sandwich (TAS)-ELISA. The results indicate that a bait plant bioassay, infectivity assay, ELISA and RT-PCR can detect PMTV in roots and leaves of baited plants, field samples, zoospores of S. subterranea and tubers of 10 potato cultivars commercially grown in the region. PMID:24161813

  5. Development of a real-time quantitative RT-PCR to detect REV contamination in live vaccine.

    PubMed

    Luan, Huaibiao; Wang, Yixin; Li, Yang; Cui, Zhizhong; Chang, Shuang; Zhao, Peng

    2016-09-01

    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). PMID:27122388

  6. Cross-Platform Evaluation of Commercial Real-Time SYBR Green RT-PCR Kits for Sensitive and Rapid Detection of European Bat Lyssavirus Type 1

    PubMed Central

    Picard-Meyer, Evelyne; Peytavin de Garam, Carine; Schereffer, Jean Luc; Marchal, Clotilde; Robardet, Emmanuelle; Cliquet, Florence

    2015-01-01

    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, R2 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. PMID:25785274

  7. Cross-platform evaluation of commercial real-time SYBR green RT-PCR kits for sensitive and rapid detection of European bat Lyssavirus type 1.

    PubMed

    Picard-Meyer, Evelyne; Peytavin de Garam, Carine; Schereffer, Jean Luc; Marchal, Clotilde; Robardet, Emmanuelle; Cliquet, Florence

    2015-01-01

    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. PMID:25785274

  8. Patterned Plasmonic Nanoparticle Arrays for Microfluidic and Multiplexed Biological Assays.

    PubMed

    He, Jie; Boegli, Michelle; Bruzas, Ian; Lum, William; Sagle, Laura

    2015-11-17

    For applications ranging from medical diagnostics and drug screening to chemical and biological warfare detection, inexpensive, rapid-readout, portable devices are required. Localized surface plasmon resonance (LSPR) technologies show substantial promise toward meeting these goals, but the generation of portable, multiplexed and/or microfluidic devices incorporating sensitive nanoparticle arrays is only in its infancy. Herein, we have combined photolithography with Hole Mask Colloidal lithography to pattern uniform nanoparticle arrays for both microfluidic and multiplexed devices. The first proof-of-concept study is carried out with 5- and 7-channel microfluidic devices to acquire one-shot binding curves and protein binding kinetic data. The second proof-of-concept study involved the fabrication of a 96-spot plate that can be inserted into a standard plate reader for the multiplexed detection of protein binding. This versatile fabrication technique should prove useful in next generation chips for bioassays and genetic screening. PMID:26494412

  9. Opportunities for bead-based multiplex assays in veterinary diagnostic laboratories

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bead based multiplex assays (BBMA) also referred to as Luminex, MultiAnalyte Profiling or cytometric bead array (CBA) assays, are applicable for high throughput, simultaneous detection of multiple analytes in solution (from several, up to 50-500 analytes within a single, small sample volume). Curren...

  10. Rapid Staphylococcus aureus agr type determination by a novel multiplex real-time quantitative PCR assay.

    PubMed

    Francois, Patrice; Koessler, Thibaud; Huyghe, Antoine; Harbarth, Stephan; Bento, Manuela; Lew, Daniel; Etienne, Jérôme; Pittet, Didier; Schrenzel, Jacques

    2006-05-01

    The accessory gene regulator (agr) is a crucial regulatory component of Staphylococcus aureus involved in the control of bacterial virulence factor expression. We developed a real-time multiplex quantitative PCR assay for the rapid determination of S. aureus agr type. This assay represents a rapid and affordable alternative to sequence-based strategies for assessing relevant epidemiological information. PMID:16672433

  11. Rapid Staphylococcus aureus agr Type Determination by a Novel Multiplex Real-Time Quantitative PCR Assay

    PubMed Central

    Francois, Patrice; Koessler, Thibaud; Huyghe, Antoine; Harbarth, Stephan; Bento, Manuela; Lew, Daniel; Etienne, Jérôme; Pittet, Didier; Schrenzel, Jacques

    2006-01-01

    The accessory gene regulator (agr) is a crucial regulatory component of Staphylococcus aureus involved in the control of bacterial virulence factor expression. We developed a real-time multiplex quantitative PCR assay for the rapid determination of S. aureus agr type. This assay represents a rapid and affordable alternative to sequence-based strategies for assessing relevant epidemiological information. PMID:16672433

  12. Microarray and RT-PCR screening for white spot syndrome virus immediate-early genes in cycloheximide-treated shrimp

    SciTech Connect

    Liu Wangjing; Chang Yunshiang; Wang Chunghsiung; Kou, Guang-Hsiung; Lo Chufang . E-mail: gracelow@ntu.edu.tw

    2005-04-10

    Here, we report for the first time the successful use of cycloheximide (CHX) as an inhibitor to block de novo viral protein synthesis during WSSV (white spot syndrome virus) infection. Sixty candidate IE (immediate-early) genes were identified using a global analysis microarray technique. RT-PCR showed that the genes corresponding to ORF126, ORF242 and ORF418 in the Taiwan isolate were consistently CHX-insensitive, and these genes were designated ie1, ie2 and ie3, respectively. The sequences for these IE genes also appear in the two other WSSV isolates that have been sequenced. Three corresponding ORFs were identified in the China WSSV isolate, but only an ORF corresponding to ie1 was predicted in the Thailand isolate. In a promoter activity assay in Sf9 insect cells using EGFP (enhanced green fluorescence protein) as a reporter, ie1 showed very strong promoter activity, producing higher EGFP signals than the insect Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) ie2 promoter.

  13. Development of multiplex PCR assay for simultaneous detection of five bacterial fish pathogens.

    PubMed

    Altinok, Ilhan; Capkin, Erol; Kayis, Sevki

    2008-10-15

    A multiplex polymerase chain reaction (PCR) method was designed for the simultaneous detection of the five major fish pathogens, Aeromonas hydrophila, Aeromonas salmonicida subsp. salmonicida, Flavobacterium columnare, Renibacterium salmoninarum, and Yersinia ruckeri. Each of the five pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism. The detection limits of the multiplex PCR was in the range of 2, 1, 1, 3, and 1CFU for A. hydrophila, A. salmonicida, F. columnare, R. salmoninarum, and Y. ruckeri, respectively. Multiplex PCR did not produce any nonspecific amplification products when tested against 23 related species of bacteria. The multiplex PCR assay was useful for the detection of the bacteria in naturally infected fish. This assay is a sensitive and specific and reproducible diagnostic tool for the simultaneous detection of five pathogenic bacteria that cause disease in fish. Therefore, it could be a useful alternative to the conventional culture based method. PMID:18499358

  14. Development of universal primers for detection of potato carlaviruses by RT-PCR.

    PubMed

    Nie, Xianzhou; Bai, Yanju; Molen, Teresa A; Desjardins, David C

    2008-05-01

    To facilitate efficient and accurate detection of potato-infecting carlaviruses, degenerated universal primers were designed based on conserved amino acid and nucleotide sequences. Two sense primers, Car-F1 and Car-F2, were based on the amino acid sequences "SNNMA" and "GLGVPTE", respectively, in the coat protein. The reverse primer, Car-R, which was located at the border of the nucleic acid binding protein gene and the 3' untranslated region, and dT-B, which was derived from the oligo-dT targeting the poly(A) tail, were selected. Successful application of fragments within the predicted size range of carlaviruses was obtained using Car-F1 paired with either Car-R or dT-B from tested carlaviruses (Potato virus S, M and latent) by RT-PCR. The Car-F2 failed to yield clear-cut fragments within the predicted size range when paired with either Car-R or dT-B in RT-PCR. However, a less degenerated version of the primer, Car-F2b, resulted in amplicons within the predicted size range when paired with either Car-R or dT-B. Sequencing of the tentative carlavirus-fragments resulting from Car-F1/Car-R and Car-F2b/dT-B proved their carlavirus-origin, thus indicating the high specificity of these primers. The sensitivity of Car-F1/Car-R or Car-F2b/Car-R mediated RT-PCR for the detection of carlavirus-infected potato tubers were assessed using composite samples containing one carlavirus-infected-potato-tuber RNA sample with up to 49 virus-free-potato-tuber RNA samples under the optimal annealing temperature. The target carlaviruses were detected readily from all composites, demonstrating a high sensitivity. The method was further evaluated using presumed virus-free or carlavirus-infected potatoes of several cultivars, and reliable results were obtained. PMID:18353450

  15. Design and validation of a real-time RT-PCR for the simultaneous detection of enteroviruses and parechoviruses in clinical samples.

    PubMed

    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

    2014-11-01

    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. PMID:25152526

  16. Sensitive and specific detection of strains of Japanese encephalitis virus using a one-step TaqMan RT-PCR technique.

    PubMed

    Huang, Jau-Ling; Lin, Hui-Tsu; Wang, Yu-Ming; Weng, Ming-Hui; Ji, Da-Der; Kuo, Ming-Der; Liu, Huan-Wun; Lin, Chang-Shen

    2004-12-01

    A rapid, sensitive, and accurate laboratory diagnostic test is needed for distinguishing Japanese encephalitis virus (JEV) from other diseases featuring similar clinical symptoms and also for preventing potential outbreaks. In this study, a TaqMan reverse transcription (RT)-polymerase chain reaction (PCR) assay was developed for rapid detection and quantification of the viral RNA of various JEV strains. A consensus JEV NS3 region was chosen to design the primers and the TaqMan probe. The JEV TaqMan assay used the EZ-rTtH RT-PCR system featuring advantages such as a one-step, high-temperature RT reaction modality and preventing carry-over contamination. The sensitivity of the JEV TaqMan assay for detecting in vitro-transcribed JEV NS3 RNA was estimated to be one to five copies of RNA per reaction. For cultured JE virions, less than 40 plaque forming unit (PFU)/ml of virus load (corresponding to 0.07 PFU/test) could be detected. In addition, the JEV TaqMan assay could detect all seven strains of JEV tested, but provided negative results for nine other flaviviruses and encephalitis viruses tested. The JEV TaqMan assay demonstrated greater sensitivity and specificity than traditional RT-PCR methods as has been previously reported. The application of the JEV TaqMan assay herein has been shown to the sensitive detection of the JEV from both mosquito pools and also JEV-spiking human blood. The assay should be of use in diagnostic laboratory conduct and could be used to replace or complement time-consuming viral-culture methods, thus achieving more rapid, sensitive, and highly specific identification of JEV infection. PMID:15484282

  17. RT-PCR analysis of Tecta, Coch, Eya4 and Strc in mouse cochlear explants.

    PubMed

    Maeda, Yukihide; Fukushima, Kunihiro; Kakiuchi, Masashi; Orita, Yorihisa; Nishizaki, Kazunori; Smith, Richard J H

    2005-03-15

    Tecta, Coch, Eya4 and Strc are mouse orthologs of four human deafness-associated genes. Their expression is markedly restricted to specific cell types in cochleae. Cochleae were dissected on embryonic day 15 and cultured in vitro. Relative messenger RNA abundance of each gene was quantified by RT-PCR and compared in-vivo cochleae of equivalent embryonic age. After 48 h in culture, in-vivo and explant Strc expression levels were equivalent, Eya4 level reduced in explanted tissues, and expression of Tecta and Coch did not show the expected temporal rise. Expression of these genes was detectable even after 96 h. These results suggest that it is feasible to test the expression of inner ear specific genes in explanted cochleae. PMID:15729138

  18. Emulating a crowded intracellular environment in vitro dramatically improves RT-PCR performance

    SciTech Connect

    Lareu, Ricky R.; Harve, Karthik S.; Raghunath, Michael

    2007-11-09

    The polymerase chain reaction's (PCR) phenomenal success in advancing fields as diverse as Medicine, Agriculture, Conservation, or Paleontology is based on the ability of using isolated prokaryotic thermostable DNA polymerases in vitro to copy DNA irrespective of origin. This process occurs intracellularly and has evolved to function efficiently under crowded conditions, namely in an environment packed with macromolecules. However, current in vitro practice ignores this important biophysical parameter of life. In order to more closely emulate conditions of intracellular biochemistry in vitro we added inert macromolecules into reverse transcription (RT) and PCR. We show dramatic improvements in all parameters of RT-PCR including 8- to 10-fold greater sensitivity, enhanced polymerase processivity, higher specific amplicon yield, greater primer annealing and specificity, and enhanced DNA polymerase thermal stability. The faster and more efficient reaction kinetics was a consequence of the cumulative molecular and thermodynamic effects of the excluded volume effect created by macromolecular crowding.

  19. [RT-PCR detecting NUP98-HOX fusion gene in leukemia].

    PubMed

    Zhang, Yan; Li, Ling; Wen, Bing-Zhao; Lin, Ren-Yong; Cao, Xu; Wang, Ning; Ha Li Da, Ya Seng; Jiang, Ming; Wen, Hao; Lu, Xiao-Mei; Feng, Xiao-Hui; Wang, Xin

    2005-02-01

    To investigate whether there are NUP98-HOXA, NUP98-HOXB, NUP98-HOXC, NUP98-HOXD fusion genes in leukemia patients in Xinjiang, cellular total RNA was extracted from the bone marrow mononuclear cells, the formaldehyde-agarose gel electrophoresis was used to judge whether RNA was intact, the 17 RT-PCR primers were designed to amplify the predicted fusion junctions and 412 bp GAPDH was used as an internal control, NUP98-HOXA fusion genes were amplified by nested-PCR following reverse transcription. One-step PCR was performed to amplify the other predicted fusion genes. The results showed that RNA was proved to be intact and expression of GAPDH was found in every sample. However, no predicted fusion transcripts were detected in leukemia patients. In conclusion, no NUP98-HOX fusion genes were detected in the samples from Xinjiang. PMID:15748441

  20. Functional multiplex reporter assay using tagged Gaussia luciferase

    PubMed Central

    van Rijn, Sjoerd; Nilsson, Jonas; Noske, David P.; Vandertop, W. Peter; Tannous, Bakhos A.; Würdinger, Thomas

    2013-01-01

    We have developed a multiplex reporter system to monitor multiple biological variables in real-time. The secreted Gaussia luciferase was fused to ten different epitope tags (Gluctag), each expressed in different tumor cells. By immunobinding of the tags followed by Gluctag detection, this system allowed the independent and real-time monitoring of mixed cell cultures in vitro and of mixed subcutaneous and intracranial tumor subpopulations in vivo. PMID:23308339

  1. Quantitative Evaluation and Selection of Reference Genes for Quantitative RT-PCR in Mouse Acute Pancreatitis

    PubMed Central

    Yan, Zhaoping; Gao, Jinhang; Lv, Xiuhe; Yang, Wenjuan; Wen, Shilei; Tong, Huan; Tang, Chengwei

    2016-01-01

    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

  2. Heminested reverse-transcriptase polymerase chain reaction (hnRT-PCR) as a tool for rabies virus detection in stored and decomposed samples

    PubMed Central

    Araújo, Danielle B; Langoni, Helio; Almeida, Marilene F; Megid, Jane

    2008-01-01

    Background The use of methods, both sensitive and specific, for rabies diagnosis are important tools for the control and prophylaxis of the disease. Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) has been used in rabies diagnosis with good results, even in decomposed materials. Additionally, molecular techniques have been used for epidemiological studies and to gain a better knowledge of viral epidemiology. Findings The aim of this work was to evaluate the RT-PCR and hnRT-PCR for rabies virus detection in original tissues stored at -20°C for different periods considering their use for rabies virus detection in stored and decomposed samples. RT-PCR and hnRT-PCR were evaluated in 151 brain samples from different animal species, thawed and left at room temperature for 72 hours for decomposition. The RT-PCR and hnRT-PCR results were compared with previous results from Direct Fluorescent Antibody Test and Mouse Inoculation Test. From the 50 positive fresh samples, 26 (52%) were positive for RT-PCR and 45 (90%) for hnRT-PCR. From the 48 positive decomposed samples, 17 (34, 3%) were positive for RT-PCR and 36 (75%) for hnRT-PCR. No false-positives results were found in the negatives samples evaluated to the molecular techniques. Conclusion These results show that the hnRT-PCR was more sensitive than RT-PCR, and both techniques presented lower sensibility in decomposed samples. The hnRT-PCR demonstrated efficacy in rabies virus detection in stored and decomposed materials suggesting it's application for rabies virus retrospective epidemiological studies. PMID:18710536

  3. A multiplex-PCR assay for identification of the quarantine plant pathogen Xanthomonas axonopodis pv. phaseoli.

    PubMed

    Boureau, T; Kerkoud, M; Chhel, F; Hunault, G; Darrasse, A; Brin, C; Durand, K; Hajri, A; Poussier, S; Manceau, C; Lardeux, F; Saubion, F; Jacques, M-A

    2013-01-01

    In this study we developed an algorithm to screen for all exact molecular signatures of the quarantine pathogen Xanthomonas axonopodis pv. phaseoli (Xap), based on available data of the presence or absence of virulence-associated genes. The simultaneous presence of genes avrBsT and xopL is specific to Xap. Therefore we developed a multiplex PCR assay targeting avrBsT and xopL for the molecular identification of Xap. The specificity of this multiplex was validated by comparison to that of other molecular identification assays aimed at Xap, on a wide collection of reference strains. This multiplex was further validated on a blind collection of Xanthomonas isolates for which pathogenicity was assayed by stem wounding and by dipping leaves into calibrated inocula. This multiplex was combined to the previously described X4c/X4e molecular identification assay for Xap. Such a combination enables the molecular identification of all strains of Xanthomonas pathogenic on bean. Results also show that assay by stem wounding does not give reliable results in the case of Xap, and that pathogenicity assays by dipping should be preferred. PMID:23142341

  4. Identification of shared TCR sequences from T cells in human breast cancer using emulsion RT-PCR.

    PubMed

    Munson, Daniel J; Egelston, Colt A; Chiotti, Kami E; Parra, Zuly E; Bruno, Tullia C; Moore, Brandon L; Nakano, Taizo A; Simons, Diana L; Jimenez, Grecia; Yim, John H; Rozanov, Dmitri V; Falta, Michael T; Fontenot, Andrew P; Reynolds, Paul R; Leach, Sonia M; Borges, Virginia F; Kappler, John W; Spellman, Paul T; Lee, Peter P; Slansky, Jill E

    2016-07-19

    Infiltration of T cells in breast tumors correlates with improved survival of patients with breast cancer, despite relatively few mutations in these tumors. To determine if T-cell specificity can be harnessed to augment immunotherapies of breast cancer, we sought to identify the alpha-beta paired T-cell receptors (TCRs) of tumor-infiltrating lymphocytes shared between multiple patients. Because TCRs function as heterodimeric proteins, we used an emulsion-based RT-PCR assay to link and amplify TCR pairs. Using this assay on engineered T-cell hybridomas, we observed ∼85% accurate pairing fidelity, although TCR recovery frequency varied. When we applied this technique to patient samples, we found that for any given TCR pair, the dominant alpha- or beta-binding partner comprised ∼90% of the total binding partners. Analysis of TCR sequences from primary tumors showed about fourfold more overlap in tumor-involved relative to tumor-free sentinel lymph nodes. Additionally, comparison of sequences from both tumors of a patient with bilateral breast cancer showed 10% overlap. Finally, we identified a panel of unique TCRs shared between patients' tumors and peripheral blood that were not found in the peripheral blood of controls. These TCRs encoded a range of V, J, and complementarity determining region 3 (CDR3) sequences on the alpha-chain, and displayed restricted V-beta use. The nucleotides encoding these shared TCR CDR3s varied, suggesting immune selection of this response. Harnessing these T cells may provide practical strategies to improve the shared antigen-specific response to breast cancer. PMID:27307436

  5. Identification of shared TCR sequences from T cells in human breast cancer using emulsion RT-PCR

    PubMed Central

    Munson, Daniel J.; Egelston, Colt A.; Chiotti, Kami E.; Parra, Zuly E.; Bruno, Tullia C.; Moore, Brandon L.; Nakano, Taizo A.; Simons, Diana L.; Jimenez, Grecia; Yim, John H.; Rozanov, Dmitri V.; Falta, Michael T.; Fontenot, Andrew P.; Reynolds, Paul R.; Leach, Sonia M.; Borges, Virginia F.; Kappler, John W.; Spellman, Paul T.; Slansky, Jill E.

    2016-01-01

    Infiltration of T cells in breast tumors correlates with improved survival of patients with breast cancer, despite relatively few mutations in these tumors. To determine if T-cell specificity can be harnessed to augment immunotherapies of breast cancer, we sought to identify the alpha–beta paired T-cell receptors (TCRs) of tumor-infiltrating lymphocytes shared between multiple patients. Because TCRs function as heterodimeric proteins, we used an emulsion-based RT-PCR assay to link and amplify TCR pairs. Using this assay on engineered T-cell hybridomas, we observed ∼85% accurate pairing fidelity, although TCR recovery frequency varied. When we applied this technique to patient samples, we found that for any given TCR pair, the dominant alpha- or beta-binding partner comprised ∼90% of the total binding partners. Analysis of TCR sequences from primary tumors showed about fourfold more overlap in tumor-involved relative to tumor-free sentinel lymph nodes. Additionally, comparison of sequences from both tumors of a patient with bilateral breast cancer showed 10% overlap. Finally, we identified a panel of unique TCRs shared between patients’ tumors and peripheral blood that were not found in the peripheral blood of controls. These TCRs encoded a range of V, J, and complementarity determining region 3 (CDR3) sequences on the alpha-chain, and displayed restricted V-beta use. The nucleotides encoding these shared TCR CDR3s varied, suggesting immune selection of this response. Harnessing these T cells may provide practical strategies to improve the shared antigen-specific response to breast cancer. PMID:27307436

  6. Validation of Reference Genes for Gene Expression by Quantitative Real-Time RT-PCR in Stem Segments Spanning Primary to Secondary Growth in Populus tomentosa.

    PubMed

    Wang, Ying; Chen, Yajuan; Ding, Liping; Zhang, Jiewei; Wei, Jianhua; Wang, Hongzhi

    2016-01-01

    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

  7. Detection of bovine group a rotavirus using rapid antigen detection kits, rt-PCR and next-generation DNA sequencing.

    PubMed

    Minami-Fukuda, Fujiko; Nagai, Makoto; Takai, Hikaru; Murakami, Toshiaki; Ozawa, Tadashi; Tsuchiaka, Shinobu; Okazaki, Sachiko; Katayama, Yukie; Oba, Mami; Nishiura, Naomi; Sassa, Yukiko; Omatsu, Tsutomu; Furuya, Tetsuya; Koyama, Satoshi; Shirai, Junsuke; Tsunemitsu, Hiroshi; Fujii, Yoshiki; Katayama, Kazuhiko; Mizutani, Tetsuya

    2013-12-30

    We investigated the sensitivity of human rotavirus rapid antigen detection (RAD) kits, RT-PCR and next-generation DNA sequencing (NGS) for detection of bovine group A rotavirus (RVA). The Dipstick 'Eiken' Rota (Dipstick) showed the highest sensitivity out of the seven RAD kits against all selected strains in limited dilution analyses, which was consistent with the results for equine rotavirus previously reported. RT-PCR had 10⁰-10³-fold higher sensitivity than the Dipstick. NGS using thirteen RT-PCR-negative fecal samples revealed that all samples yielded RVA reads and especially that two of them covered all 11 genome segments. Moreover, mapping reads to reference sequences allowed genotyping. The NGS would be sensitive and useful for analysis of less dependent on specific primers and screening of genotypes. PMID:23912876

  8. Development of a novel multiplex beads-based assay for autoantibody detection for colorectal cancer diagnosis.

    PubMed

    Villar-Vázquez, Roi; Padilla, Guillermo; Fernández-Aceñero, María Jesús; Suárez, Adolfo; Fuente, Eduardo; Pastor, Carlos; Calero, Miguel; Barderas, Rodrigo; Casal, J Ignacio

    2016-04-01

    Humoral response in cancer patients can be used for early cancer detection. By screening high-density protein microarrays with sera from colorectal cancer (CRC) patients and controls, we identified 16 tumor-associated antigens (TAAs) exhibiting high diagnostic value. This high number of TAAs requires the development of multiplex assays combining different antigens for a faster and more accurate prediction of CRC. Here, we have developed and optimized a bead-based assay using nine selected TAAs and two controls to provide a multiplex test for early CRC diagnosis. We screened a collection of 307 CRC patients' and control sera with the beads assay to identify and validate the best TAA combination for CRC detection. The multiplex bead-based assay exhibited a similar diagnostic performance to detect the humoral response in comparison to multiple ELISA analyses. After multivariate analysis, a panel composed of GTF2B, EDIL3, HCK, PIM1, STK4, and p53, together with gender and age, was identified as the best combination of TAAs for CRC diagnosis, achieving an AUC of 89.7%, with 66% sensitivity at 90.0% fixed specificity. The model was validated using bootstrapping analysis. In summary, we have developed a novel multiplex bead assay that after validation with a larger independent cohort of sera could be utilized in a high-throughput manner for population screening to facilitate the detection of early CRC patients. PMID:26915739

  9. Genome-Wide Identification and Validation of Reference Genes in Infected Tomato Leaves for Quantitative RT-PCR Analyses

    PubMed Central

    Müller, Oliver A.; Grau, Jan; Thieme, Sabine; Prochaska, Heike; Adlung, Norman; Sorgatz, Anika; Bonas, Ulla

    2015-01-01

    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

  10. Genome-Wide Identification and Validation of Reference Genes in Infected Tomato Leaves for Quantitative RT-PCR Analyses.

    PubMed

    Müller, Oliver A; Grau, Jan; Thieme, Sabine; Prochaska, Heike; Adlung, Norman; Sorgatz, Anika; Bonas, Ulla

    2015-01-01

    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

  11. Development and Evaluation of an Enterovirus D68 Real-Time Reverse Transcriptase PCR Assay

    PubMed Central

    Wylie, Todd N.; Wylie, Kristine M.; Buller, Richard S.; Cannella, Maria

    2015-01-01

    We have developed and evaluated a real-time reverse transcriptase PCR (RT-PCR) assay for the detection of human enterovirus D68 (EV-D68) in clinical specimens. This assay was developed in response to the unprecedented 2014 nationwide EV-D68 outbreak in the United States associated with severe respiratory illness. As part of our evaluation of the outbreak, we sequenced and published the genome sequence of the EV-D68 virus circulating in St. Louis, MO. This sequence, along with other GenBank sequences from past EV-D68 occurrences, was used to computationally select a region of EV-D68 appropriate for targeting in a strain-specific RT-PCR assay. The RT-PCR assay amplifies a segment of the VP1 gene, with an analytic limit of detection of 4 copies per reaction, and it was more sensitive than commercially available assays that detect enteroviruses and rhinoviruses without distinguishing between the two, including three multiplex respiratory panels approved for clinical use by the FDA. The assay did not detect any other enteroviruses or rhinoviruses tested and did detect divergent strains of EV-D68, including the first EV-D68 strain (Fermon) identified in California in 1962. This assay should be useful for identifying and studying current and future outbreaks of EV-D68 viruses. PMID:26063859

  12. Development and Evaluation of an Enterovirus D68 Real-Time Reverse Transcriptase PCR Assay.

    PubMed

    Wylie, Todd N; Wylie, Kristine M; Buller, Richard S; Cannella, Maria; Storch, Gregory A

    2015-08-01

    We have developed and evaluated a real-time reverse transcriptase PCR (RT-PCR) assay for the detection of human enterovirus D68 (EV-D68) in clinical specimens. This assay was developed in response to the unprecedented 2014 nationwide EV-D68 outbreak in the United States associated with severe respiratory illness. As part of our evaluation of the outbreak, we sequenced and published the genome sequence of the EV-D68 virus circulating in St. Louis, MO. This sequence, along with other GenBank sequences from past EV-D68 occurrences, was used to computationally select a region of EV-D68 appropriate for targeting in a strain-specific RT-PCR assay. The RT-PCR assay amplifies a segment of the VP1 gene, with an analytic limit of detection of 4 copies per reaction, and it was more sensitive than commercially available assays that detect enteroviruses and rhinoviruses without distinguishing between the two, including three multiplex respiratory panels approved for clinical use by the FDA. The assay did not detect any other enteroviruses or rhinoviruses tested and did detect divergent strains of EV-D68, including the first EV-D68 strain (Fermon) identified in California in 1962. This assay should be useful for identifying and studying current and future outbreaks of EV-D68 viruses. PMID:26063859

  13. Genotyping of Toxoplasma gondii isolates with 15 microsatellite markers in a single multiplex PCR assay.

    PubMed

    Ajzenberg, Daniel; Collinet, Frédéric; Mercier, Aurélien; Vignoles, Philippe; Dardé, Marie-Laure

    2010-12-01

    We developed an easy-to-use method for genotyping Toxoplasma gondii isolates in a single multiplex PCR assay with 15 microsatellite markers. This method was validated by testing 26 reference isolates that had been characterized with other sets of markers. PMID:20881166

  14. Genotyping of Toxoplasma gondii Isolates with 15 Microsatellite Markers in a Single Multiplex PCR Assay

    PubMed Central

    Ajzenberg, Daniel; Collinet, Frédéric; Mercier, Aurélien; Vignoles, Philippe; Dardé, Marie-Laure

    2010-01-01

    We developed an easy-to-use method for genotyping Toxoplasma gondii isolates in a single multiplex PCR assay with 15 microsatellite markers. This method was validated by testing 26 reference isolates that had been characterized with other sets of markers. PMID:20881166

  15. A Protocol for a High-Throughput Multiplex Cell Viability Assay.

    PubMed

    Gilbert, Daniel F; Boutros, Michael

    2016-01-01

    High-throughput cell viability assays are broadly used in RNAi and small molecule screening experiments to identify compounds that selectively kill cancer cells or as counter screens to exclude the compounds that have a generic effect on cell growth. While there are several assaying techniques available, cellular fitness is often assessed on the basis of one single and often rather indirect physiological indicator. This can lead to inconsistencies and poor correspondence between cell viability screening experiments, conducted under comparable conditions but with different viability indicators. Multiplexing, i.e., the combination of different individual assaying techniques in one experiment and subsequent comparative analysis of multiparametric data can decrease inter-assay variability and increase dataset concordance. Here, we describe a protocol for a multiplexing approach for high-throughput cell viability screening to address the issues encountered in the classical strategy using a single fitness indicator described above. The method combines a biochemical, luminescence-based approach and two fluorescence-based assay types. The biochemical method assesses cellular fitness by quantifying intracellular ATP concentration. Calcein labeling reflects cell fitness through membrane integrity and indirect measurement of ATP-dependent enzymatic esterase activity. Hoechst DNA stain correlates cell fitness with cellular DNA content. The presented multiplexing approach is suitable for low, medium and high-throughput screening and has the potential to decrease inter-assay variability and increase dataset concordance as well as reproducibility of experimental results. PMID:27581285

  16. Evaluation of Multiplexed Foot-and-Mouth Disease Nonstructural Protein Antibody Assay Against Standardized Bovine Serum Panel

    SciTech Connect

    Perkins, J; Parida, S; Clavijo, A

    2007-05-14

    Liquid array technology has previously been used to show proof-of-principle of a multiplexed non structural protein serological assay to differentiate foot-and-mouth infected and vaccinated animals. The current multiplexed assay consists of synthetically produced peptide signatures 3A, 3B and 3D and recombinant protein signature 3ABC in combination with four controls. To determine diagnostic specificity of each signature in the multiplex, the assay was evaluated against a naive population (n = 104) and a vaccinated population (n = 94). Subsequently, the multiplexed assay was assessed using a panel of bovine sera generated by the World Reference Laboratory for foot-and-mouth disease in Pirbright, UK. This sera panel has been used to assess the performance of other singleplex ELISA-based non-structural protein antibody assays. The 3ABC signature in the multiplexed assay showed comparative performance to a commercially available non-structural protein 3ABC ELISA (Cedi test{reg_sign}) and additional information pertaining to the relative diagnostic sensitivity of each signature in the multiplex is acquired in one experiment. The encouraging results of the evaluation of the multiplexed assay against a panel of diagnostically relevant samples promotes further assay development and optimization to generate an assay for routine use in foot-and-mouth disease surveillance.

  17. Quantitative RT-PCR Gene Evaluation and RNA Interference in the Brown Marmorated Stink Bug

    PubMed Central

    Bansal, Raman; Mittapelly, Priyanka; Chen, Yuting; Mamidala, Praveen; Zhao, Chaoyang; Michel, Andy

    2016-01-01

    The brown marmorated stink bug (Halyomorpha halys) has emerged as one of the most important invasive insect pests in the United States. Functional genomics in H. halys remains unexplored as molecular resources in this insect have recently been developed. To facilitate functional genomics research, we evaluated ten common insect housekeeping genes (RPS26, EF1A, FAU, UBE4A, ARL2, ARP8, GUS, TBP, TIF6 and RPL9) for their stability across various treatments in H. halys. Our treatments included two biotic factors (tissues and developmental stages) and two stress treatments (RNAi injection and starvation). Reference gene stability was determined using three software algorithms (geNorm, NormFinder, BestKeeper) and a web-based tool (RefFinder). The qRT-PCR results indicated ARP8 and UBE4A exhibit the most stable expression across tissues and developmental stages, ARL2 and FAU for dsRNA treatment and TBP and UBE4A for starvation treatment. Following the dsRNA treatment, all genes except GUS showed relatively stable expression. To demonstrate the utility of validated reference genes in accurate gene expression analysis and to explore gene silencing in H. halys, we performed RNAi by administering dsRNA of target gene (catalase) through microinjection. A successful RNAi response with over 90% reduction in expression of target gene was observed. PMID:27144586

  18. Selection and Validation of Reference Genes for qRT-PCR in Cycas elongata

    PubMed Central

    Deng, Tian; Chen, Letian; Wu, Hong; Zhang, Shouzhou

    2016-01-01

    Quantitative reverse transcription PCR (qRT-PCR) is a sensitive technique used in gene expression studies. To achieve a reliable quantification of transcripts, appropriate reference genes are required for comparison of transcripts in different samples. However, few reference genes are available for non-model taxa, and to date, reliable reference genes in Cycas elongata have not been well characterized. In this study, 13 reference genes (ACT7, TUB, UBQ, EIF4, EF1, CLATHRIN1, PP2A, RPB2, GAPC2, TIP41, MAPK, SAMDC and CYP) were chosen from the transcriptome database of C. elongata, and these genes were evaluated in 8 different organ samples. Three software programs, NormFinder, GeNorm and BestKeeper, were used to validate the stability of the potential reference genes. Results obtained from these three programs suggested that CeGAPC2 and CeRPB2 are the most stable reference genes, while CeACT7 is the least stable one among the 13 tested genes. Further confirmation of the identified reference genes was established by the relative expression of AGAMOUSE gene of C. elongata (CeAG). While our stable reference genes generated consistent expression patterns in eight tissues, we note that our results indicate that an inappropriate reference gene might cause erroneous results. Our systematic analysis for stable reference genes of C. elongata facilitates further gene expression studies and functional analyses of this species. PMID:27124298

  19. Survey and RT-PCR Based Detection of Cardamom mosaic virus Affecting Small Cardamom in India.

    PubMed

    Biju, C N; Siljo, A; Bhat, A I

    2010-10-01

    Mosaic or marble or katte disease caused by Cardamom mosaic virus (CdMV) is an important production constraint in all cardamom growing regions of the world. In the present study, 84 cardamom plantations in 44 locations of Karnataka and Kerala were surveyed. The incidence of the disease ranged from 0 to 85%. The incidence was highest in Madikeri (Karnataka) while no incidence was recorded in Peermade (Kerala). In general, incidence and severity of the disease was higher in cardamom plantations of Karnataka. A procedure for total RNA isolation from cardamom and detection of CdMV through reverse transcription-polymerase chain reaction (RT-PCR) using primers targeting the conserved region of coat protein was standardized and subsequently validated by testing more than 50 field cardamom samples originating from Karnataka and Kerala states. The method can be used for indexing the planting material and identifying resistant lines/cultivars before either they are further multiplied in large scale or incorporated in breeding. PMID:23637495

  20. The use of relative quantitative RT-PCR for expression analysis in azalea flower color sports.

    PubMed

    De Keyser, E; De Riek, J; Van Bockstaele, E

    2003-01-01

    The fastest way to create new azalea (Rhododendron simsii hybrids) cultivars is by making use of flower colour sports, which appear spontaneously on azalea plants. Unfortunately, there is still very little known on how bud sport induction occurs. Therefore, genes coding for two key enzymes of the azalea flavonoid biosynthesis pathway, chalcon synthase (chs) and dihydroflavonol 4-reductase (dfr) that were reported before to be apt for modification by the action of bud sporting, were isolated and characterized. The expression of these two flower colour genes in the petals of azalea flowers will be compared between all 'Hellmut Vogel' flower colour sports. To measure the expression levels of both genes, relative quantitative RT-PCR analysis will be worked out on a real-time PCR machine. The expression of housekeeping genes, which is expected to be the same for all sports, will be used to calculate the relative expression level of the two genes of interest. The optimisation of this technique will be discussed. PMID:24757769

  1. Absolute mRNA quantification of Pseudomonas fluorescens Pf-5 by qRT-PCR using universal RNA controls

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real time quantitative RT-PCR is considered as standard for gene expression and mRNA estimate. As a calibration standard, a conserved control gene such as housekeeping genes is commonly used for data normalization and analysis. A significant problem has been observed with increased applications; h...

  2. Detection of EML4-ALK in Lung Adenocarcinoma Using Pleural Effusion with FISH, IHC, and RT-PCR Methods

    PubMed Central

    Zhou, Xiaodie; Song, Yong; Zhou, Xiaojun; Yu, Like; Wang, Jiandong

    2015-01-01

    Anaplastic lymphoma kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) gene rearrangements occur in approximately 5% of non-small-cell lung cancers (NSCLC), leading to the overexpression of anaplastic lymphoma kinase and predicting a response to the targeted inhibitor, crizotinib. Malignant pleural effusion occurs in most patients with advanced lung cancer, especially adenocarcinoma, and tissue samples are not always available from these patients. We attempted to clarify the feasibility of detecting the EML4-ALK fusion gene in pleural effusion cells using different methods. We obtained 66 samples of pleural effusion from NSCLC patients. The pleural effusion fluid was centrifuged, and the cellular components obtained were formalin fixed and paraffin embedded. The EML4-ALK fusion gene status was determined with fluorescent in situ hybridization (FISH), reverse transcription—polymerase chain reaction (RT-PCR), and immunohistochemistry (IHC). EML4-ALK was detected in three of 66 patient samples (4.5%) with RT-PCR. When the RT-PCR data were used as the standard, one false positive and one false negative samples were identified with IHC; and one false negative sample was identified with FISH. These results suggest that a block of pleural effusion cells can be used to detect the EML4-ALK fusion gene. IHC had good sensitivity, but low specificity. FISH had low sensitivity, but high specificity. RT-PCR is a good candidate method for detecting EML4-ALK in blocks of pleural effusion cells from lung cancer patients. PMID:25785456

  3. Evaluation of an updated real-time RT-PCR test for the identification of the H7 subtype

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rapid detection of avian influenza (AI) virus and identification of the H5 and H7 subtypes is critical for wild bird monitoring programs. A real-time RT-PCR test for identification of the H7 subtype in North America was first reported in 2002. With the recent surveillance in wild birds it was disc...

  4. The Use of Collagenase to Improve the Detection of Plant Viruses in Vector Nematodes by RT/PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tomato ringspot virus (ToRSV) Tobacco ringspot virus (TRSV) and Tobacco rattle virus (TRV) are transmitted to healthy plants by viruliferous nematodes in the soil. We developed a method for extraction of genomic viral RNA from virus particles carried within nematodes and a sensitive nested RT/PCR ...

  5. A Robust Plant RNA Isolation Method for Affymetrix Genechip® Analysis and Quantitative Real-Time RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  6. Characterization of cytokine expression induced by avian influenza virus infection with real-time RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  7. Standardized RT-PCR conditions for detection and identification of eleven viruses of potato and Potato spindle tuber viroid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Standardized RT-PCR procedures were developed and validated for detection of Alfalfa mosaic virus (AMV), Impatiens necrotic spot virus (INSV), Tobacco rattle virus (TRV), Tomato spotted wilt virus (TSWV), Potato leaf roll virus (PLRV), Potato mop top virus (PMTV), Potato virus A (PVA), Potato viru...

  8. Bluetongue virus RNA detection by real-time rt-PCR in post-vaccination samples from cattle.

    PubMed

    De Leeuw, I; Garigliany, M; Bertels, G; Willems, T; Desmecht, D; De Clercq, K

    2015-04-01

    Bluetongue virus serotype 8 (BTV-8) was responsible for a large outbreak among European ruminant populations in 2006-2009. In spring 2008, a massive vaccination campaign was undertaken, leading to the progressive disappearance of the virus. During surveillance programmes in Western Europe in 2010-2011, a low but significant number of animals were found weakly positive using BTV-specific real-time RT-PCR, raising questions about a possible low level of virus circulation. An interference of the BTV-8 inactivated vaccine on the result of the real-time RT-PCR was also hypothesized. Several studies specifically addressed the potential association between a recent vaccination and BTV-8 RNA detection in the blood of sheep. Results were contradictory and cattles were not investigated. To enlighten this point, a large study was performed to determine the risks of detection of bluetongue vaccine-associated RNA in the blood and spleen of cattle using real-time RT-PCR. Overall, the results presented clearly demonstrate that vaccine viral RNA can reach the blood circulation in sufficient amounts to be detected by real-time RT-PCR in cattle. This BTV-8 vaccine RNA carriage appears as short lasting. PMID:23611408

  9. Multiplexed chemiluminescent assays in ArrayPlates for high-throughput measurement of gene expression

    NASA Astrophysics Data System (ADS)

    Martel, Ralph R.; Rounseville, Matthew P.; Botros, Ihab W.; Seligmann, Bruce E.

    2002-06-01

    Multiplexed Molecular Profiling (MMP) assays for drug discovery are performed in ArrayPlates. ArrayPlates are 96- well microtiter plates that contain a 16-element array at the bottom of each well. Each element within an array measures one analyte in a sample. A CCD imager records the quantitative chemiluminescent readout of all 1,536 elements in a 96-well plate simultaneously. Since array elements are reagent modifiable by the end-user, ArrayPlates can be adapted to a broad range of nucleic acid- and protein-based assays. Such multiplexed assays are rapidly established, flexible, robust, automation-friendly and cost-effective. Nucleic acid assays in ArrayPlates can detect DNA and RNA, including SNPs and ESTs. A multiplexed mRNA assay to measure the expression of 16 genes is described. The assay combines a homogeneous nuclease protection assay with subsequent probe immobilization to the array by means of a sandwich hybridization followed with chemiluminescent detection. This assay was used to examine cells grown and treated in microplates and avoided cloning, transfection, RNA insolation, reverse transcription, amplification and fluorochrome labeling. Standard deviations for the measurement of 16 genes ranged from 3 percent to 13 percent in samples of 30,000 cells. Such ArrayPlates transcription assays are useful in drug discovery and development for target validation, screening, lead optimization, metabolism and toxicity profiling. Chemiluminescent detection provides ArrayPlates assays with high signal-to-noise readout and simplifies imager requirements. Imaging a 2D surface that contains arrays simplifies lens requirements relative to imaging columns of liquid in microtiter plate wells. The Omix imager for ArrayPlates is described.

  10. Identification by a Digital Gene Expression Displayer (DGED) and test by RT-PCR analysis of new mRNA candidate markers for colorectal cancer in peripheral blood.

    PubMed

    Lauriola, Mattia; Ugolini, Giampaolo; Rosati, Giancarlo; Zanotti, Simone; Montroni, Isacco; Manaresi, Alessio; Zattoni, Davide; Rivetti, Stefano; Mattei, Gabriella; Coppola, Domenico; Strippoli, Pierluigi; Taffurelli, Mario; Solmi, Rossella

    2010-08-01

    Evidence from the literature widely supports the efficacy of screening for colorectal cancer (CRC) in reducing mortality. A blood-based assay, potentially, represents a more accessible early detection tool for the identification of circulating tumour cells originating from a primary tumour site in the body. The present work aimed at identifying a set of specific mRNAs expressed in colon tissue but not in blood cells. These mRNAs may represent useful markers for early detection of circulating colon cancer cells by a simple, qualitative RT-PCR assay, following RNA extraction from peripheral blood samples. Using a data-mining tool called cDNA digital gene expression displayer (DGED), based on serial analysis of gene expression (SAGE) from the Cancer Genome Anatomy Project (CGAP) database, 4-colon and 14-blood cDNA libraries were analyzed. We selected 7 genes expressed in colon tissue but not in blood and were able to test 6 of them by RT-PCR in peripheral blood of CRC patients and healthy controls. We present a relatively easy and highly reproducible technique for the detection of mRNA expression of genes as candidate markers of malignancy in blood samples of patients with colon cancer. SAGE DGED provided a list of the best candidate mRNAs predicted to detect colon cells in the blood, namely those encoding the following proteins: hypothetical protein LOC644844 (LOC644844, whose cDNA was not amplifiable), fatty acid binding protein 1 (FABP1), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), mucin 13 cell surface associated (MUC13), guanylate cyclase activator 2A (GUCA2A), amiloride binding protein 1 (ABP1), galactoside-binding, solute carrier family 26, member 3 (SLC26A3). The mRNA expression of these genes was evaluated in 8 samples from subjects diagnosed with CRC and 9 from healthy controls. We observed the expression of 2 of the 6 investigated genes in the blood samples of the vast majority of patients considered, but also in a subset of the

  11. Comparison and optimization of detection methods for noroviruses in frozen strawberries containing different amounts of RT-PCR inhibitors.

    PubMed

    Bartsch, Christina; Szabo, Kathrin; Dinh-Thanh, Mai; Schrader, Christina; Trojnar, Eva; Johne, Reimar

    2016-12-01

    Frozen berries have been repeatedly identified as vehicles for norovirus (NoV) transmission causing large gastroenteritis outbreaks. However, virus detection in berries is often hampered by the presence of RT-PCR-inhibiting substances. Here, several virus extraction methods for subsequent real-time RT-PCR-based NoV-RNA detection in strawberries were compared and optimized. NoV recovery rates (RRs) between 0.21 ± 0.13% and 10.29 ± 6.03% were found when five different artificially contaminated strawberry batches were analyzed by the ISO/TS15216-2 method indicating the presence of different amounts of RT-PCR inhibitors. A comparison of five different virus extraction methods using artificially contaminated strawberries containing high amounts of RT-PCR inhibitors revealed the best NoV RRs for the ISO/TS15216 method. Further improvement of NoV RRs from 2.83 ± 2.92% to 15.28 ± 9.73% was achieved by the additional use of Sephacryl(®)-based columns for RNA purification. Testing of 22 frozen strawberry samples from a batch involved in a gastroenteritis outbreak resulted in 5 vs. 13 NoV GI-positive and in 9 vs. 20 NoV GII-positive samples using the original ISO/TS15216 method vs. the extended protocol, respectively. It can be concluded that the inclusion of an additional RNA purification step can increase NoV detection by the ISO/TS15216-2 method in frozen berries containing high amounts of RT-PCR inhibitors. PMID:27554153

  12. Coupled reverse transcription-polymerase chain reaction (RT-PCR) as a sensitive and rapid method for isozyme genotyping.

    PubMed

    Mocharla, H; Mocharla, R; Hodes, M E

    1990-09-14

    We have developed a highly sensitive and rapid coupled reverse transcription-polymerase chain reaction (RT-PCR) technique for detection of alpha-amylase-encoding gene transcripts and for distinguishing between the human salivary (AMY1) and pancreatic (AMY2) gene transcripts. The two genes are 93-94% homologous. However, the AMY1 gene has an additional exon known as exon S, and an extra 32 bp in exon 1. Genotyping of the different AMYs by RT-PCR was based on this unique feature of the AMY1 mRNA sequence. Detection of AMY gene (AMY1 and AMY2) transcripts in cellular RNA was achieved with a set of primers common to both human AMY1 and AMY2 genes and derived from the exon 3-4 regions. In contrast, AMY1 gene transcripts were distinguished from the pancreatic AMY2 gene transcripts by use of primers specific to the exon S-1 regions of the AMY1 gene. To distinguish AMY1 transcripts from a mixture of AMY1 and AMY2, use was made of the differences in the ethidium bromide-stained agarose gel patterns obtained after digestion of the amplified exon 3-4 fragments with TaqI. AMY gene transcripts were detectable by autoradiography in RT-PCR amplified DNA obtained from as little as 5 pg of human pancreatic or parotid total RNA. A comparison of sensitivity of Northern blotting vs. RT-PCR suggested that the RT-PCR method is about 3-6 x 10(3)-fold more sensitive than Northern blotting in detecting AMY gene transcripts in human pancreatic total RNA. PMID:1699848

  13. Cloning of fox (Vulpes vulpes) Il2, Il6, Il10 and IFNgamma and analysis of their expression by quantitative RT-PCR in fox PBMC after in vitro stimulation by Concanavalin A.

    PubMed

    Rolland-Turner, Magali; Farré, Guillaume; Boué, Franck

    2006-04-15

    The immune response in the fox (Vulpes vulpes), despite the success of the oral rabies vaccine is not well characterised, and specific immunological tools are needed. A quantitative RT-PCR using SyBR Green to investigate fox cytokine expression after antigen PBMC in vitro re-stimulation is presented here. First, we cloned by homology with dog cytokine sequences the fox IL2, IL6, IL10, IFNgamma and a partial 18S sequence. Fox specific primers were then defined and used to set up a species-specific quantitative RT-PCR assay using SyBR Green and 18S housekeeping gene as internal standard. The technique was validated using total RNA from fox PBMC stimulated with a polyclonal activator, Concanavaline A. PMID:16321447

  14. Rapid Detection of Hepatitis B Virus Variants Associated with Lamivudine and Adefovir Resistance by Multiplex Ligation-Dependent Probe Amplification Combined with Real-Time PCR

    PubMed Central

    Jia, Shuangrong; Wang, Feng; Li, Fake; Chang, Kai; Yang, Shaojun; Zhang, Kejun; Jiang, Wenbin; Shang, Ya

    2014-01-01

    Drug-resistant mutations of hepatitis B virus (HBV) are the major obstacles to successful therapy for chronic hepatitis B infection. Although there are many methods for detecting the antiviral drug-resistant mutations of HBV, their applications are restricted because of their shortcomings, such as low sensitivity, the time required, and the high cost. For this study, a multiplex ligation-dependent probe real-time PCR (MLP-RT-PCR) method was developed to simultaneously detect lamivudine (LAM)- and adefovir (ADV)-resistant HBV mutants (those with the mutations rtM204V/I, rtA181V/T, and rtN236T). The new method combined the high-throughput nature of multiplex ligation-dependent probe amplification (MLPA) with the rapid and sensitive detection of real-time PCR. In this report, MLP-RT-PCR was evaluated by detecting drug-resistant mutants in 116 patients with chronic hepatitis B infection. By MLP-RT-PCR analysis, LAM-resistant mutations were detected in 41 patients (35.3%), ADV-resistant mutations were detected in 17 patients (14.7%), and LAM- and-ADV-resistant mutations were detected in 5 patients (4.3%). Based on the results of MLP-RT-PCR, the mutations rtM204V, rtM204I, rtA181T, rtA181V, and rtN236T were 95.7% (111/116 patients), 98.3% (114/116 patients), 99.1% (115/116 patients), 98.3% (114/116 patients), and 99.1% (115/116 patients) concordant, respectively, with those of direct sequencing. The MLP-RT-PCR assay was more sensitive than direct sequencing for detecting mutations with low frequencies. Four samples containing the low-frequency (<10%) mutants were identified by MLP-RT-PCR and further confirmed by clonal sequencing. MLP-RT-PCR is a rapid and sensitive method that enables the detection of multidrug-resistant HBV mutations in clinical practice. PMID:24478474

  15. A comparative study of digital RT-PCR and RT-qPCR for quantification of Hepatitis A virus and Norovirus in lettuce and water samples.

    PubMed

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

    2015-05-18

    Sensitive and quantitative detection of foodborne enteric viruses is classically achieved by quantitative RT-PCR (RT-qPCR). Recently, digital PCR (dPCR) was described as a novel approach to genome quantification without need for a standard curve. The performance of microfluidic digital RT-PCR (RT-dPCR) was compared to RT-qPCR for detecting the main viruses responsible for foodborne outbreaks (human Noroviruses (NoV) and Hepatitis A virus (HAV)) in spiked lettuce and bottled water. Two process controls (Mengovirus and Murine Norovirus) were used and external amplification controls (EAC) were added to examine inhibition of RT-qPCR and RT-dPCR. For detecting viral RNA and cDNA, the sensitivity of the RT-dPCR assays was either comparable to that of RT-qPCR (RNA of HAV, NoV GI, Mengovirus) or slightly (around 1 log10) decreased (NoV GII and MNV-1 RNA and of HAV, NoV GI, NoV GII cDNA). The number of genomic copies determined by dPCR was always from 0.4 to 1.7 log10 lower than the expected numbers of copies calculated by using the standard qPCR curve. Viral recoveries calculated by RT-dPCR were found to be significantly higher than by RT-qPCR for NoV GI, HAV and Mengovirus in water, and for NoV GII and HAV in lettuce samples. The RT-dPCR assay proved to be more tolerant to inhibitory substances present in lettuce samples. This absolute quantitation approach may be useful to standardize quantification of enteric viruses in bottled water and lettuce samples and may be extended to quantifying other human pathogens in food samples. PMID:25725459

  16. Reference Gene Validation for Quantitative RT-PCR during Biotic and Abiotic Stresses in Vitis vinifera

    PubMed Central

    Borges, Alexandre Filipe; Fonseca, Catarina; Ferreira, Ricardo Boavida; Lourenço, Ana Maria; Monteiro, Sara

    2014-01-01

    Grapevine is one of the most cultivated fruit crop worldwide with Vitis vinifera being the species with the highest economical importance. Being highly susceptible to fungal pathogens and increasingly affected by environmental factors, it has become an important agricultural research area, where gene expression analysis plays a fundamental role. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is currently amongst the most powerful techniques to perform gene expression studies. Nevertheless, accurate gene expression quantification strongly relies on appropriate reference gene selection for sample normalization. Concerning V. vinifera, limited information still exists as for which genes are the most suitable to be used as reference under particular experimental conditions. In this work, seven candidate genes were investigated for their stability in grapevine samples referring to four distinct stresses (Erysiphe necator, wounding and UV-C irradiation in leaves and Phaeomoniella chlamydospora colonization in wood). The expression stability was evaluated using geNorm, NormFinder and BestKeeper. In all cases, full agreement was not observed for the three methods. To provide comprehensive rankings integrating the three different programs, for each treatment, a consensus ranking was created using a non-weighted unsupervised rank aggregation method. According to the last, the three most suitable reference genes to be used in grapevine leaves, regardless of the stress, are UBC, VAG and PEP. For the P. chlamydospora treatment, EF1, CYP and UBC were the best scoring genes. Acquaintance of the most suitable reference genes to be used in grapevine samples can contribute for accurate gene expression quantification in forthcoming studies. PMID:25340748

  17. RT-PCR analysis of Deformed wing virus in honeybees (Apis mellifera) and mites (Varroa destructor).

    PubMed

    Yue, Constanze; Genersch, Elke

    2005-12-01

    Deformed wing virus (DWV) is a honeybee viral pathogen either persisting as an inapparent infection or resulting in wing deformity. The occurrence of deformity is associated with the transmission of DWV through Varroa destructor during pupal stages. Such infections with DWV add to the pathology of V. destructor and play a major role in colony collapse in the course of varroosis. Using a recently developed RT-PCR protocol for the detection of DWV, individual bees and mites originating from hives differing in Varroa infestation levels and the occurrence of crippled bees were analysed. It was found that 100 % of both crippled and asymptomatic bees were positive for DWV. However, a significant difference in the spatial distribution of DWV between asymptomatic and crippled bees could be demonstrated: when analysing head, thorax and abdomen of crippled bees, all body parts were always strongly positive for viral sequences. In contrast, for asymptomatic bees viral sequences could be detected in RNA extracted from the thorax and/or abdomen but never in RNA extracted from the head. DWV replication was demonstrated in almost all DWV-positive body parts of infected bees. Analysing individual mites for the presence of DWV revealed that the percentage of DWV-positive mites differed between mite populations. In addition, it was demonstrated that DWV was able to replicate in some but not all mites. Interestingly, virus replication in mites was correlated with wing deformity. DWV was also detected in the larval food, implicating that in addition to transmission by V. destructor DWV is also transmitted by feeding. PMID:16298989

  18. Proteomic Analysis and qRT-PCR Verification of Temperature Response to Arthrospira (Spirulina) platensis

    PubMed Central

    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

    2013-01-01

    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

  19. Rapid discrimination of rabies viruses isolated from various host species in Brazil by multiplex reverse transcription-polymerase chain reaction.

    PubMed

    Sato, Go; Tanabe, Hitomi; Shoji, Youko; Itou, Takuya; Ito, Fumio H; Sato, Tetsuo; Sakai, Takeo

    2005-08-01

    Rabies is carried mainly by mammalian carnivores and vampire bats in Latin America. However, rabies virus (RV) has been isolated in recent years from not only vampire bats in rural areas but also from several non-vampire bat species in urban areas, respectively. Therefore, rapid molecular screening is necessary for efficient epidemiology of these RVs. In this study, we investigated the usefulness of multiplex reverse transcription-polymerase chain reaction (RT-PCR) for determining the origins of 54 RV isolates from various host species in Brazil. And to evaluate the multiplex RT-PCR as a potential diagnostic tool, we investigated the sensitivity of this method. In addition, we compared the results with a phylogenetic tree developed from sequences of the RV glycoprotein (G protein) gene. Multiplex RT-PCR products showed five different sizes of products, whereas the phylogenic tree showed six groups. Of these six groups, four corresponded with the four sizes of the multiplex RT-PCR products. The other two groups showed correspondance with another one size of the multiplex RT-PCR products, indicating that multiplex RT-PCR results reflected the lineage of the 54 isolates. This study also showed that this method can detect trace amounts of RNA. In conclusion, this multiplex RT-PCR method allows the rapid, specific, and simultaneous detection of RVs isolated from various host species in Brazil. PMID:16036175

  20. Reproducibility of quantitative RT-PCR array in miRNA expression profiling and comparison with microarray analysis

    PubMed Central

    Chen, Yongxin; Gelfond, Jonathan AL; McManus, Linda M; Shireman, Paula K

    2009-01-01

    Background MicroRNAs (miRNAs) have critical functions in various biological processes. MiRNA profiling is an important tool for the identification of differentially expressed miRNAs in normal cellular and disease processes. A technical challenge remains for high-throughput miRNA expression analysis as the number of miRNAs continues to increase with in silico prediction and experimental verification. Our study critically evaluated the performance of a novel miRNA expression profiling approach, quantitative RT-PCR array (qPCR-array), compared to miRNA detection with oligonucleotide microchip (microarray). Results High reproducibility with qPCR-array was demonstrated by comparing replicate results from the same RNA sample. Pre-amplification of the miRNA cDNA improved sensitivity of the qPCR-array and increased the number of detectable miRNAs. Furthermore, the relative expression levels of miRNAs were maintained after pre-amplification. When the performance of qPCR-array and microarrays were compared using different aliquots of the same RNA, a low correlation between the two methods (r = -0.443) indicated considerable variability between the two assay platforms. Higher variation between replicates was observed in miRNAs with low expression in both assays. Finally, a higher false positive rate of differential miRNA expression was observed using the microarray compared to the qPCR-array. Conclusion Our studies demonstrated high reproducibility of TaqMan qPCR-array. Comparison between different reverse transcription reactions and qPCR-arrays performed on different days indicated that reverse transcription reactions did not introduce significant variation in the results. The use of cDNA pre-amplification increased the sensitivity of miRNA detection. Although there was variability associated with pre-amplification in low abundance miRNAs, the latter did not involve any systemic bias in the estimation of miRNA expression. Comparison between microarray and q

  1. Multiplex Assay for Protein Profiling and Potency Measurement of German Cockroach Allergen Extracts

    PubMed Central

    Khurana, Taruna; Dobrovolskaia, Ekaterina; Shartouny, Jessica R.; Slater, Jay E.

    2015-01-01

    Background German cockroach (GCr) allergens induce IgE responses and may cause asthma. Commercial GCr allergen extracts are variable and existing assays may not be appropriate for determining extract composition and potency. Objective Our aim was to develop a multiplex antibody/bead-based assay for assessment of GCr allergen extracts. Methods Single chain fragment variable (scFv) antibodies against GCr were obtained by screening libraries derived from naïve human lymphocytes and hyperimmunized chicken splenocytes and bone marrow. Selected clones were sequenced and characterized by immunoblotting. Eighteen scFv antibodies (17 chicken, 1 human) coupled to polystyrene beads were used in this suspension assay; binding of targeted GCr allergens to antibody-coated beads was detected using rabbit antisera against GCr, and against specific allergens rBla g 1, rBla g 2, and rBla g 4. The assay was tested for specificity, accuracy, and precision. Extracts were also compared by IgE competition ELISA. Results Chicken scFv’s generated eight different binding patterns to GCr proteins from 14 to 150 kDa molecular weight. Human scFv’s recognized a 100 kDa GCr protein. The multiplex assay was found to be specific and reproducible with intra-assay coefficient of variation (CV) of 2.64% and inter-assay CV of 10.0%. Overall potencies of various GCr extracts were calculated using mean logEC50s for eight selected scFvs. Overall potency measures were also analyzed by assessing the contributions to potency of each target. Conclusions An scFv antibody-based multiplex assay has been developed capable of simultaneously measuring different proteins in a complex mixture, and to determine the potencies and compositions of allergen extracts. PMID:26444288

  2. mPSQed: a software for the design of multiplex pyrosequencing assays.

    PubMed

    Dabrowski, Piotr Wojtek; Nitsche, Andreas

    2012-01-01

    Molecular-based diagnostic assays are the gold standard for infectious diseases today, since they allow a rapid and sensitive identification and typing of various pathogens. While PCR can be designed to be specific for a certain pathogen, a subsequent sequence analysis is frequently required for confirmation or typing. The design of appropriate PCR-based assays is a complex task, especially when conserved discriminating polymorphisms are rare or if the number of types which need to be differentiated is high. One extremely useful but underused method for this purpose is the multiplex pyrosequencing technique. Unfortunately there is no software available to aid researchers in designing multiplex pyrosequencing assays. Here, we present mPSQed (Multiplex PyroSeQuencing EDitor), a program targeted at closing this gap. We also present the design of an exemplarily theoretical assay for the differentiation of human adenovirus types A-F using two pyrosequencing primers on two distinct PCR products, designed quickly and easily using our software. PMID:22675516

  3. mPSQed: A Software for the Design of Multiplex Pyrosequencing Assays

    PubMed Central

    Dabrowski, Piotr Wojtek; Nitsche, Andreas

    2012-01-01

    Molecular-based diagnostic assays are the gold standard for infectious diseases today, since they allow a rapid and sensitive identification and typing of various pathogens. While PCR can be designed to be specific for a certain pathogen, a subsequent sequence analysis is frequently required for confirmation or typing. The design of appropriate PCR-based assays is a complex task, especially when conserved discriminating polymorphisms are rare or if the number of types which need to be differentiated is high. One extremely useful but underused method for this purpose is the multiplex pyrosequencing technique. Unfortunately there is no software available to aid researchers in designing multiplex pyrosequencing assays. Here, we present mPSQed (Multiplex PyroSeQuencing EDitor), a program targeted at closing this gap. We also present the design of an exemplarily theoretical assay for the differentiation of human adenovirus types A–F using two pyrosequencing primers on two distinct PCR products, designed quickly and easily using our software. PMID:22675516

  4. Final Report Nucleic Acid System - PCR, Multiplex Assays and Sample Preparation Project

    SciTech Connect

    Koopman, R.P.; Langlois, R.G.; Nasarabadi, S.; Benett, W.J.; Richards, J.B.; Hadley, D.R.; Miles, R.R.; Brown, S.B.; Stratton, P.L.; Milanovich, F.P.

    2001-04-20

    The objective of this project was to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction). This entailed not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This project had two principal deliverables: (1) design, construct, test and deliver a 24 chamber, multiplex capable suitcase sized PCR instrument, and (2) develop and reduce to practice a multiplex assay for the detection of PCR product by flow cytometry. In addition, significant resources were allocated to test and evaluation of the Hand-held Advanced Nucleic Acid Analyzer (HANAA). This project helps provide the signature and intelligence gathering community the ability to perform, on-site or remote, rapid analysis of environmental or like samples for the presence of a suite of biological warfare pathogens.

  5. A Multiplex Polymerase Chain Reaction Microarray Assay to Detect Bioterror Pathogens in Blood

    PubMed Central

    Tomioka, Keiko; Peredelchuk, Michael; Zhu, Xiangyang; Arena, Roberto; Volokhov, Dmitri; Selvapandiyan, Angamuthu; Stabler, Katie; Mellquist-Riemenschneider, Jenny; Chizhikov, Vladimir; Kaplan, Gerardo; Nakhasi, Hira; Duncan, Robert

    2005-01-01

    Heightened concern about the dangers of bioterrorism requires that measures be developed to ensure the safety of the blood supply. Multiplex detection of such agents using a blood-screening DNA microarray is a sensitive and specific method to screen simultaneously for a number of suspected agents. We have developed and optimized a multiplex polymerase chain reaction microarray assay to screen blood for three potential bioterror bacterial pathogens and a human ribosomal RNA gene internal control. The analytical sensitivity of the assay was demonstrated to be 50 colony-forming units/ml for Bacillus anthracis, Francisella tularensis, and Yersinia pseudotuberculosis (surrogate for Yersinia pestis). The absence of any false-positives demonstrated high analytical specificity. Screening B. anthracis-infected mouse blood samples and uninfected controls demonstrated effectiveness and specificity in a preclinical application. This study represents proof of the concept of microarray technology to screen simultaneously for multiple bioterror pathogens in blood samples. PMID:16237218

  6. Differentiation of five tuna species by a multiplex primer-extension assay.

    PubMed

    Bottero, Maria Teresa; Dalmasso, Alessandra; Cappelletti, Marco; Secchi, Camillo; Civera, Tiziana

    2007-05-01

    A novel methodology based on analysis of mtDNA-cytb diagnostic sites was performed to discriminate four closely related species of Thunnus (Thunnus alalunga, Thunnus albacares, Thunnus obesus and Thunnus thynnus) and one species of Euthynnus (Katsuwonus pelamis) genus in raw and canned tuna. The primers used in the preliminary PCR designed in well conserved region upstream and downstream of the diagnosis sites successfully amplified a 132bp region from the cytb gene of all the species taken into consideration. The sites of diagnosis have been interrogate simultaneously using a multiplex primer-extension assay (PER) and the results were confirmed by fragment sequencing. The applicability of the multiplex PER assay to commercial canned tuna samples was also demonstrated. The proposed test could be useful for detection of fraud and for seafood traceability. PMID:17353060

  7. Single cell multiplexed assay for proteolytic activity using droplet microfluidics.

    PubMed

    Ng, Ee Xien; Miller, Miles A; Jing, Tengyang; Chen, Chia-Hung

    2016-07-15

    Cellular enzymes interact in a post-translationally regulated fashion to govern individual cell behaviors, yet current platform technologies are limited in their ability to measure multiple enzyme activities simultaneously in single cells. Here, we developed multi-color Förster resonance energy transfer (FRET)-based enzymatic substrates and use them in a microfluidics platform to simultaneously measure multiple specific protease activities from water-in-oil droplets that contain single cells. By integrating the microfluidic platform with a computational analytical method, Proteolytic Activity Matrix Analysis (PrAMA), we are able to infer six different protease activity signals from individual cells in a high throughput manner (~100 cells/experimental run). We characterized protease activity profiles at single cell resolution for several cancer cell lines including breast cancer cell line MDA-MB-231, lung cancer cell line PC-9, and leukemia cell line K-562 using both live-cell and in-situ cell lysis assay formats, with special focus on metalloproteinases important in metastasis. The ability to measure multiple proteases secreted from or expressed in individual cells allows us to characterize cell heterogeneity and has potential applications including systems biology, pharmacology, cancer diagnosis and stem cell biology. PMID:26995287

  8. Development of multiplex serological assay for the detection of human African trypanosomiasis.

    PubMed

    Nzou, Samson Muuo; Fujii, Yoshito; Miura, Masashi; Mwau, Matilu; Mwangi, Anne Wanjiru; Itoh, Makoto; Salam, Md Abdus; Hamano, Shinjiro; Hirayama, Kenji; Kaneko, Satoshi

    2016-04-01

    Human African trypanosomiasis (HAT) is a disease caused by Kinetoplastid infection. Serological tests are useful for epidemiological surveillance. The aim of this study was to develop a multiplex serological assay for HAT to assess the diagnostic value of selected HAT antigens for sero-epidemiological surveillance. We cloned loci encoding eight antigens from Trypanosoma brucei gambiense, expressed the genes in bacterial systems, and purified the resulting proteins. Antigens were subjected to Luminex multiplex assays using sera from HAT and VL patients to assess the antigens' immunodiagnostic potential. Among T. b. gambiense antigens, the 64-kDa and 65-kDa invariant surface glycoproteins (ISGs) and flagellar calcium binding protein (FCaBP) had high sensitivity for sera from T. b. gambiense patients, yielding AUC values of 0.871, 0.737 and 0.858 respectively in receiver operating characteristics (ROC) analysis. The ISG64, ISG65, and FCaBP antigens were partially cross-reactive to sera from Trypanosoma brucei rhodesiense patients. The GM6 antigen was cross-reactive to sera from T. b. rhodesiense patients as well as to sera from VL patients. Furthermore, heterogeneous antibody responses to each individual HAT antigen were observed. Testing for multiple HAT antigens in the same panel allowed specific and sensitive detection. Our results demonstrate the utility of applying multiplex assays for development and evaluation of HAT antigens for use in sero-epidemiological surveillance. PMID:26519611

  9. DEVELOPMENT OF MULTIPLEX RT-PCR FOR THE DETECTION OF REOVIRUS, HEPATITIS A VIRUS, POLIOVIRUS, NORWALK VIRUS AND ROTAVIRUS

    EPA Science Inventory

    Water sources are often found to be contaminated by enteric viruses. This is a public health concern as food and waterborne outbreaks caused by enteric viruses such as noroviruses, rotaviruses, hepatitis A virus (HAV) and enteroviruses are a common occurrence. All of these viru...

  10. Simultaneous detection and differentiation of four closely related sweet potato potyviruses by a multiplex one-step RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Four closely related potyviruses, Sweet potato feathery mottle virus (SPFMV), Sweet potato virus C (SPVC), Sweet potato virus G (SPVG) and/or Sweet potato virus 2 (SPV2), are involved in Sweet Potato Viral Disease, the most devastating disease of sweet potato worldwide. Identification and detection ...

  11. Global RT-PCR and RT-qPCR Analysis of the mRNA Expression of the Human PTPome.

    PubMed

    Nunes-Xavier, Caroline E; Pulido, Rafael

    2016-01-01

    Comprehensive comparative gene expression analysis of the tyrosine phosphatase superfamily members (PTPome) under cell- or tissue-specific growth conditions may help to define their individual and specific role in physiology and disease. Semi-quantitative and quantitative PCR are commonly used methods to analyze and measure gene expression. Here, we describe technical aspects of PTPome mRNA expression analysis by semi-quantitative RT-PCR and quantitative RT-PCR (RT-qPCR). We provide a protocol for each method consisting in reverse transcription followed by PCR using a global platform of specific PTP primers. The chapter includes aspects from primer validation to the setup of the PTPome RT-qPCR platform. Examples are given of PTP-profiling gene expression analysis using a human breast cancer cell line upon long-term or short-term treatment with cell signaling-activation agents. PMID:27514798

  12. Validation of reference genes in Penicillium echinulatum to enable gene expression study using real-time quantitative RT-PCR.

    PubMed

    Zampieri, Denise; Nora, Luísa C; Basso, Vanessa; Camassola, Marli; Dillon, Aldo J P

    2014-08-01

    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. PMID:24509829

  13. A MULTIPLEXED ASSAY FOR DETERMINATION OF NEUROTOXICANT EFFECTS ON SPONTANEOUS NETWORK ACTIVITY AND CELL VIABILITY FROM MICROELECTRODE ARRAYS

    EPA Science Inventory

    AbstractTITLE: A MULTIPLEXED ASSAY FOR DETERMINATION OF NEUROTOXICANT EFFECTS ON SPONTANEOUS NETWORK ACTIVITY AND CELL VIABILITY FROM MICROELECTRODE ARRAYSABSTRACT BODY: Microelectrode array (MEA) recordings are increasingly being used as an in vitro method to detect and characte...

  14. Differentiation of infectious bursal disease virus strains using real-time RT-PCR and high resolution melt curve analysis.

    PubMed

    Ghorashi, Seyed A; O'Rourke, Denise; Ignjatovic, Jagoda; Noormohammadi, Amir H

    2011-01-01

    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. PMID:21111004

  15. Molecular characterization and clinical impact of TMPRSS2-ERG rearrangement on prostate cancer: comparison between FISH and RT-PCR.

    PubMed

    Fernández-Serra, A; Rubio, L; Calatrava, A; Rubio-Briones, J; Salgado, R; Gil-Benso, R; Espinet, B; García-Casado, Z; López-Guerrero, J A

    2013-01-01

    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

  16. Validation of reference genes for quantitative gene expression studies in Volvox carteri using real-time RT-PCR.

    PubMed

    Kianianmomeni, Arash; Hallmann, Armin

    2013-12-01

    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. PMID:24057254

  17. Recommendations for Use and Fit-for-Purpose Validation of Biomarker Multiplex Ligand Binding Assays in Drug Development.

    PubMed

    Jani, Darshana; Allinson, John; Berisha, Flora; Cowan, Kyra J; Devanarayan, Viswanath; Gleason, Carol; Jeromin, Andreas; Keller, Steve; Khan, Masood U; Nowatzke, Bill; Rhyne, Paul; Stephen, Laurie

    2016-01-01

    Multiplex ligand binding assays (LBAs) are increasingly being used to support many stages of drug development. The complexity of multiplex assays creates many unique challenges in comparison to single-plexed assays leading to various adjustments for validation and potentially during sample analysis to accommodate all of the analytes being measured. This often requires a compromise in decision making with respect to choosing final assay conditions and acceptance criteria of some key assay parameters, depending on the intended use of the assay. The critical parameters that are impacted due to the added challenges associated with multiplexing include the minimum required dilution (MRD), quality control samples that span the range of all analytes being measured, quantitative ranges which can be compromised for certain targets, achieving parallelism for all analytes of interest, cross-talk across assays, freeze-thaw stability across analytes, among many others. Thus, these challenges also increase the complexity of validating the performance of the assay for its intended use. This paper describes the challenges encountered with multiplex LBAs, discusses the underlying causes, and provides solutions to help overcome these challenges. Finally, we provide recommendations on how to perform a fit-for-purpose-based validation, emphasizing issues that are unique to multiplex kit assays. PMID:26377333

  18. Multiplex PCR Assay Targeting a Diguanylate Cyclase-Encoding Gene, cgcA, To Differentiate Species within the Genus Cronobacter

    PubMed Central

    Carter, L.; Lindsey, L. A.; Grim, C. J.; Sathyamoorthy, V.; Jarvis, K. G.; Gopinath, G.; Lee, C.; Sadowski, J. A.; Trach, L.; Pava-Ripoll, M.; McCardell, B. A.; Tall, B. D.

    2013-01-01

    In a comparison to the widely used Cronobacter rpoB PCR assay, a highly specific multiplexed PCR assay based on cgcA, a diguanylate cyclase gene, that identified all of the targeted six species among 305 Cronobacter isolates was designed. This assay will be a valuable tool for identifying suspected Cronobacter isolates from food-borne investigations. PMID:23144142

  19. Development of a SYBR green I-based quantitative RT-PCR for Ross River virus: Application in vector competence studies and antiviral drug evaluation.

    PubMed

    Dash, Paban Kumar; Agarwal, Ankita; Sharma, Shashi; Saha, Amrita; Joshi, Gaurav; Gopalan, Natarajan; Sukumaran, Devanathan; Parida, Man Mohan

    2016-08-01

    Ross River virus (RRV) is an emerging Alphavirus and is presently endemic in many parts of Oceania. Keeping in mind its emergence, we developed a molecular detection system and utilized it to study vector competence and evaluate activity of antiviral compounds against RRV. A SYBR Green I-based quantitative RT-PCR for detection of RRV was developed targeting the E2 gene, with a detection limit of 100 RNA copies/reaction. The specificity was confirmed with closely related Alphaviruses and Flaviviruses. The assay was applied to study the vector competence of Indian Aedes aegypti for RRV, which revealed 100% infection and dissemination rate with 75% transmission rate. Viral RNA was found in saliva as early as 3day post infection (dpi). Further application of the assay in antiviral drug evaluation revealed the superior in vitro activity of ribavirin compared to chloroquine in Vero cells. Successful demonstration of this assay to detect RRV in low titre mosquito samples makes it a sensitive tool in vector surveillance. This study also showed that Indian Ae. aegypti are well competent to transmit RRV highlighting the risk of its introduction to naïve territories across continents. Further validation of this assay, revealed its utility in screening of potential antivirals against RRV. PMID:27105737

  20. Rapid detection of foot-and-mouth disease virus, influenza A virus and classical swine fever virus by high-speed real-time RT-PCR.

    PubMed

    Wernike, Kerstin; Beer, Martin; Hoffmann, Bernd

    2013-10-01

    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. PMID:23702025

  1. Multiplex Real-Time PCR Assay for Rapid Detection of Methicillin-Resistant Staphylococci Directly from Positive Blood Cultures

    PubMed Central

    Wang, Hye-young; Kim, Sunghyun; Kim, Jungho; Park, Soon-Deok

    2014-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent cause of bloodstream infections (BSIs) and is recognized as a major nosocomial pathogen. This study aimed to evaluate a newly designed multiplex real-time PCR assay capable of the simultaneous detection of mecA, S. aureus, and coagulase-negative staphylococci (CoNS) in blood culture specimens. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays (M&D, Republic of Korea) use the TaqMan probes 16S rRNA for Staphylococcus spp., the nuc gene for S. aureus, and the mecA gene for methicillin resistance. The detection limit of the multiplex real-time PCR assay was 103 CFU/ml per PCR for each gene target. The multiplex real-time PCR assay was evaluated using 118 clinical isolates from various specimen types and a total of 350 positive blood cultures from a continuous monitoring blood culture system. The results obtained with the multiplex real-time PCR assay for the three targets were in agreement with those of conventional identification and susceptibility testing methods except for one organism. Of 350 positive bottle cultures, the sensitivities of the multiplex real-time PCR kit were 100% (166/166 cultures), 97.2% (35/36 cultures), and 99.2% (117/118 cultures) for the 16S rRNA, nuc, and mecA genes, respectively, and the specificities for all three targets were 100%. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays are very useful for the rapid accurate diagnosis of staphylococcal BSIs. In addition, the Real-MRSA and Real-MRCoNS multiplex real-time PCR assays could have an important impact on the choice of appropriate antimicrobial therapy, based on detection of the mecA gene. PMID:24648566

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

    PubMed Central

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

    2016-01-01

    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

  3. Multiplex PCR assay for the detection of five meat species forbidden in Islamic foods.

    PubMed

    Ali, Md Eaqub; Razzak, Md Abdur; Hamid, Sharifah Bee Abd; Rahman, Md Mahfujur; Amin, Md Al; Rashid, Nur Raifana Abd; Asing

    2015-06-15

    Food falsification has direct impact on public health, religious faith, fair-trades and wildlife. For the first time, here we described a multiplex polymerase chain reaction assay for the accurate identification of five meat species forbidden in Islamic foods in a single assay platform. Five pairs of species-specific primers were designed targeting mitochondrial ND5, ATPase 6, and cytochrome b genes to amplify 172, 163, 141, 129 and 108 bp DNA fragments from cat, dog, pig, monkey and rat meats, respectively. All PCR products were identified in gel-images and electrochromatograms obtained from Experion Bioanalyzer. Species-specificity checking against 15 important meat and fish and 5 plant species detected no cross-species amplification. Screening of target species in model and commercial meatballs reflected its application to detect target species in process foods. The assay was tested to detect 0.01-0.02 ng DNA under raw states and 1% suspected meats in meatball formulation. PMID:25660879

  4. Simultaneous detection and differentiation of Rice black streaked dwarf virus (RBSDV) and Southern rice black streaked dwarf virus (SRBSDV) by duplex real time RT-PCR

    PubMed Central

    2013-01-01

    Background The diseases caused by Rice black streaked dwarf virus (RBSDV) and Southern rice black streaked dwarf virus (SRBSDV) have been occurring epidemically in China and southeastern Asia in recent years. A sensitive, reliable and quantitative method is required to detect and distinguish for RBSDV and SRBSDV in rice and vector insects. Results We developed a sensitive and lineage-specific duplex real time RT-qPCR for detection of RBSDV and SRBSDV in a single or/and double infection in rice samples. The duplex RT-qPCR was optimized using standard samples transcribed by T7 Large Scale RNA Production System in vitro. We developed a reliable system for duplex RT-qPCR, in which its co-efficiency of RBSDV and SRBSDV, were 91.6% and 90.7%, respectively. The coefficient of determination was more than 0.990; the slope of linear equation was −3.542, and −3.567, respectively. Out of 30 samples collected in North and Central China, which were suspected to be infected with these two viruses, 10 samples were detected RBSDV positive by RT-PCR and 12 samples by RT-qPCR. No mixed infections were found. Simultaneously, out of total 60 samples collected from Southern China, which were also suspected to be infected with these two viruses, 41 samples were determined SRBSDV positive by RT-PCR and 47 samples by RT-qPCR. Also in this case no mixed infections were found. The rice genes eEF-1a and UBQ5 were selected as internal controls for quantification assay also performed as good expression stability. Conclusion The duplex RT-qPCR assay provided as a sufficiently sensitive, specific, accurate, reproducible and rapid tool for the detection and differentiation of RBSDV and SRBSDV. The RT-qPCR assay can be used in routine diagnostic of these two viruses in order to study the disease epidemiology in rice crops. PMID:23331990

  5. Multiplex assays for biomarker research and clinical application: translational science coming of age.

    PubMed

    Fu, Qin; Schoenhoff, Florian S; Savage, William J; Zhang, Pingbo; Van Eyk, Jennifer E

    2010-03-01

    Over the last decade, translational science has come into the focus of academic medicine, and significant intellectual and financial efforts have been made to initiate a multitude of bench-to-bedside projects. The quest for suitable biomarkers that will significantly change clinical practice has become one of the biggest challenges in translational medicine. Quantitative measurement of proteins is a critical step in biomarker discovery. Assessing a large number of potential protein biomarkers in a statistically significant number of samples and controls still constitutes a major technical hurdle. Multiplexed analysis offers significant advantages regarding time, reagent cost, sample requirements and the amount of data that can be generated. The two contemporary approaches in multiplexed and quantitative biomarker validation, antibody-based immunoassays and MS-based multiple (or selected) reaction monitoring, are based on different assay principles and instrument requirements. Both approaches have their own advantages and disadvantages and therefore have complementary roles in the multi-staged biomarker verification and validation process. In this review, we discuss quantitative immunoassay and multiple reaction monitoring/selected reaction monitoring assay principles and development. We also discuss choosing an appropriate platform, judging the performance of assays, obtaining reliable, quantitative results for translational research and clinical applications in the biomarker field. PMID:21137048

  6. Detection of four important Eimeria species by multiplex PCR in a single assay.

    PubMed

    You, Myung-Jo

    2014-06-01

    The oocysts of some of the recognized species of chicken coccidiosis are difficult to distinguish morphologically. Diagnostic laboratories are increasingly utilizing DNA-based technologies for the specific identification of Eimeria species. This study reports a multiplex polymerase chain reaction (PCR) assay based on internal transcribed spacer-1 (ITS-1) for the simultaneous diagnosis of the Eimeria tenella, Eimeria acervulina, Eimeria maxima, and Eimeria necatrix species, which infect domestic fowl. Primer pairs specific to each species were designed in order to generate a ladder of amplification products ranging from 20 to 25 bp, and a common optimum annealing temperature for these species was determined to be 52.5 °C. Sensitivity tests were performed for each species, showing a detection threshold of 1-5 pg. All the species were amplified homogeneously, and a homogenous band ladder was observed, indicating that the assay permitted the simultaneous detection of all the species in a single-tube reaction. In the phylogenic study, there was a clear species clustering, which was irrespective of geographical location, for all the ITS-1 sequences used. This multiplex PCR assay represents a rapid and potential cost-effective diagnostic method for the detection of some key Eimeria species that infect domestic fowl. PMID:24495953

  7. Identification of Novel Reference Genes Suitable for qRT-PCR Normalization with Respect to the Zebrafish Developmental Stage

    PubMed Central

    Hu, Yu; Xie, Shuying; Yao, Jihua

    2016-01-01

    Reference genes used in normalizing qRT-PCR data are critical for the accuracy of gene expression analysis. However, many traditional reference genes used in zebrafish early development are not appropriate because of their variable expression levels during embryogenesis. In the present study, we used our previous RNA-Seq dataset to identify novel reference genes suitable for gene expression analysis during zebrafish early developmental stages. We first selected 197 most stably expressed genes from an RNA-Seq dataset (29,291 genes in total), according to the ratio of their maximum to minimum RPKM values. Among the 197 genes, 4 genes with moderate expression levels and the least variation throughout 9 developmental stages were identified as candidate reference genes. Using four independent statistical algorithms (delta-CT, geNorm, BestKeeper and NormFinder), the stability of qRT-PCR expression of these candidates was then evaluated and compared to that of actb1 and actb2, two commonly used zebrafish reference genes. Stability rankings showed that two genes, namely mobk13 (mob4) and lsm12b, were more stable than actb1 and actb2 in most cases. To further test the suitability of mobk13 and lsm12b as novel reference genes, they were used to normalize three well-studied target genes. The results showed that mobk13 and lsm12b were more suitable than actb1 and actb2 with respect to zebrafish early development. We recommend mobk13 and lsm12b as new optimal reference genes for zebrafish qRT-PCR analysis during embryogenesis and early larval stages. PMID:26891128

  8. Detection and differentiation of pigeon paramyxovirus serotype-1 (PPMV-1) isolates by RT-PCR and restriction enzyme analysis.

    PubMed

    Naveen, Kuttanda A; Singh, Shambhu Dayal; Kataria, Jag Mohan; Barathidasan, Rajamani; Dhama, Kuldeep

    2013-06-01

    Detection and pathotyping of Newcastle disease virus (NDV) is extremely important because the appearance of virulent virus has significant economic consequences. During 1981 to 1985, infections of racing and show pigeons with an avian paramyxovirus serotype-1 (APMV-1) hit worldwide, and a panzootic occurred due to a variant form of classical NDV. On the basis of pathogenicity and monoclonal antibody binding studies, the virus was termed 'pigeon PMV-1' (PPMV-1). In the past, number of Newcastle disease outbreaks in poultry and other birds has been attributed to PPMV-1. PPMV-1 viruses are known to present difficulty when assessed by conventional in vivo pathogenicity tests. In this study, the technique of reverse transcription-polymerase chain reaction (RT-PCR) and restriction enzyme (RE) analysis was used to detect and differentiate PPMV-1 isolates of Indian origin. Restriction enzyme digestion analysis of RT-PCR-amplified fusion protein (F) gene, encoding for the cleavage activation sites of fusion protein, was carried out with restriction enzymes BglI, HhaI, HaeIII, HinfI, MboI, MspI, PvuII and StyI. A set of only four enzymes HhaI, MspI or HaeIII, MboI and BglI alone were sufficient to differentially detect APMV-1 and PPMV-1 viruses and their pathotypes. In conclusion, RT-PCR followed by RE analysis proved to be useful for detection and differentiation of APMV-1 and PPMV-1 isolates at genomic level. PMID:23334380

  9. Development of an in situ magnetic beads based RT-PCR method for electrochemiluminescent detection of rotavirus

    NASA Astrophysics Data System (ADS)

    Zhan, Fangfang; Zhou, Xiaoming

    2012-12-01

    Rotaviruses are double-stranded RNA viruses belonging to the family of enteric pathogens. It is a major cause of diarrhoeal disease in infants and young children worldwide. Consequently, rapid and accurate detection of rotaviruses is of great importance in controlling and preventing food- and waterborne diseases and outbreaks. Reverse transcription-polymerase chain reaction (RT-PCR) is a reliable method that possesses high specificity and sensitivity. It has been widely used to detection of viruses. Electrochemiluminescence (ECL) can be considered as an important and powerful tool in analytical and clinical application with high sensitivity, excellent specificity, and low cost. Here we have developed a method for the detection of rotavirus by combining in situ magnetic beads (MBs) based RT-PCR with ECL. RT of rotavirus RNA was carried out in a traditional way and the resulting cDNA was directly amplified on MBs. Forward primers were covalently bounded to MBs and reverse primers were labeled with tris-(2, 2'-bipyridyl) ruthenium (TBR). During the PCR cycling, the TBR labeled products were directly loaded and enriched on the surface of MBs. Then the MBs-TBR complexes could be analyzed by a magnetic ECL platform without any post-modification or post-incubation which avoid some laborious manual operations and achieve rapid yet sensitive detection. In this study, rotavirus from fecal specimens was successfully detected within 2 h, and the limit of detection was estimated to be 104copies/μL. This novel in situ MBs based RT-PCR with ECL detection method can be used for pathogen detection in food safety field and clinical diagnosis.

  10. Identification of Novel Reference Genes Suitable for qRT-PCR Normalization with Respect to the Zebrafish Developmental Stage.

    PubMed

    Hu, Yu; Xie, Shuying; Yao, Jihua

    2016-01-01

    Reference genes used in normalizing qRT-PCR data are critical for the accuracy of gene expression analysis. However, many traditional reference genes used in zebrafish early development are not appropriate because of their variable expression levels during embryogenesis. In the present study, we used our previous RNA-Seq dataset to identify novel reference genes suitable for gene expression analysis during zebrafish early developmental stages. We first selected 197 most stably expressed genes from an RNA-Seq dataset (29,291 genes in total), according to the ratio of their maximum to minimum RPKM values. Among the 197 genes, 4 genes with moderate expression levels and the least variation throughout 9 developmental stages were identified as candidate reference genes. Using four independent statistical algorithms (delta-CT, geNorm, BestKeeper and NormFinder), the stability of qRT-PCR expression of these candidates was then evaluated and compared to that of actb1 and actb2, two commonly used zebrafish reference genes. Stability rankings showed that two genes, namely mobk13 (mob4) and lsm12b, were more stable than actb1 and actb2 in most cases. To further test the suitability of mobk13 and lsm12b as novel reference genes, they were used to normalize three well-studied target genes. The results showed that mobk13 and lsm12b were more suitable than actb1 and actb2 with respect to zebrafish early development. We recommend mobk13 and lsm12b as new optimal reference genes for zebrafish qRT-PCR analysis during embryogenesis and early larval stages. PMID:26891128

  11. Hyaluronidase treatment of synovial fluid to improve assay precision for biomarker research using multiplex immunoassay platforms.

    PubMed

    Jayadev, Chethan; Rout, Raj; Price, Andrew; Hulley, Philippa; Mahoney, David

    2012-12-14

    Synovial fluid (SF) is a difficult biological matrix to analyse due to its complex non-Newtonian nature. This can result in poor assay repeatability and potentially inefficient use of precious samples. This study assessed the impact of SF treatment by hyaluronidase and/or dilution on intra-assay precision using the Luminex and Meso Scale Discovery (MSD) multiplex platforms. SF was obtained from patients with knee osteoarthritis at the time of joint replacement surgery. Aliquots derived from the same sample were left untreated (neat), 2-fold diluted, 4-fold diluted or treated with 2mg/ml testicular hyaluronidase (with 2-fold dilution). Preparation methods were compared in a polysterene-bead Luminex 10-plex (N=16), magnetic-bead Luminex singleplex (N=7) and MSD 4-plex (N=7). Each method was assessed for coefficient of variation (CV) of replicate measurements, number of bead events (for Luminex assays) and dilution-adjusted analyte concentration. Percentage recovery was calculated for dilutions and HAse treatment. Hyaluronidase treatment significantly increased the number of wells with satisfactory bead events/region (95%) compared to neat (48%, p<0.001) in the polystyrene-bead Luminex assay, but the magnetic-bead Luminex assay achieved ≥50 bead events irrespective of treatment method. Hyaluronidase treatment resulted in lower intra-assay CVs for detectable ligands (group average CV<10%) than neat, 2-fold and 4-fold dilution (CV~25% for all, p<0.05) in both polystyrene- and magnetic-bead Luminex assays. In addition, measured sample concentrations were higher and recovery was poor (elevated) after hyaluronidase treatment. In the MSD 4-plex, within-group comparison of the intra-assay CV or concentration was not conclusively influenced by SF preparation. However, only hyaluronidase treatment resulted in CV<25% for all samples for TNF-α. There was no effect on analyte concentrations or recovery. Hyaluronidase treatment can improve intra-assay precision and assay signal

  12. Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process

    PubMed Central

    Expósito-Rodríguez, Marino; Borges, Andrés A; Borges-Pérez, Andrés; Pérez, José A

    2008-01-01

    Background The elucidation of gene expression patterns leads to a better understanding of biological processes. Real-time quantitative RT-PCR has become the standard method for in-depth studies of gene expression. A biologically meaningful reporting of target mRNA quantities requires accurate and reliable normalization in order to identify real gene-specific variation. The purpose of normalization is to control several variables such as different amounts and quality of starting material, variable enzymatic efficiencies of retrotranscription from RNA to cDNA, or differences between tissues or cells in overall transcriptional activity. The validity of a housekeeping gene as endogenous control relies on the stability of its expression level across the sample panel being analysed. In the present report we describe the first systematic evaluation of potential internal controls during tomato development process to identify which are the most reliable for transcript quantification by real-time RT-PCR. Results In this study, we assess the expression stability of 7 traditional and 4 novel housekeeping genes in a set of 27 samples representing different tissues and organs of tomato plants at different developmental stages. First, we designed, tested and optimized amplification primers for real-time RT-PCR. Then, expression data from each candidate gene were evaluated with three complementary approaches based on different statistical procedures. Our analysis suggests that SGN-U314153 (CAC), SGN-U321250 (TIP41), SGN-U346908 ("Expressed") and SGN-U316474 (SAND) genes provide superior transcript normalization in tomato development studies. We recommend different combinations of these exceptionally stable housekeeping genes for suited normalization of different developmental series, including the complete tomato development process. Conclusion This work constitutes the first effort for the selection of optimal endogenous controls for quantitative real-time RT-PCR studies of gene

  13. Evaluation of optimal extracellular vesicle small RNA isolation and qRT-PCR normalisation for serum and urine.

    PubMed

    Crossland, Rachel E; Norden, Jean; Bibby, Louis A; Davis, Joanna; Dickinson, Anne M

    2016-02-01

    MicroRNAs are small regulatory molecules that demonstrate useful biomarker potential. They have been recognised in biofluids, where they are protected from degradation by encapsulation into extracellular vesicles (EVs). A number of commercial products are available for the isolation of EVs and their RNA content; however, extensive protocol comparisons are lacking. Furthermore, robust qRT-PCR assessment of microRNA expression within EVs is problematic, as endogenous controls (ECs) previously used in cellular samples may not be present. This study compares EV isolation and RNA extraction methods (EV precipitation reagents, RNA isolation kits and ultracentrifugation) from serum or urine samples and evaluates suitable ECs for incorporation into qRT-PCR analysis. Results were assessed by electron microscopy, nanoparticle tracking analysis and bioanalyzer concentrations. The stability of 8 ECs was compared for both serum and urine EV RNA and retrospectively validated in independent cohorts (serum n=55, urine n=50). The Life Technologies precipitation reagent gave superior serum EV recovery compared to SBI reagent, as assessed by NTA size distribution, increased RNA concentration, and lower small RNA Ct values. Similarly, the Norgen Biotek Urine Exosome RNA Isolation Kit gave improved results for urine EV isolation compared to ultracentrifugation, when determined by the same parameters. The Qiagen miRNeasy™ RNA isolation kit gave suitable serum EV RNA concentrations compared to other kits, as assessed by Bioanalyzer and small RNA qRT-PCR. Small RNAs HY3 (S.D=1.77, CoV=6.2%) and U6 (S.D=2.14, CoV=8.6%) were selected as optimal ECs for serum EV microRNA expression analysis, while HY3 (S.D=1.67, CoV=6.5%) and RNU48 (S.D=1.85, CoV=5.3%) were identified as suitable for urine studies. In conclusion, this study identifies optimal methods for isolation of serum and urine EV RNA, and suitable ECs for normalisation of qRT-PCR studies. Such reports should aid in the

  14. From SOMAmer-Based Biomarker Discovery to Diagnostic and Clinical Applications: A SOMAmer-Based, Streamlined Multiplex Proteomic Assay

    PubMed Central

    Kraemer, Stephan; Vaught, Jonathan D.; Bock, Christopher; Gold, Larry; Katilius, Evaldas; Keeney, Tracy R.; Kim, Nancy; Saccomano, Nicholas A.; Wilcox, Sheri K.; Zichi, Dom; Sanders, Glenn M.

    2011-01-01

    Recently, we reported a SOMAmer-based, highly multiplexed assay for the purpose of biomarker identification. To enable seamless transition from highly multiplexed biomarker discovery assays to a format suitable and convenient for diagnostic and life-science applications, we developed a streamlined, plate-based version of the assay. The plate-based version of the assay is robust, sensitive (sub-picomolar), rapid, can be highly multiplexed (upwards of 60 analytes), and fully automated. We demonstrate that quantification by microarray-based hybridization, Luminex bead-based methods, and qPCR are each compatible with our platform, further expanding the breadth of proteomic applications for a wide user community. PMID:22022604

  15. Multiplex PCR assay for immediate identification of the infecting species in patients with mycobacterial disease.

    PubMed Central

    Kox, L F; Jansen, H M; Kuijper, S; Kolk, A H

    1997-01-01

    Rapid identification of infecting mycobacterial species enables appropriate medical care decisions to be made. Our aim was to demonstrate the clinical usefulness of the multiplex PCR assay, a test based on PCR, which permits direct identification of 12 mycobacterial species in clinical specimens. A total of 259 specimens from 177 patients who had clinical symptoms of mycobacterial disease but for whom there were difficulties in diagnosis were tested. Specimens were analyzed within 48 h of receipt of the sample. Mycobacteria were identified in 102 specimens; 66 specimens contained nontuberculous mycobacteria, and 36 specimens contained Mycobacterium tuberculosis complex mycobacteria. The PCR assay identified the mycobacterial species in 43 (97.7%) of 44 microscopy- and culture-positive specimens and in 15 (93.8%) of 16 culture-positive, microscopy-negative specimens. It also permitted species identification in infections caused by more than one mycobacterial species. For 56 (96.5%) of the 58 specimens from patients with infections caused by opportunistic mycobacteria, the organisms were identified with the PCR assay. The test was useful also for the identification of fastidious mycobacteria, e.g., M. genavense, and those that cannot be cultured, e.g., M. leprae. After resolution of discrepant results, the sensitivity of the PCR assay was 97.9%, the specificity was 96.9%, the positive predictive value was 95.0%, and the negative predictive value was 98.7%. For culture these values were 60.8, 100, 100, and 81.0%, respectively. Thus, the multiplex PCR assay enables prompt diagnosis when rapid identification of infecting mycobacteria is necessary. PMID:9163468

  16. How can we reduce costs of solid-phase multiplex-bead assays used to determine anti-HLA antibodies?

    PubMed

    Kamburova, E G; Wisse, B W; Joosten, I; Allebes, W A; van der Meer, A; Hilbrands, L B; Baas, M C; Spierings, E; Hack, C E; van Reekum, F E; van Zuilen, A D; Verhaar, M; Bots, M L; Drop, A C A D; Plaisier, L; Seelen, M A J; Sanders, J S F; Hepkema, B G; Lambeck, A J; Bungener, L B; Roozendaal, C; Tilanus, M G J; Vanderlocht, J; Voorter, C E; Wieten, L; van Duijnhoven, E M; Gelens, M; Christiaans, M H L; van Ittersum, F J; Nurmohamed, A; Lardy, N M; Swelsen, W; van der Pant, K A; van der Weerd, N C; Ten Berge, I J M; Bemelman, F J; Hoitsma, A; van der Boog, P J M; de Fijter, J W; Betjes, M G H; Heidt, S; Roelen, D L; Claas, F H; Otten, H G

    2016-09-01

    Solid-phase multiplex-bead assays are widely used in transplantation to detect anti-human leukocyte antigen (HLA) antibodies. These assays enable high resolution detection of low levels of HLA antibodies. However, multiplex-bead assays are costly and yield variable measurements that limit the comparison of results between laboratories. In the context of a Dutch national Consortium study we aimed to determine the inter-assay and inter-machine variability of multiplex-bead assays, and we assessed how to reduce the assay reagents costs. Fifteen sera containing a variety of HLA antibodies were used yielding in total 7092 median fluorescence intensities (MFI) values. The inter-assay and inter-machine mean absolute relative differences (MARD) of the screening assay were 12% and 13%, respectively. The single antigen bead (SAB) inter-assay MARD was comparable, but showed a higher lot-to-lot variability. Reduction of screening assay reagents to 50% or 40% of manufacturers' recommendations resulted in MFI values comparable to 100% of the reagents, with an MARD of 12% or 14%, respectively. The MARD of the 50% and 40% SAB assay reagent reductions were 11% and 22%, respectively. From this study, we conclude that the reagents can be reliably reduced at least to 50% of manufacturers' recommendations with virtually no differences in HLA antibody assignments. PMID:27534609

  17. Acoustic trapping as a generic non-contact incubation site for multiplex bead-based assays.

    PubMed

    Tenje, Maria; Xia, Hongyan; Evander, Mikael; Hammarström, Björn; Tojo, Axel; Belák, Sándor; Laurell, Thomas; LeBlanc, Neil

    2015-01-01

    In this study, we show a significantly reduced assay time and a greatly increased bead recovery for a commercial Luminex-based multiplex diagnostic immunoassay by performing all liquid handling steps of the assay protocol in a non-contact acoustic trapping platform. The Luminex assay is designed for detecting antibodies in poultry serum for infectious bursal disease virus, infectious bronchitis virus, Newcastle disease virus and avian reovirus. Here, we show proof-of-concept of a microfluidic system capable of being fully automated and handling samples in a parallel format with a miniature physical footprint where the affinity beads are retained in a non-contact levitated mode in a glass capillary throughout the assay protocol. The different steps are: incubation with the serum sample, secondary antibodies and fluorescent reporters and finally washing to remove any non-specifically bound species. A Luminex 200 instrument was used for the readout. The flow rates applied to the capillary during the initial trapping event and the wash steps were optimised for maximum bead recovery, resulting in a bead recovery of 75% for the complete assay. This can be compared to a bead recovery of approximately 30% when an automatic wash station was used when the assay was performed in the conventional manual format. The time for the incubation steps for a single assay was reduced by more than 50%, without affecting assay performance, since intermediate wash steps became redundant in the continuously perfused bead trapping capillary. We analyzed seven samples, in triplicates, and we can show that the readout of the assay performed in the acoustic trap compared 100% to the control ELISAs (positive or negative readout) and resulted in comparable S/P values as the conventional manual protocol. As the acoustic trapping does not require the particles to have magnetic properties, a greater degree of freedom in selecting microparticles can be provided. In extension, this can provide an

  18. Utility of automated real-time RT-PCR for the detection of foot-and-mouth disease virus excreted in milk.

    PubMed

    Reid, Scott M; Parida, Satya; King, Donald P; Hutchings, Geoffrey H; Shaw, Andrew E; Ferris, Nigel P; Zhang, Zhidong; Hillerton, J Eric; Paton, David J

    2006-01-01

    Foot-and-mouth disease virus (FMDV) can be excreted in milk and thereby spread infection to susceptible animals in other holdings. The feasibility of using real-time reverse transcription polymerase chain reaction (rRT-PCR) as a diagnostic tool for detection of FMDV in milk was assessed by studying the excretion of virus from experimentally-infected cattle. Fore- and machine milk samples were collected over a 4-week period from two dairy cows infected with FMDV and from two in-contact cows held in the same pen. The whole, skim, cream and cellular debris components of the milks were tested by automated rRT-PCR and results compared to virus isolation (VI) in cell culture. The onset of clinical signs of FMD in all four cows correlated with viraemia, and the presence of FMDV in other clinical samples. rRT-PCR results matched closely with VI in detecting FMDV in all milk components and generally coincided with, but did not consistently precede, the onset of clinical signs. rRT-PCR detected FMDV in milk up to 23 days post inoculation which was longer than VI. Furthermore, the detection limit of FMDV in milk was greater by rRT-PCR than VI and, in contrast to VI, rRT-PCR detected virus genome following heat treatment that simulated pasteurisation. rRT-PCR was also able to detect FMDV in preservative-treated milk. In conclusion, this study showed that automated rRT-PCR is quicker and more sensitive than VI and can be used to detect FMDV in whole milk as well as milk fractions from infected animals. PMID:16336929

  19. Development of 2, 7-Diamino-1, 8-Naphthyridine (DANP) Anchored Hairpin Primers for RT-PCR Detection of Chikungunya Virus Infection.

    PubMed

    Chen, Huixin; Parimelalagan, Mariya; Takei, Fumie; Hapuarachchi, Hapuarachchige Chanditha; Koay, Evelyn Siew-Chuan; Ng, Lee Ching; Ho, Phui San; Nakatani, Kazuhiko; Chu, Justin Jang Hann

    2016-08-01

    A molecular diagnostic platform with DANP-anchored hairpin primer was developed and evaluated for the rapid and cost-effective detection of Chikungunya virus (CHIKV) with high sensitivity and specificity. The molecule 2, 7-diamino-1, 8-naphthyridine (DANP) binds to a cytosine-bulge and emits fluorescence at 450 nm when it is excited by 400 nm light. Thus, by measuring the decline in fluorescence emitted from DANP-primer complexes after PCR reaction, we could monitor the PCR progress. By adapting this property of DANP, we have previously developed the first generation DANP-coupled hairpin RT-PCR assay. In the current study, we improved the assay performance by conjugating the DANP molecule covalently onto the hairpin primer to fix the DANP/primer ratio at 1:1; and adjusting the excitation emission wavelength to 365/430 nm to minimize the background signal and a 'turn-on' system is achieved. After optimizing the PCR cycle number to 30, we not only shortened the total assay turnaround time to 60 minutes, but also further reduced the background fluorescence. The detection limit of our assay was 0.001 PFU per reaction. The DANP-anchored hairpin primer, targeting nsP2 gene of CHIKV genome, is highly specific to CHIKV, having no cross-reactivity to a panel of other RNA viruses tested. In conclusion, we report here a molecular diagnostic assay that is sensitive, specific, rapid and cost effective for CHIKV detection and can be performed where no real time PCR instrumentation is required. Our results from patient samples indicated 93.62% sensitivity and 100% specificity of this method, ensuring that it can be a useful tool for rapid detection of CHIKV for outbreaks in many parts of the world. PMID:27571201

  20. Development of 2, 7-Diamino-1, 8-Naphthyridine (DANP) Anchored Hairpin Primers for RT-PCR Detection of Chikungunya Virus Infection

    PubMed Central

    Chen, Huixin; Parimelalagan, Mariya; Takei, Fumie; Hapuarachchi, Hapuarachchige Chanditha; Koay, Evelyn Siew-Chuan; Ng, Lee Ching; Ho, Phui San; Nakatani, Kazuhiko; Chu, Justin Jang Hann

    2016-01-01

    A molecular diagnostic platform with DANP-anchored hairpin primer was developed and evaluated for the rapid and cost-effective detection of Chikungunya virus (CHIKV) with high sensitivity and specificity. The molecule 2, 7-diamino-1, 8-naphthyridine (DANP) binds to a cytosine-bulge and emits fluorescence at 450 nm when it is excited by 400 nm light. Thus, by measuring the decline in fluorescence emitted from DANP—primer complexes after PCR reaction, we could monitor the PCR progress. By adapting this property of DANP, we have previously developed the first generation DANP-coupled hairpin RT-PCR assay. In the current study, we improved the assay performance by conjugating the DANP molecule covalently onto the hairpin primer to fix the DANP/primer ratio at 1:1; and adjusting the excitation emission wavelength to 365/430 nm to minimize the background signal and a ‘turn-on’ system is achieved. After optimizing the PCR cycle number to 30, we not only shortened the total assay turnaround time to 60 minutes, but also further reduced the background fluorescence. The detection limit of our assay was 0.001 PFU per reaction. The DANP-anchored hairpin primer, targeting nsP2 gene of CHIKV genome, is highly specific to CHIKV, having no cross-reactivity to a panel of other RNA viruses tested. In conclusion, we report here a molecular diagnostic assay that is sensitive, specific, rapid and cost effective for CHIKV detection and can be performed where no real time PCR instrumentation is required. Our results from patient samples indicated 93.62% sensitivity and 100% specificity of this method, ensuring that it can be a useful tool for rapid detection of CHIKV for outbreaks in many parts of the world. PMID:27571201

  1. Antigen sandwich ELISA predicts RT-PCR detection of dengue virus genome in infected culture fluids of Aedes albopictus C6/36 cells.

    PubMed

    Buerano, Corazon C; Natividad, Filipinas F; Contreras, Rodolfo C; Ibrahim, Ima Nurisa; Mangada, Marlou Noel M; Hasebe, Futoshi; Inoue, Shingo; Matias, Ronald R; Igarashi, Akira

    2008-09-01

    Antigen detection by sandwich ELISA was evaluated to predict RT-PCR detection of dengue viral genome in infected culture fluid of Aedes albopictus clone C6/36 cells. Serum specimens collected from dengue patients within 5 days from onset of fever in 2 hospitals in Metro Manila, Philippines, were inoculated into C6/36 cells, and incubated at 28 degrees C. A total of 282 infected culture fluid specimens were harvested and examined by sandwich ELISA and RT-PCR to detect dengue viral antigen and genome, respectively. In the sandwich ELISA, the P/N ratio was calculated by dividing optical density (OD) of a given test specimen by the OD of the standard negative specimen. Samples with a P/N ratio > or = 4.001 were positive for viral genome detection by RT-PCR. The sensitivity and specificity of antigen sandwich ELISA with RT-PCR as the standard, were 90.4% and 100%, respectively. Although antigen sandwich ELISA is less sensitive than RT-PCR, its usefulness lies in its capability to screen a large number of samples at a minimum cost, especially during an outbreak. Samples that meet a set cutoff value can undergo confirmation by RT-PCR for further epidemiological studies. PMID:19058574

  2. High Throughput Flow Cytometry Bead-based Multiplex Assay for Identification of Rho GTPase Inhibitors

    PubMed Central

    Surviladze, Zurab; Young, Susan M; Sklar, Larry A

    2015-01-01

    Summary Rho family GTPases and their effector proteins regulate a wide range of cell signaling pathways. In normal physiological conditions their activity is tightly controlled and it is not surprising that their aberrant activation contributes to tumorigenesis or other diseases. For this reason, the identification of small, cell permeable molecules capable of inhibition of Rho GTPases can be extraordinarily useful, particularly if they are specific and act reversibly. Herein we describe a flow cytometric assay, which allows us to measure the activity of six small GTPases simultaneously. GST-tagged small GTPases are bound to six glutathione bead sets each set having a different intensity of red fluorescence at a fixed wavelength. The coated bead sets were washed, combined, and dispensed into 384-well plates with test compounds, and fluorescent-GTP binding was used as the read-out. This multiplex bead-based assay was successfully used for to identify both general and selective inhibitors of Rho family GTPases. PMID:22144280

  3. Detection of Nicotiana DNA in Tobacco Products Using a Novel Multiplex Real-Time PCR Assay.

    PubMed

    Korchinski, Katie L; Land, Adrian D; Craft, David L; Brzezinski, Jennifer L

    2016-07-01

    Establishing that a product contains tobacco is a requirement for the U.S. Food and Drug Administration's regulation and/or prosecution of tobacco products. Therefore, a multiplex real-time PCR method was designed to determine if Nicotiana (tobacco) DNA is present in tobacco products. The PCR method simultaneously amplifies a 73 bp fragment of the cytochrome P450 monoxygenase CYP82E4 gene and 66 bp fragment in the nia-1 gene for nitrate reductase, which are detected using dual-labeled TaqMan probes. The assay is capable of detecting approximately 7.8 pg purified tobacco DNA, with a similar sensitivity for either gene target while incorporating an internal positive control (IPC). DNA was extracted from prepared tobacco products-including chewing tobacco, pipe tobacco, and snuff-or from the cut fill (no wrapper) of cigarettes and cigars. Of the 13 products analyzed, 12 were positive for both tobacco-specific markers and the IPC. DNA was also extracted from the fill of five varieties of herbal cigarettes, which were negative for both tobacco-specific gene targets and positive for the IPC. Our method expands on current assays by introducing a multiplex reaction, targeting two sequences in two different genes of interest, incorporating an IPC into the reaction, and lowering the LOD and LOQ while increasing the efficiency of the PCR. PMID:27143320

  4. Wide spectral-range imaging spectroscopy of photonic crystal microbeads for multiplex biomolecular assay applications

    NASA Astrophysics Data System (ADS)

    Li, Jianping

    2014-05-01

    Suspension assay using optically color-encoded microbeads is a novel way to increase the reaction speed and multiplex of biomolecular detection and analysis. To boost the detection speed, a hyperspectral imaging (HSI) system is of great interest for quickly decoding the color codes of the microcarriers. Imaging Fourier transform spectrometer (IFTS) is a potential candidate for this task due to its advantages in HSI measurement. However, conventional IFTS is only popular in IR spectral bands because it is easier to track its scanning mirror position in longer wavelengths so that the fundamental Nyquist criterion can be satisfied when sampling the interferograms; the sampling mechanism for shorter wavelengths IFTS used to be very sophisticated, high-cost and bulky. In order to overcome this handicap and take better usage of its advantages for HSI applications, a new wide spectral range IFTS platform is proposed based on an optical beam-folding position-tracking technique. This simple technique has successfully extended the spectral range of an IFTS to cover 350-1000nm. Test results prove that the system has achieved good spectral and spatial resolving performances with instrumentation flexibilities. Accurate and fast measurement results on novel colloidal photonic crystal microbeads also demonstrate its practical potential for high-throughput and multiplex suspension molecular assays.

  5. Longitudinal Evaluation of Enteric Protozoa in Haitian Children by Stool Exam and Multiplex Serologic Assay

    PubMed Central

    Moss, Delynn M.; Priest, Jeffrey W.; Hamlin, Kathy; Derado, Gordana; Herbein, Joel; Petri, William A.; Lammie, Patrick J.

    2014-01-01

    Haitian children were monitored longitudinally in a filariasis study. Included were stool samples examined for Giardia intestinalis and Entamoeba histolytica cysts, and serum specimens analyzed for immunoglobulin G (IgG) responses to eight recombinant antigens from G. intestinalis (variant-specific surface protein [VSP1–VSP5]), E. histolytica (lectin adhesion molecule [LecA]), and Cryptosporidium parvum (17- and 27-kDa) using a multiplex bead assay. The IgG responses to VSP antigens peaked at 2 years of age and then diminished and were significantly lower (P < 0.002) in children > 4.5 years than in children < 4.5 years. The IgG responses to Cryptosporidium tended to increase with age. The IgG responses to LecA and VSP antigens and the prevalence of stools positive for cysts were significantly higher (P < 0.037 and P < 0.035, respectively) in the rainy season than in the dry season. The multiplex bead assay provides a powerful tool for analyzing serologic responses to multiple pathogens. PMID:24591430

  6. Demonstration of Array-Based Analysis for Highly Multiplexed PCR Assays

    PubMed Central

    Spence, Janice M.; Rothberg, Paul G.; Wang, Nancy; Burack, W. Richard

    2011-01-01

    We demonstrate an approach that allowed rapid development of a robust assay for the detection of chromosomal translocations. The method includes highly multiplexed PCR with analysis of the PCR products performed by array detection. As proof of principle, we applied this approach to the detection of IGH@-BCL2 translocations in DNA prepared from FFPE specimens. This translocation and specimen type were chosen because of the known difficulties associated with PCR-based detection of this lesion and the additional loss of sensitivity associated with FFPE samples. The multiplex PCR with array detection method detected the IGH@-BCL2 translocation in 26 of 36 FFPE follicular lymphoma specimens, whereas the BIOMED-2 assay detected 13 of 36 specimens. This increased sensitivity was the result of both the increased density of BCL2 primers and identification of PCR products by low-density array. The method was specific and allowed mapping of the BCL2 break point in all cases. The method detected the IGH@-BCL2 lesion when the tumor DNA was diluted more than 1:20 in normal DNA but not when it was diluted more than 1:100. This sensitivity allows detection of diagnostically relevant levels of IGH@-BCL2 but will not detect the rare cells with IGH@-BCL2 translocations in healthy individuals. PMID:21497287

  7. Selection of reference genes for qRT-PCR analysis of gene expression in sea cucumber Apostichopus japonicus during aestivation

    NASA Astrophysics Data System (ADS)

    Zhao, Ye; Chen, Muyan; Wang, Tianming; Sun, Lina; Xu, Dongxue; Yang, Hongsheng

    2014-11-01

    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.

  8. Evaluation of reference genes for real-time RT-PCR expression studies in the plant pathogen Pectobacterium atrosepticum

    PubMed Central

    Takle, Gunnhild W; Toth, Ian K; Brurberg, May B

    2007-01-01

    Background Real-time RT-PCR has become a powerful technique to monitor low-abundance mRNA expression and is a useful tool when examining bacterial gene expression inside infected host tissues. However, correct evaluation of data requires accurate and reliable normalisation against internal standards. Thus, the identification of reference genes whose expression does not change during the course of the experiment is of paramount importance. Here, we present a study where manipulation of cultural growth conditions and in planta experiments have been used to validate the expression stability of reference gene candidates for the plant pathogen Pectobacterium atrosepticum, belonging to the family Enterobacteriaceae. Results Of twelve reference gene candidates tested, four proved to be stably expressed both in six different cultural growth conditions and in planta. Two of these genes (recA and ffh), encoding recombinase A and signal recognition particle protein, respectively, proved to be the most stable set of reference genes under the experimental conditions used. In addition, genes proC and gyrA, encoding pyrroline-5-carboxylate reductase and DNA gyrase, respectively, also displayed relatively stable mRNA expression levels. Conclusion Based on these results, we suggest recA and ffh as suitable candidates for accurate normalisation of real-time RT-PCR data for experiments investigating the plant pathogen P. atrosepticum and potentially other related pathogens. PMID:17888160

  9. Tick-borne encephalitis in dogs: application of "nested real-time RT-PCR" for intravital virus detection.

    PubMed

    Hekrlová, Alena; Kubíček, Oldfich; Lány, Petr; Rosenbergová, Kateřina; Schánilec, Pavel

    2015-01-01

    Tick-borne encephalitis (TBE) virus is a tick-transmitted virus causing disorders of the nervous system in humans, monkeys, dogs and horses (rarely). At present the detection of TBE infection in dogs is performed by confirmation of seroconversion in paired samples of serum in clinical practice. The intention of the study was the assessment of the possible application of nested real-time RT-PCR for detection of TBE virus in canine blood. The study was carried out in 2011-2012 using samples originating in the Czech Republic, South Moravian region (region with endemic occurrence of TBE). The dogs were randomly selected from the patients visiting the clinic during this time period. Of the total amount of 159 canine blood samples, 20 samples were tested with a PCR-positive result (12.6%). Out of these 20 animals, the neurological clinical symptoms typical of TBE were detected in seven dogs. PCR-positive results were found between March and November. Three dogs were tested with a competitive ELISA-positive result and a "nested real-time RT-PCR"-positive result concurrently. In the group of 159 dogs the value of seroprevalence was found to be 11.3%. PMID:26591386

  10. Further development of multiplex single nucleotide polymorphism typing method, the DigiTag2 assay.

    PubMed

    Nishida, Nao; Tanabe, Tetsuya; Takasu, Miwa; Suyama, Akira; Tokunaga, Katsushi

    2007-05-01

    A number of single nucleotide polymorphisms (SNPs) are considered to be candidate susceptibility or resistance genetic factors for multifactorial disease. Genome-wide searches for disease susceptibility regions followed by high-resolution mapping of primary genes require cost-effective and highly reliable technology. To accomplish successful and low-cost typing for candidate SNPs, new technologies must be developed. We previously reported a multiplex SNP typing method, designated the DigiTag assay, that has the potential to analyze nearly any SNP with high accuracy and reproducibility. However, the DigiTag assay requires multiple washing steps in manipulation and uses genotyping probes modified with biotin for each target SNP. Here we describe the next version of the assay, DigiTag2, which works with simple protocols and uses unmodified genotyping probes. We investigated the feasibility of the DigiTag2 assay by genotyping 96 target SNPs spanning a 610-kb region of human chromosome 5. The DigiTag2 assay is suitable for genotyping an intermediate number of SNPs (tens to hundreds of sites) with a high conversion rate (>90%), high accuracy, and low cost. PMID:17359929

  11. Rapid and sensitive detection of major uropathogens in a single-pot multiplex PCR assay.

    PubMed

    Padmavathy, B; Vinoth Kumar, R; Patel, Amee; Deepika Swarnam, S; Vaidehi, T; Jaffar Ali, B M

    2012-07-01

    Urinary tract infection (UTI) is among the most common bacterial infections and poses a significant healthcare burden. Escherichia coli is the most common cause of UTI accounting for up to 70 % and a variable contribution from Proteus mirabilis, Pseudomonas aeruginosa and Klebsiella pneumoniae. To establish a complete diagnostic system, we have developed a single-tube multiplex PCR assay (mPCR) for the detection of the above-mentioned four major uropathogens. The sensitivity of the assay was found to be as low as 10(2) cfu/ml of cells. The mPCR evaluated on 280 clinical isolates detected 100 % of E. coli, P. aeruginosa, P. mirabilis and 95 % of K. pneumonia. The assay was performed on 50 urine samples and found to be specific and sensitive for clinical diagnosis. In addition, the mPCR was also validated on spiked urine samples using 40 clinical isolates to demonstrate its application under different strain used in this assay. In total, mPCR reported here is a rapid and simple screening tool that can compete with conventional biochemical-based screening assays that may require 2-3 days for detection. PMID:22526571

  12. Real-Time Reverse Transcription-PCR Assay for Detection of Mumps Virus RNA in Clinical Specimens▿

    PubMed Central

    Boddicker, Jennifer D.; Rota, Paul A.; Kreman, Trisha; Wangeman, Andrea; Lowe, Louis; Hummel, Kimberly B.; Thompson, Robert; Bellini, William J.; Pentella, Michael; DesJardin, Lucy E.

    2007-01-01

    The mumps virus is a negative-strand RNA virus in the family Paramyxoviridae. Mumps infection results in an acute illness with symptoms including fever, headache, and myalgia, followed by swelling of the salivary glands. Complications of mumps can include meningitis, deafness, pancreatitis, orchitis, and first-trimester abortion. Laboratory confirmation of mumps infection can be made by the detection of immunoglobulin M-specific antibodies to mumps virus in acute-phase serum samples, the isolation of mumps virus in cell culture, or by detection of the RNA of the mumps virus by reverse transcription (RT)-PCR. We developed and validated a multiplex real-time RT-PCR assay for rapid mumps diagnosis in a clinical setting. This assay used oligonucleotide primers and a TaqMan probe targeting the mumps SH gene, as well as primers and a probe that targeted the human RNase P gene to assess the presence of PCR inhibitors and as a measure of specimen quality. The test was specific, since it did not amplify a product from near-neighbor viruses, as well as sensitive and accurate. Real-time RT-PCR results showed 100% correlation with results from viral culture, the gold standard for mumps diagnostic testing. Assay efficiency was over 90% and displayed good precision after performing inter- and intraassay replicates. Thus, we have developed and validated a molecular method for rapidly diagnosing mumps infection that may be used to complement existing techniques. PMID:17652480

  13. Discrimination between E. granulosus sensu stricto, E. multilocularis and E. shiquicus Using a Multiplex PCR Assay

    PubMed Central

    Li, Li; Yan, Hong-Bin; Blair, David; Lei, Meng-Tong; Cai, Jin-Zhong; Fan, Yan-Lei; Li, Jian-Qiu; Fu, Bao-Quan; Yang, Yu-Rong; McManus, Donald P.; Jia, Wan-Zhong

    2015-01-01

    Background Infections of Echinococcus granulosus sensu stricto (s.s), E. multilocularis and E. shiquicus are commonly found co-endemic on the Qinghai-Tibet plateau, China, and an efficient tool is needed to facilitate the detection of infected hosts and for species identification. Methodology/Principal Findings A single-tube multiplex PCR assay was established to differentiate the Echinococcus species responsible for infections in intermediate and definitive hosts. Primers specific for E. granulosus, E. multilocularis and E. shiquicus were designed based on sequences of the mitochondrial NADH dehydrogenase subunit 1 (nad1), NADH dehydrogenase subunit 5 (nad5) and cytochrome c oxidase subunit 1 (cox1) genes, respectively. This multiplex PCR accurately detected Echinococcus DNA without generating nonspecific reaction products. PCR products were of the expected sizes of 219 (nad1), 584 (nad5) and 471 (cox1) bp. Furthermore, the multiplex PCR enabled diagnosis of multiple infections using DNA of protoscoleces and copro-DNA extracted from fecal samples of canine hosts. Specificity of the multiplex PCR was 100% when evaluated using DNA isolated from other cestodes. Sensitivity thresholds were determined for DNA from protoscoleces and from worm eggs, and were calculated as 20 pg of DNA for E. granulosus and E. shiquicus, 10 pg of DNA for E. multilocularis, 2 eggs for E. granulosus, and 1 egg for E. multilocularis. Positive results with copro-DNA could be obtained at day 17 and day 26 after experimental infection of dogs with larval E. multilocularis and E. granulosus, respectively. Conclusions/Significance The multiplex PCR developed in this study is an efficient tool for discriminating E. granulosus, E. multilocularis and E. shiquicus from each other and from other taeniid cestodes. It can be used for the detection of canids infected with E. granulosus s.s. and E. multilocularis using feces collected from these definitive hosts. It can also be used for the identification

  14. Development of a multiplex PCR assay to detect the major clonal complexes of Streptococcus suis relevant to human infection.

    PubMed

    Hatrongjit, Rujirat; Kerdsin, Anusak; Gottschalk, Marcelo; Hamada, Shigeyuki; Oishi, Kazunori; Akeda, Yukihiro

    2016-05-01

    Multilocus sequence typing (MLST) is considered a reliable method for providing insight into the Streptococcus suis population structure, clonal relationships and the potential of particular clones to cause disease. Indeed, MLST has revealed the presence of several clonal complexes (CCs) within the Streptococcus suis population. However, the method is costly, time-consuming and difficult to use for screening large numbers of isolates. In this study, a multiplex PCR assay was developed to identify Streptococcus suis CCs that are relevant to human infections. The multiplex PCR assay was capable of simultaneously distinguishing CC1, CC25, CC28, CC104, CC221/234 and CC233/379, which are related to human infections in Thailand, in a single reaction. The multiplex PCR assay is useful for low-cost screening of large numbers of isolates with rapid analytical capacity and could be utilized in most laboratories. PMID:26932590

  15. Simultaneous typing of nine avian respiratory pathogens using a novel GeXP analyzer-based multiplex PCR assay.

    PubMed

    Xie, Zhixun; Luo, Sisi; Xie, Liji; Liu, Jiabo; Pang, Yaoshan; Deng, Xianwen; Xie, Zhiqin; Fan, Qing; Khan, Mazhar I

    2014-10-01

    A new, rapid, and high-throughput GenomeLab Gene Expression Profiler (GeXP) analyzer-based multiplex PCR method was developed for simultaneous detection and differentiation of nine avian respiratory pathogens. The respiratory pathogens included in this study were avian influenza subtypes H5, H7, and H9, infectious bronchitis virus (IBV), Newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS) and Haemophilus paragallinarum (HPG). Ten pairs of primers were designed using conserved and specific sequence genes of AIV subtypes and respiratory pathogens from GenBank. Single and mixed pathogen cDNA/DNA templates were used to evaluate the specificity of the GeXP-multiplex assay. The corresponding specific DNA products were amplified for each pathogen. The specific DNA product amplification peaks of nine respiratory pathogens were observed on the GeXP analyzer. Non-respiratory avian pathogens, including chicken infectious anemia virus, fowl adenovirus, avian reovirus and infectious bursal disease virus, did not produce DNA products. The detection limit for the GeXP-multiplex assay was determined to be 100 copies/μl using various pre-mixed plasmids/ssRNAs containing known target genes of the respiratory pathogens. Further, GeXP-multiplex PCR assay was 100% specific when 24 clinical samples with respiratory infections were tested in comparison with conventional PCR method. The GeXP-multiplex PCR assay provides a novel tool for simultaneous detection and differentiation of nine avian respiratory pathogens. PMID:25025815

  16. Validation of Reference Genes for Gene Expression by Quantitative Real-Time RT-PCR in Stem Segments Spanning Primary to Secondary Growth in Populus tomentosa

    PubMed Central

    Wang, Ying; Chen, Yajuan; Ding, Liping; Zhang, Jiewei; Wei, Jianhua

    2016-01-01

    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

  17. Validation of two real-time RT-PCR methods for foot-and-mouth disease diagnosis: RNA-extraction, matrix effect, uncertainty of measurement and precision.

    PubMed

    Goris, Nesya; Vandenbussche, Frank; Herr, Cécile; Villers, Jérôme; Van der Stede, Yves; De Clercq, Kris

    2009-09-01

    Real-time reverse transcription polymerase chain reaction (rRT-PCR) assays are being used routinely for diagnosing foot-and-mouth disease virus (FMDV). Although most laboratories determine analytical and diagnostic sensitivity and specificity, a thorough validation in terms of establishing optimal RNA-extraction conditions, matrix effect, uncertainty of measurement and precision is not performed or reported generally. In this study, different RNA-extraction procedures were compared for two FMDV rRT-PCRs. The NucleoSpin columns available commercially combined high extraction efficiency with ease-of-automation. Furthermore, six different FMDV-negative matrices were spiked with a dilution series of FMDV SAT1 ZIM 25/89. Compared to cell-culture-spiked viral control samples, no matrix effect on the analytical sensitivity was found for blood or foot epithelium. Approximately 1log(10) reduction in detection limit was noted for faecal and tongue epithelium samples, whereas a 3log(10) decrease was observed for spleen samples. By testing the same dilution series in duplicate on 10 different occasions, an estimation of uncertainty of measurement and precision was obtained using blood as matrix. Both rRT-PCRs produced highly precise results emphasising their potential to replace conventional virological methods. The uncertainty measurement, as described in this study, proved to be a useful tool to evaluate the probability of making a wrong decision. PMID:19447138

  18. Analysis of myosin heavy chain mRNA expression by RT-PCR

    NASA Technical Reports Server (NTRS)

    Wright, C.; Haddad, F.; Qin, A. X.; Baldwin, K. M.

    1997-01-01

    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.

  19. Multiplexed homogeneous assays of proteolytic activity using a smartphone and quantum dots.

    PubMed

    Petryayeva, Eleonora; Algar, W Russ

    2014-03-18

    Semiconductor quantum dot (QD) bioconjugates, with their unique and highly advantageous physicochemical and optical properties, have been extensively utilized as probes for bioanalysis and continue to generate widespread interest for these applications. An important consideration for expanding the utility of QDs and making their use routine is to make assays with QDs more accessible for laboratories that do not specialize in nanomaterials. Here, we show that digital color imaging of QD photoluminescence (PL) with a smartphone camera is a viable, easily accessible readout platform for quantitative, multiplexed, and real-time bioanalyses. Red-, green-, and blue-emitting CdSeS/ZnS QDs were conjugated with peptides that were labeled with a deep-red fluorescent dye, Alexa Fluor 647, and the dark quenchers, QSY9 and QSY35, respectively, to generate Förster resonance energy transfer (FRET) pairs sensitive to proteolytic activity. Changes in QD PL caused by the activity of picomolar to nanomolar concentrations of protease were detected as changes in the red-green-blue (RGB) channel intensities in digital color images. Importantly, measurements of replicate samples made with smartphone imaging and a sophisticated fluorescence plate reader yielded the same quantitative results, including initial proteolytic rates and specificity constants. Homogeneous two-plex and three-plex assays for the activity of trypsin, chymotrypsin, and enterokinase were demonstrated with RGB imaging. Given the ubiquity of smartphones, this work largely removes any instrumental impediments to the adoption of QDs as routine tools for bioanalysis in research laboratories and is a critical step toward the use of QDs for point-of-care diagnostics. This work also adds to the growing utility of smartphones in analytical methods by enabling multiplexed fluorimetric assays within a single sample volume and across multiple samples in parallel. PMID:24571675

  20. Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

    PubMed Central

    O'Sullivan, Matthew V. N.; Zhou, Fei; Sintchenko, Vitali; Kong, Fanrong; Gilbert, Gwendolyn L.

    2011-01-01

    Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction followed by probe hybridization on a nylon membrane, which is re-usable. Probes are 5' amine modified to allow fixation to the membrane. Primers are 5' biotin modified which allows detection of hybridized PCR products using streptavidin-peroxidase and a chemiluminescent substrate via photosensitive film. With low setup and consumable costs, this technique is inexpensive (approximately US$2 per sample), high throughput (multiple membranes can be processed simultaneously) and has a short turnaround time (approximately 10 hours). The technique can be utilized in a number of ways. Multiple probes can be designed to detect sequence variation within a single amplified product, or multiple products can be amplified simultaneously, with one (or more) probes used for subsequent detection. A combination of both approaches can also be used within a single assay. The ability to include multiple probes for a single target sequence makes the assay highly specific. Published applications of mPCR/RLB include detection of antibiotic resistance genes1,2, typing of methicillin-resistant Staphylococcus aureus3-5 and Salmonella sp6, molecular serotyping of Streptococcus pneumoniae7,8, Streptococcus agalactiae9 and enteroviruses10,11, identification of Mycobacterium sp12, detection of genital13-15 and respiratory tract16 and other17 pathogens and detection and identification of mollicutes18. However, the versatility of the technique means the applications are virtually limitless and not restricted to molecular analysis of micro-organisms. The five steps in mPCR/RLB are a) Primer and Probe design, b) DNA extraction and PCR amplification c) Preparation of the membrane, d) Hybridization and detection, and e) Regeneration of the Membrane. PMID:21847083

  1. Sensitive multiplex PCR assay to differentiate Lyme spirochetes and emerging pathogens Anaplasma phagocytophilum and Babesia microti

    PubMed Central

    2013-01-01

    Background The infection with Borrelia burgdorferi can result in acute to chronic Lyme disease. In addition, coinfection with tick-borne pathogens, Babesia species and Anaplasma phagocytophilum has been increasing in endemic regions of the USA and Europe. The currently used serological diagnostic tests are often difficult to interpret and, moreover, antibodies against the pathogens persist for a long time making it difficult to confirm the cure of the disease. In addition, these tests cannot be used for diagnosis of early disease state before the adaptive immune response is established. Since nucleic acids of the pathogens do not persist after the cure, DNA-based diagnostic tests are becoming highly useful for detecting infectious diseases. Results In this study, we describe a real-time multiplex PCR assay to detect the presence of B. burgdorferi, B. microti and A. phagocytophilum simultaneously even when they are present in very low copy numbers. Interestingly, this quantitative PCR technique is also able to differentiate all three major Lyme spirochete species, B. burgdorferi, B. afzelii, and B. garinii by utilizing a post-PCR denaturation profile analysis and a single molecular beacon probe. This could be very useful for diagnosis and discrimination of various Lyme spirochetes in European countries where all three Lyme spirochete species are prevalent. As proof of the principle for patient samples, we detected the presence of low number of Lyme spirochetes spiked in the human blood using our assay. Finally, our multiplex assay can detect all three tick-borne pathogens in a sensitive and specific manner irrespective of the level of each pathogen present in the sample. We anticipate that this novel diagnostic method will be able to simultaneously diagnose early to chronic stages of Lyme disease, babesiosis and anaplasmosis using the patients’ blood samples. Conclusion Real-time quantitative PCR using specific primers and molecular beacon probes for the selected

  2. Detection of toxigenic strains of Aeromonas species in foods by a multiplex PCR assay.

    PubMed

    Balakrishna, K; Murali, H S; Batra, H V

    2010-06-01

    Aeromonas hydrophila and other aeromonads are ubiquitous organisms found in meat, vegetables, drinking water and various other food items. They cause diarrhea and extra-intestinal infections in normal and immunocompromised patients. The aim of the study was to develop a multiplex PCR assay for the detection of virulence-associated genes of Aeromonas including hemolysin (hlyA), aerolysin (aerA), glycerophospholipid-cholesterol acyl transferase (GCAT) alongwith a 16S rRNA gene. Internal amplification control (IAC), which was coamplified with aerA primers, was also included in this study. The results showed that all cultures of Aeromonas were accurately identified by the assay without showing non-specificity. A. hydrophila could be detected at a range of 10-50 CFU ml(-1) from experimentally spiked fish, chicken and milk samples following overnight enrichment in alkaline peptone water supplemented with 10 μg/ml ampicillin (APW-A) by this multiplex PCR (mPCR). When evaluated on a total of 74 naturally occurring food samples, four samples were identified to contain Aeromonas by mPCR. All these results were compared to the conventional culture, isolation and biochemical identification procedures. The high throughput and cost-effective mPCR method developed in this study could provide a powerful tool for detection of pathogenic Aeromonas spp. from food and environmental samples and in addition, the method has advantages in terms of specificity, sensitivity and ease of use compared to other reported PCR methods and DNA hybridization assays. PMID:23100820

  3. Simultaneous quantification of mitochondrial DNA copy number and deletion ratio: A multiplex real-time PCR assay

    PubMed Central

    Phillips, Nicole R.; Sprouse, Marc L.; Roby, Rhonda K.

    2014-01-01

    Mitochondrial dysfunction is implicated in a vast array of diseases and conditions, such as Alzheimer's disease, cancer, and aging. Alterations in mitochondrial DNA (mtDNA) may provide insight into the processes that either initiate or propagate this dysfunction. Here, we describe a unique multiplex assay which simultaneously provides assessments of mtDNA copy number and the proportion of genomes with common large deletions by targeting two mitochondrial sites and one nuclear locus. This probe-based, single-tube multiplex provides high specificity while eliminating well-to-well variability that results from assaying nuclear and mitochondrial targets individually. PMID:24463429

  4. Multiplexed lateral flow microarray assay for detection of citrus pathogens Xylella fastidiosa and Xanthomonas axonopodis pv citri

    DOEpatents

    Cary; R. Bruce; Stubben, Christopher J.

    2011-03-22

    The invention provides highly sensitive and specific assays for the major citrus pathogens Xylella fastidiosa and Xanthomonas axonopodis, including a field deployable multiplexed assay capable of rapidly assaying for both pathogens simultaneously. The assays are directed at particular gene targets derived from pathogenic strains that specifically cause the major citrus diseases of citrus variegated chlorosis (Xylella fastidiosa 9a5c) and citrus canker (Xanthomonas axonopodis pv citri). The citrus pathogen assays of the invention offer femtomole sensitivity, excellent linear dynamic range, and rapid and specific detection.

  5. Development of a rapid multiplexed assay for the direct screening of antimicrobial residues in raw milk.

    PubMed

    McGrath, Terry F; McClintock, Laura; Dunn, John S; Husar, Gregory M; Lochhead, Michael J; Sarver, Ronald W; Klein, Frank E; Rice, Jennifer A; Campbell, Katrina; Elliott, Christopher T

    2015-06-01

    Antimicrobial residues found to be present in milk can have both health and economic impacts. For these reasons, the widespread routine testing of milk is required. Due to delays with sample handling and test scheduling, laboratory-based tests are not always suited for making decisions about raw material intake and product release, especially when samples require shipping to a central testing facility. Therefore, rapid on-site screening tests that can produce results within a matter of minutes are required to facilitate rapid intake and product release processes. Such tests must be simple for use by non-technical staff. There is increasing momentum towards the development and implementation of multiplexing tests that can detect a range of important antimicrobial residues simultaneously. A simple in situ multiplexed planar waveguide device that can simultaneously detect chloramphenicol, streptomycin and desfuroylceftiofur in raw dairy milk, without sample preparation, has been developed. Samples are simply mixed with antibody prior to an aliquot being passed through the detection cartridge for 5 min before reading on a field-deployable portable instrument. Multiplexed calibration curves were produced in both buffer and raw milk. Buffer curves, for chloramphenicol, streptomycin and desfuroylceftiofur, showed linear ranges (inhibitory concentration (IC)20-IC80) of 0.1-0.9, 3-129 and 12-26 ng/ml, whilst linear range in milk was 0.13-0.74, 11-376 and 2-12 ng/ml, respectively, thus meeting European legislated concentration requirements for both chloramphenicol and streptomycin, in milk, without the need for any sample preparation. Desfuroylceftiofur-contaminated samples require only simple sample dilution to bring positive samples within the range of quantification. Assay repeatability and reproducibility were lower than 12 coefficient of variation (%CV), whilst blank raw milk samples (n = 9) showed repeatability ranging between 4.2 and 8.1%CV when measured on all

  6. Identification of Candidate Reference Genes in Perennial Ryegrass for Quantitative RT-PCR under Various Abiotic Stress Conditions

    PubMed Central

    Jiang, Xiaomei; Yin, Guohua; Zhang, Xinquan; Qi, Xiao; Zhang, Yu; Yan, Yanhong; Ma, Xiao; Peng, Yan

    2014-01-01

    Background Quantitative real-time reverse-transcriptase PCR (qRT-PCR) is an important technique for analyzing differences in gene expression due to its sensitivity, accuracy and specificity. However, the stability of the expression of reference genes is necessary to ensure accurate qRT-PCR assessment of expression in genes of interest. Perennial ryegrass (Lolium perenne L.) is important forage and turf grass species in temperate regions, but the expression stability of its reference genes under various stresses has not been well-studied. Methodology/Principal Findings In this study, 11 candidate reference genes were evaluated for use as controls in qRT-PCR to quantify gene expression in perennial ryegrass under drought, high salinity, heat, waterlogging, and ABA (abscisic acid) treatments. Four approaches – Delta CT, geNorm, BestKeeper and Normfinder were used to determine the stability of expression in these reference genes. The results are consistent with the idea that the best reference genes depend on the stress treatment under investigation. Eukaryotic initiation factor 4 alpha (eIF4A), Transcription elongation factor 1 (TEF1) and Tat binding protein-1 (TBP-1) were the three most stably expressed genes under drought stress and were also the three best genes for studying salt stress. eIF4A, TBP-1, and Ubiquitin-conjugating enzyme (E2) were the most suitable reference genes to study heat stress, while eIF4A, TEF1, and E2 were the three best reference genes for studying the effects of ABA. Finally, Ubiquitin (UBQ), TEF1, and eIF4A were the three best reference genes for waterlogging treatments. Conclusions/Significance These results will be helpful in choosing the best reference genes for use in studies related to various abiotic stresses in perennial ryegrass. The stability of expression in these reference genes will enable better normalization and quantification of the transcript levels for studies of gene expression in such studies. PMID:24699822

  7. Cloning of a chub metallothionein cDNA and development of competitive RT-PCR of chub metallothionein mRNA as a potential biomarker of heavy metal exposure.

    PubMed

    Hayes, Ruth A; Regondi, Simona; Winter, Matthew J; Butler, Patrick J; Agradi, Elisabetta; Taylor, Edwin W; Kevin Chipman, J

    2004-01-01

    Metallothionein has been assayed in a range of aquatic animal tissues as an indicator of metal exposure. We sequenced chub (Leuciscus cephalus) metallothionein cDNA which showed over 90% homology to common carp, goldfish and stone loach and 77% homology to rainbow trout sequences for metallothionein. We then used the extended primer method to develop an accurate quantitative competitive RT-PCR assay for metallothionein mRNA. RT-PCR was used to measure metallothionein mRNA in feral chub from a range of field sites, with different levels of heavy metal pollution, in the West Midlands, UK. Measurements were complemented by analysis of liver and gill metallothionein protein by capillary electrophoresis. There was no significant difference in the metallothionein protein levels between fish of different rivers and there was no evidence of elevation of mRNA at the sites of highest metal exposure. The level of metal exposure (e.g. zinc, nickel and cadmium each ranging between 15 and 28 microg/l ) at the pH (7.5-8.5) of these rivers appears insufficient to elevate hepatic or gill metallothionein in chub. A lack of elevation of hepatic metallothionein mRNA in chub exposed to zinc, copper and manganese for 24 h and 10 days in the laboratory also suggests a non-responsiveness of this species. PMID:15178096

  8. RT-PCR detection of CYP1A1, 1A2, and 2E1 mRNAs in rat nasal tissue

    SciTech Connect

    Reddy, S.L.; Kim, S.G.; States, J.C.; Dahl, A.R.; Hotchkiss, J.; Novak, R.F. Lovelace Biomedical and Environmental Research Inst., Albuquerque, NM )

    1991-03-15

    The expression of P450 in nasal tissue is of considerable importance given the exposure of these tissues to xenobiotics and the role of P450s in xenobiotic metabolism. CYP1A1, 1A2 and 2E1 mRNA expression was examined in olfactory tissue of rats exposed to 5 ppm pyridine 6 h daily for 4 d. RT-PCR was performed on poly(A){sup +} RNA using gene specific primers selected from published rat liver 1A1, 1A2 and 2E1 cDNAs. RT-PCR products derived from nasal mRNAs were detected and co-migrated with liver 1A1, 1A2 and 2W1 Rt-PCR products. Identical restriction patterns were obtained from HinfI and HpaII digests of nasal and liver 1A1 RT-PCR products; restriction digest patterns of nasal and liver 1A2 RT-PCR products were also identical. Southern analyses of nasal RT-PCR products, using liver 1A1 and 12 DNA probes, showed a single band suggesting considerable homology between nasal and liver 1A1 and 1A2 fragments. Cloning and sequencing of nasal 1A1, 1A2 and 2E1 RT-PCR products will confirm the identity of these gene products. These results show that 1A1, 1A2 and 2E1 mRNAs are expressed in rat olfactory tissue and suggest that the fragments examined share homology with those expressed in liver.

  9. Development of a Rift Valley fever real-time RT-PCR assay that can detect all three genome segments

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  10. Identification and Differentiation of Verticillium Species and V. longisporum Lineages by Simplex and Multiplex PCR Assays

    PubMed Central

    Inderbitzin, Patrik; Davis, R. Michael; Bostock, Richard M.; Subbarao, Krishna V.

    2013-01-01

    Accurate species identification is essential for effective plant disease management, but is challenging in fungi including Verticillium sensu stricto (Ascomycota, Sordariomycetes, Plectosphaerellaceae), a small genus of ten species that includes important plant pathogens. Here we present fifteen PCR assays for the identification of all recognized Verticillium species and the three lineages of the diploid hybrid V. longisporum. The assays were based on DNA sequence data from the ribosomal internal transcribed spacer region, and coding and non-coding regions of actin, elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase and tryptophan synthase genes. The eleven single target (simplex) PCR assays resulted in amplicons of diagnostic size for V. alfalfae, V. albo-atrum, V. dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii, V. nonalfalfae, V. nubilum, V. tricorpus, V. zaregamsianum, and Species A1 and Species D1, the two undescribed ancestors of V. longisporum. The four multiple target (multiplex) PCR assays simultaneously differentiated the species or lineages within the following four groups: Verticillium albo-atrum, V. alfalfae and V. nonalfalfae; Verticillium dahliae and V. longisporum lineages A1/D1, A1/D2 and A1/D3; Verticillium dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii and V. tricorpus; Verticillium isaacii, V. klebahnii and V. tricorpus. Since V. dahliae is a parent of two of the three lineages of the diploid hybrid V. longisporum, no simplex PCR assay is able to differentiate V. dahliae from all V. longisporum lineages. PCR assays were tested with fungal DNA extracts from pure cultures, and were not evaluated for detection and quantification of Verticillium species from plant or soil samples. The DNA sequence alignments are provided and can be used for the design of additional primers. PMID:23823707

  11. Automated Microchromatography Enables Multiplexing of Immunoaffinity Enrichment of Peptides to Greater than 150 for Targeted MS-Based Assays.

    PubMed

    Ippoliti, Paul J; Kuhn, Eric; Mani, D R; Fagbami, Lola; Keshishian, Hasmik; Burgess, Michael W; Jaffe, Jacob D; Carr, Steven A

    2016-08-01

    Immunoaffinity enrichment of peptides coupled with analysis by stable isotope dilution multiple reaction mass spectrometry has been shown to have analytical performance and detection limits suitable for many biomarker verification studies and biological applications. Prior studies have shown that antipeptide antibodies can be multiplexed up to 50 in a single assay without significant loss of performance. Achieving higher multiplex levels is relevant to all studies involving precious biological material as this minimizes the amount of sample that must be consumed to measure a given set of analytes and reduces the assay cost per analyte. Here we developed automated methods employing the Agilent AssayMAP Bravo microchromatography platform and used these methods to characterize the performance of immunoaffinity enrichment of peptides up to multiplex levels of 172. Median capture efficiency for the target peptides remained high (88%) even at levels of 150-plex and declined to 70% at 172-plex compared to antibody performance observed at standard lower multiplex levels (n = 25). Subsequently, we developed and analytically characterized a multiplexed immuno-multiple reaction monitoring-mass spectrometry (immuno-MRM-MS) assay (n = 110) and applied it to measure candidate protein biomarkers of cardiovascular disease in plasma of patients undergoing planned myocardial infarction. The median lower limit of detection of all peptides was 71.5 amol/μL (nM), and the coefficient of variation (CV) was less than 15% at the lower limit of quantification. The results demonstrate that high multiplexed immuno-MRM-MS assays are readily achievable using the optimized sample processing and peptide capture methods described here. PMID:27321643

  12. Performance of a one-step quantitative duplex RT-PCR for detection of rotavirus A and noroviruses GII during two periods of high viral circulation.

    PubMed

    Fumian, Tulio M; Leite, José Paulo G; Rocha, Mônica S; de Andrade, Juliana S R; Fioretti, Julia M; de Assis, Rosane M S; Assis, Matheus R S; Fialho, Alexandre M; Miagostovich, Marize P

    2016-02-01

    Rotavirus A (RVA) and noroviruses (NoV) are the major viral agents of acute gastroenteritis (AGE) worldwide. In the present study, we aimed to evaluate the performance of a one-step duplex quantitative RT-PCR (dRT-qPCR) assay, established for detection and quantification of RVA and NoV genogroup II (GII) using a single DNA standard curve (SC), as well as to investigate the association between fecal viral load and optical density (OD) values, and viruses' genotyping. The results obtained by dRT-qPCR in 530 fecal samples from AGE cases were compared with methods employed for the diagnosis of those viruses as follows: enzyme immunoassay (EIA) and polyacrylamide gel electrophoresis (PAGE) for RVA; and qualitative PCR for NoV. By using dRT-qPCR, we detected RVA and NoV in 353 (66%), increasing the positivity rate by 22.5% for RVA and 11.5% NoV, comparing the number of positive samples. RVA and NoV GII were detected in a range of 5.17 × 10(3) to 6.56 × 10(9) and 3.76 × 10(3) to 9.13 × 10(10) genome copies per gram of feces, respectively. We observed a significant direct correlation between genome copies values and optical density, using dRT-qPCR and EIA assays, respectively (Spearman ρ=0.41; p<0.0001). Viruses characterization demonstrated a predominance of NoV GII.4 Sidney 2012 variant during October 2013 to February 2014, followed by the emergence of RVA genotype G12P[8] in 2014. The established assay using a single SC provides an early feedback concerning detection and quantification, with the advantage of detecting simultaneously RVA and NoV GII, reducing time and reagent costs. PMID:26611226

  13. Image decoding of photonic crystal beads array in the microfluidic chip for multiplex assays.

    PubMed

    Yuan, Junjie; Zhao, Xiangwei; Wang, Xiaoxia; Gu, Zhongze

    2014-01-01

    Along with the miniaturization and intellectualization of biomedical instruments, the increasing demand of health monitoring at anywhere and anytime elevates the need for the development of point of care testing (POCT). Photonic crystal beads (PCBs) as one kind of good encoded microcarriers can be integrated with microfluidic chips in order to realize cost-effective and high sensitive multiplex bioassays. However, there are difficulties in analyzing them towards automated analysis due to the characters of the PCBs and the unique detection manner. In this paper, we propose a strategy to take advantage of automated image processing for the color decoding of the PCBs array in the microfluidic chip for multiplex assays. By processing and alignment of two modal images of epi-fluorescence and epi-white light, every intact bead in the image is accurately extracted and decoded by PC colors, which stand for the target species. This method, which shows high robustness and accuracy under various configurations, eliminates the high hardware requirement of spectroscopy analysis and user-interaction software, and provides adequate supports for the general automated analysis of POCT based on PCBs array. PMID:25341876

  14. Image Decoding of Photonic Crystal Beads Array in the Microfluidic Chip for Multiplex Assays

    NASA Astrophysics Data System (ADS)

    Yuan, Junjie; Zhao, Xiangwei; Wang, Xiaoxia; Gu, Zhongze

    2014-10-01

    Along with the miniaturization and intellectualization of biomedical instruments, the increasing demand of health monitoring at anywhere and anytime elevates the need for the development of point of care testing (POCT). Photonic crystal beads (PCBs) as one kind of good encoded microcarriers can be integrated with microfluidic chips in order to realize cost-effective and high sensitive multiplex bioassays. However, there are difficulties in analyzing them towards automated analysis due to the characters of the PCBs and the unique detection manner. In this paper, we propose a strategy to take advantage of automated image processing for the color decoding of the PCBs array in the microfluidic chip for multiplex assays. By processing and alignment of two modal images of epi-fluorescence and epi-white light, every intact bead in the image is accurately extracted and decoded by PC colors, which stand for the target species. This method, which shows high robustness and accuracy under various configurations, eliminates the high hardware requirement of spectroscopy analysis and user-interaction software, and provides adequate supports for the general automated analysis of POCT based on PCBs array.

  15. Identification of cytoplasm types in rapeseed (Brassica napus L.) accessions by a multiplex PCR assay.

    PubMed

    Zhao, H X; Li, Z J; Hu, S W; Sun, G L; Chang, J J; Zhang, Z H

    2010-08-01

    Cytoplasmic male sterility (CMS) has widely been used as an efficient pollination control system in rapeseed hybrid production. Identification of cytoplasm type of rapeseed accessions is becoming the most important basic work for hybrid-rapeseed breeding. In this study, we report a simple multiplex PCR method to distinguish the existing common cytoplasm resources, Pol, Nap, Cam, Ogu and Ogu-NWSUAF cytoplasm, in rapeseed. Cytoplasm type of 35 F(1) hybrids and 140 rapeseed open pollinated varieties or breeding lines in our rapeseed breeding programme were tested by this method. The results indicated that 10 of 35 F(1) hybrids are the Nap, and 25 the Pol cytoplasm type, which is consistent with the information provided by the breeders. Out of 140 accessions tested, 100 (71.4%), 21 (15%) and 19 (13.6%) accessions possess Nap, Cam and Pol cytoplasm, respectively. All 19 accessions with Pol cytoplasm are from China. Pedigree analysis indicated that these accessions with Pol cytoplasm were either restorers for Pol CMS, including Shaan 2C, Huiyehui, 220, etc. or derived from hybrids with Pol CMS as female parent. Our molecular results are consistent with those of the classical testcross, suggesting the reliability of this method. The multiplex PCR assay method can be applied to CMS "three-line" breeding, selection and validation of hybrid rapeseed. PMID:20401459

  16. Image Decoding of Photonic Crystal Beads Array in the Microfluidic Chip for Multiplex Assays

    PubMed Central

    Yuan, Junjie; Zhao, Xiangwei; Wang, Xiaoxia; Gu, Zhongze

    2014-01-01

    Along with the miniaturization and intellectualization of biomedical instruments, the increasing demand of health monitoring at anywhere and anytime elevates the need for the development of point of care testing (POCT). Photonic crystal beads (PCBs) as one kind of good encoded microcarriers can be integrated with microfluidic chips in order to realize cost-effective and high sensitive multiplex bioassays. However, there are difficulties in analyzing them towards automated analysis due to the characters of the PCBs and the unique detection manner. In this paper, we propose a strategy to take advantage of automated image processing for the color decoding of the PCBs array in the microfluidic chip for multiplex assays. By processing and alignment of two modal images of epi-fluorescence and epi-white light, every intact bead in the image is accurately extracted and decoded by PC colors, which stand for the target species. This method, which shows high robustness and accuracy under various configurations, eliminates the high hardware requirement of spectroscopy analysis and user-interaction software, and provides adequate supports for the general automated analysis of POCT based on PCBs array. PMID:25341876

  17. Generic and sequence-variant specific molecular assays for the detection of the highly variable Grapevine leafroll-associated virus 3.

    PubMed

    Chooi, Kar Mun; Cohen, Daniel; Pearson, Michael N

    2013-04-01

    Grapevine leafroll-associated virus 3 (GLRaV-3) is an economically important virus, which is found in all grapevine growing regions worldwide. Its accurate detection in nursery and field samples is of high importance for certification schemes and disease management programmes. To reduce false negatives that can be caused by sequence variability, a new universal primer pair was designed against a divergent sequence data set, targeting the open reading frame 4 (heat shock protein 70 homologue gene), and optimised for conventional one-step RT-PCR and one-step SYBR Green real-time RT-PCR assays. In addition, primer pairs for the simultaneous detection of specific GLRaV-3 variants from groups 1, 2, 6 (specifically NZ-1) and the outlier NZ2 variant, and the generic detection of variants from groups 1 to 5 were designed and optimised as a conventional one-step multiplex RT-PCR assay using the plant nad5 gene as an internal control (i.e. one-step hexaplex RT-PCR). Results showed that the generic and variant specific assays detected in vitro RNA transcripts from a range of 1×10(1)-1×10(8) copies of amplicon per μl diluted in healthy total RNA from Vitis vinifera cv. Cabernet Sauvignon. Furthermore, the assays were employed effectively to screen 157 germplasm and 159 commercial field samples. Thus results demonstrate that the GLRaV-3 generic and variant-specific assays are prospective tools that will be beneficial for certification schemes and disease management programmes, as well as biological and epidemiological studies of the divergent GLRaV-3 populations. PMID:23313884

  18. Nucleic acid extraction from polluted estuarine water for detection of viruses and bacteria by PCR and RT-PCR analysis.

    PubMed

    Petit, F; Craquelin, S; Guespin-Michel, J; Buffet-Janvresse, C

    1999-03-01

    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. PMID:10209769

  19. A hard microflow cytometer using groove-generated sheath flow for multiplexed bead and cell assays

    PubMed Central

    Thangawng, Abel L.; Kim, Jason S.; Golden, Joel P.; Anderson, George P.; Robertson, Kelly L.; Low, Vyechi

    2010-01-01

    With a view toward developing a rugged microflow cytometer, a sheath flow system was micromachined in hard plastic (polymethylmethacrylate) for analysis of particles and cells using optical detection. Six optical fibers were incorporated into the interrogation region of the chip, in which hydrodynamic focusing narrowed the core stream to ∼35 μm×40 μm. The use of a relatively large channel at the inlet as well as in the interrogation region (375 μm×125 μm) successfully minimized the risk of clogging. The device could withstand pressures greater than 100 psi without leaking. Assays using both coded microparticles and cells were demonstrated using the microflow cytometer. Multiplexed immunoassays detected nine different bacteria and toxins using a single mixture of coded microspheres. A549 cancer cells processed with locked nucleic acid probes were evaluated using fluorescence in situ hybridization. PMID:20658281

  20. Robustness of Equations that Define Molecular Subtypes of Glioblastoma Tumors Based on Five Transcripts Measured by RT-PCR

    PubMed Central

    Castells, Xavier; Acebes, Juan José; Majós, Carles; Boluda, Susana; Julià-Sapé, Margarida; Candiota, Ana Paula; Ariño, Joaquín; Barceló, Anna

    2015-01-01

    Abstract Glioblastoma (Gb) is one of the most deadly tumors. Its molecular subtypes are yet to be fully characterized while the attendant efforts for personalized medicine need to be intensified in relation to glioblastoma diagnosis, treatment, and prognosis. Several molecular signatures based on gene expression microarrays were reported, but the use of microarrays for routine clinical practice is challenged by attendant economic costs. Several authors have proposed discriminant equations based on RT-PCR. Still, the discriminant threshold is often incompletely described, which makes proper validation difficult. In a previous work, we have reported two Gb subtypes based on the expression levels of four genes: CHI3L1, LDHA, LGALS1, and IGFBP3. One Gb subtype presented with low expression of the four genes mentioned, and of MGMT in a large portion of the patients (with anticipated high methylation of its promoter), and mutated IDH1. Here, we evaluate the robustness of the equations fitted with these genes using RT-PCR values in a set of 64 cases and importantly, define an unequivocal discriminant threshold with a view to prognostic implications. We developed two approaches to generate the discriminant equations: 1) using the expression level of the four genes mentioned above, and 2) using those genes displaying the highest correlation with survival among the aforementioned four ones, plus MGMT, as an attempt to further reduce the number of genes. The ease of equations' applicability, reduction in cost for raw data, and robustness in terms of resampling-based classification accuracy warrant further evaluation of these equations to discern Gb tumor biopsy heterogeneity at molecular level, diagnose potential malignancy, and prognosis of individual patients with glioblastomas. PMID:25562199

  1. Evaluation and Selection of Candidate Reference Genes for Normalization of Quantitative RT-PCR in Withania somnifera (L.) Dunal

    PubMed Central

    Singh, Varinder; Kaul, Sunil C.; Wadhwa, Renu; Pati, Pratap Kumar

    2015-01-01

    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

  2. Evaluation and selection of candidate reference genes for normalization of quantitative RT-PCR in Withania somnifera (L.) Dunal.

    PubMed

    Singh, Varinder; Kaul, Sunil C; Wadhwa, Renu; Pati, Pratap Kumar

    2015-01-01

    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

  3. Overexpression of vascular endothelial growth factor and its receptors in bronchial dypslasia demonstrated by quantitative RT-PCR analysis.

    PubMed

    Merrick, Daniel T; Haney, Jerry; Petrunich, Sheila; Sugita, Michio; Miller, York E; Keith, Robert L; Kennedy, Tim C; Franklin, Wilbur A

    2005-04-01

    Neoangiogenesis is required for the growth of invasive lung carcinoma, however, the role of angiogenesis in the progression of premalignant changes to carcinoma of the lung is less clear. We have evaluated vascular endothelial growth factor (VEGF) expression and microvessel densities (MVDs) in 62 bronchoscopic biopsies from normal, reactive (basal cell hyperplasia (BCH)) and dysplastic bronchial epithelium and in tissue from twenty-seven invasive lung carcinomas in an effort to demonstrate angiogenic activity in these preneoplastic lesions and determine whether it is associated with increased bronchial epithelial VEGF expression. MVDs and VEGF RNA expression measured by quantitative RT-PCR were found to be elevated in comparison to normal bronchial tissue in bronchial dysplasias and invasive lung carcinomas but not in basal cell hyperplasias. Immunohistochemical (IHC) analyses revealed that expression of VEGF arose predominantly from bronchial epithelium. ELISA analysis of lung tumor tissue showed that elevated VEGF protein expression correlated with VEGF RNA levels (r=0.59, p=0.004). Increased expression of VEGF RNA was also found in histologically normal bronchial mucosa from patients with either dysplasia at other sites or a history of heavy tobacco use suggesting a possible field effect in regard to the elaboration of VEGF. Furthermore, analysis of VEGF isoforms and VEGF receptors by semi-quantitative RT-PCR in dysplastic and invasive lesions revealed characteristic altered patterns of expression in dysplasia and early cancer as compared to normal tissue. These results indicate that angiogenesis develops early in lung carcinogenesis and is associated with overexpression of VEGF. PMID:15777969

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  5. Development of a multiplex bead-based assay to monitor dengue virus seroconversion.

    PubMed

    Ng, Kaiting; Connolly, John E

    2014-01-01

    Dengue virus (DENV) envelope protein is responsible for viral attachment to host cells and as such is a target of neutralizing antibody responses. However, the presence of envelope-specific antibodies against a given serotype may contribute to enhanced disease during secondary infection with another serotype. There is a need therefore for a standardized, high-throughput low-volume assay which permits the simultaneous screening of reactivity to multiple DENV serotypes. Here, we describe a method of identifying DENV serotype-specific response in exposed individuals using a multiplexed bead-based immunoassay. The ED3 domain of a specific DENV serotype is cloned into pQEAM containing 6xHIS, TEV protease, and AviTag biotinylation sites. Biotinylated ED3 proteins are expressed in E. coli CVB101 and purified by sequential column fractionation followed by coupling onto fluorescent avidin-coated microspheres. Methods for determining the optimum amount of biotinylated ED3 protein coupled onto the microsphere are described. The assay demonstrates both a high degree of sensitivity and specificity using well-characterized patient plasma samples. The nature of the assay permits further development to include a variety of DENV serotypes and regionally important sub-serotypes. PMID:24696331

  6. A neutralization assay for chikungunya virus infections in a multiplex format.

    PubMed

    Weber, Christopher; König, Renate; Niedrig, Matthias; Emmerich, Petra; Schnierle, Barbara S

    2014-06-01

    Chikungunya virus (CHIKV) is a mosquito-transmitted Alphavirus that causes chikungunya fever and has infected millions of people mainly in developing countries. The associated disease is characterized by rash, high fever and severe arthritis that can persist for years. Since the epidemic on La Réunion in 2006, CHIKV has adapted to Aedes albopictus, which also inhabits temperate regions of the eastern and western hemispheres, including Europe and the United States. A. albopictus might continue migrating north with continuing climate change and CHIKV would then no longer be confined to the developing nations. No treatment or licensed CHIKV vaccine exists. A CHIKV neutralization assay in a 384-well format by using CHIKV-pseudotyped lentiviral vectors was established. This assay system can be used for entry inhibitor screening under a reduced safety level (S2). Production of CHIKV-pseudotyped lentiviral vectors and the reaction volume are optimized. A dose dependent, specific neutralization of CHIKV-pseudotyped vectors with sera of CHIKV-infected individuals could be measured in a 384-well format. A safe and simple multiplex assay for the analysis of CHIKV neutralizing activities was developed and will be able to improve drug and vaccine development as well as it would improve the understanding of CHIKV epidemics regarding antibody responses. PMID:24552952

  7. Diagnosis of Upper and Lower Respiratory Tract Bacterial Infections with the Use of Multiplex PCR Assays

    PubMed Central

    Xirogianni, Athanasia; Tsolia, Maria; Voyiatzi, Aliki; Sioumala, Maria; Makri, Antonia; Argyropoulou, Athina; Paniara, Olga; Markoulatos, Panayotis; Kourea-Kremastinou, Jenny; Tzanakaki, Georgina

    2013-01-01

    The investigation of respiratory infections by molecular techniques provides important information about the epidemiology of respiratory disease, especially during the post-vaccination era. The objective of the present study was the detection of bacterial pathogens directly in clinical samples from patients with upper and lower respiratory tract infections using multiplex polymerase chain reaction (PCR) assays developed in our laboratory. Clinical samples taken over a three-year period (2007–2009) and obtained from 349 patients (adults (n = 66); children (n = 283)) with signs and symptoms of certain upper or lower respiratory tract infections, consisted of: bronchoalveolar lavages (BAL, n = 83), pleural fluids (n = 29), and middle-ear aspirates (n = 237). Overall, 212 samples (61%) were confirmed by culture and/or PCR. Among the positive samples, Streptococcus pneumoniae (mainly serotype 3) was predominant (104/212; 49.0%), followed by non-typable Haemophilus influenzae (NTHi) 59/212; 27.8%) and Streptococcus pyogenes (47/212; 22%). Haemophilus influenzae type b was detected in only three samples. The underlying microbiology of respiratory infections is gradually changing in response to various selective pressures, such as vaccine use and antibiotic consumption. The application of multiplex PCR (mPCR) assays is particularly useful since it successfully identified the microorganisms implicated in acute otitis media or lower respiratory tract infections in nearly 75% of patients with a positive result compared to conventional cultures. Non-culture identification of the implicated pneumococcal serotypes is also an important issue for monitoring pneumococcal infections in the era of conjugate pneumococcal vaccines. PMID:26835676

  8. Development of a multiplex PCR assay for rapid virulence factor profiling of extraintestinal pathogenic Escherichia coli isolated from cattle.

    PubMed

    Ojima, Toru; Hirano, Kaori; Honda, Kohei; Kusumoto, Masahiro

    2016-09-01

    Virulence factor (VF) profiling is important for the control of extraintestinal pathogenic Escherichia coli (ExPEC) infection because VF prevalence is highly variable. We analyzed the VF profile of ExPEC isolated from cattle in Yamagata prefecture, Japan, 2000-2015 and developed a rapid VF profiling method using a multiplex PCR assay. PMID:27380962

  9. Human tear analysis with miniaturized multiplex cytokine assay on “wall-less” 96-well plate

    PubMed Central

    Quah, Joanne; Tong, Louis; Kim, Namyong

    2015-01-01

    Purpose Tears are a particularly limited body fluid and commonly used in the diagnosis of patients who have ocular diseases. A popular method for analysis of ocular inflammation in tears uses Luminex® bead multiplex technology to generate valuable multiple cytokine profile outputs with 25–50 µl tear sample volume. We propose a method for measuring tear cytokines with 5 μl tear sample volume and 80% reduced Luminex reagents compared to previous protocols. Methods Using human tears pooled from 1,000 participants, the DA-Bead-based method running at 5–20 µl volume, using manual pipetting, in conjunction with a magnetic Luminex cytokine (four-plex) panel assay in a 96-well format was performed and validated for tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-1β, and IL-6. Results Upon use of the DA-Bead method at the 5 μl volume with cytokine standards, the concentrations of each of the four cytokines were found to be linear over a range of 3.5–4 log pg/ml with an intra-assay coefficient of variation (CV) ≤5%, inter-assay %CV ≤10%, and accuracy within the 70–130% range. Upon use of a 5 µl healthy pooled tear sample, cytokine concentrations were detected with a precision intra-assay %CV ˂ 20% for IL-6, IFN-γ, or TNF-α or 30.37% with IL-1β. The inter-assay %CV with tears was ≤20.84% for all cytokines. Tear volumes run at 5 μl on DA-Bead produced a similar cytokine expression profile at a 1-month interval and were highly correlated with the larger 10 μl–based tear sample volume cytokine profile with R2 = 0.98. Conclusions DA-Bead assay is highly sensitive and reproducible and has a performance profile that is potentially suitable for use in standard clinical scenarios. Considering the use of as little as 5 µl of assay beads and 5 µl sample, this is also likely to reduce the assay cost significantly and ease diagnosis of patients with ocular diseases. PMID:26539027

  10. A Pan-Dengue Virus Reverse Transcription-Insulated Isothermal PCR Assay Intended for Point-of-Need Diagnosis of Dengue Virus Infection by Use of the POCKIT Nucleic Acid Analyzer.

    PubMed

    Go, Yun Young; Rajapakse, R P V Jayanthe; Kularatne, Senanayake A M; Lee, Pei-Yu Alison; Ku, Keun Bon; Nam, Sangwoo; Chou, Pin-Hsing; Tsai, Yun-Long; Liu, Yu-Lun; Chang, Hsiao-Fen Grace; Wang, Hwa-Tang Thomas; Balasuriya, Udeni B R

    2016-06-01

    Dengue virus (DENV) infection is considered a major public health problem in developing tropical countries where the virus is endemic and continues to cause major disease outbreaks every year. Here, we describe the development of a novel, inexpensive, and user-friendly diagnostic assay based on a reverse transcription-insulated isothermal PCR (RT-iiPCR) method for the detection of all four serotypes of DENV in clinical samples. The diagnostic performance of the newly established pan-DENV RT-iiPCR assay targeting a conserved 3' untranslated region of the viral genome was evaluated. The limit of detection with a 95% confidence was estimated to be 10 copies of in vitro-transcribed (IVT) RNA. Sensitivity analysis using RNA prepared from 10-fold serial dilutions of tissue culture fluid containing DENVs suggested that the RT-iiPCR assay was comparable to the multiplex real-time quantitative RT-PCR (qRT-PCR) assay for DENV-1, -3, and -4 detection but 10-fold less sensitive for DENV-2 detection. Subsequently, plasma collected from patients suspected of dengue virus infection (n = 220) and individuals not suspected of dengue virus infection (n = 45) were tested by the RT-iiPCR and compared to original test results using a DENV NS1 antigen rapid test and the qRT-PCR. The diagnostic agreement of the pan-DENV RT-iiPCR, NS1 antigen rapid test, and qRT-PCR tests was 93.9%, 84.5%, and 97.4%, respectively, compared to the composite reference results. This new RT-iiPCR assay along with the portable POCKIT nucleic acid analyzer could provide a highly reliable, sensitive, and specific point-of-need diagnostic assay for the diagnosis of DENV in clinics and hospitals in developing countries. PMID:27030492

  11. Development of multiplex loop-mediated isothermal amplification assays to detect medically important yeasts in dairy products.

    PubMed

    Kasahara, Kohei; Ishikawa, Hiroshi; Sato, Sumie; Shimakawa, Yasuhisa; Watanabe, Koichi

    2014-08-01

    Rapid detection of yeast contamination is important in the food industry. We have developed loop-mediated isothermal amplification (LAMP) assays to detect the emerging opportunistic pathogenic yeasts: Candida albicans, Candida glabrata, Candida tropicalis, the Candida parapsilosis group, Trichosporon asahii, and Trichosporon mucoides. These yeasts may cause deep-seated candidiasis or trichosporonosis. Four LAMP primer sets specific for Candida were designed to target the internal transcribed spacer 2 (ITS2) region between the 5.8S and 26S rRNA genes, and two LAMP primer sets specific for Trichosporon were designed to target the intergenic spacer 1 (IGS1) region between the 26S and 5S rRNA genes. The LAMP assays could detect these yeasts in a range between 10(0) and 10(3)  cells mL(-1) in a contaminated dairy product within 1 h. We also developed multiplex LAMP assays to detect these Candida or Trichosporon species in a single reaction. Multiplex LAMP assays can detect contamination if at least one of the target species is present; they are more time- and cost-efficient than conventional methods and could detect target yeasts with sensitivity close to that of the LAMP assays. Multiplex LAMP assays established in this study can be used as a primary screening method for yeast contamination in food products. PMID:24965944

  12. Comparison of a Real-Time Multiplex PCR and Sequetyping Assay for Pneumococcal Serotyping

    PubMed Central

    Robberts, Lourens; Wolter, Nicole; Nicol, Paul; Mafofo, Joseph; Africa, Samantha; Zar, Heather J.; Nicol, Mark P.

    2015-01-01

    Background Pneumococcal serotype identification is essential to monitor pneumococcal vaccine effectiveness and serotype replacement. Serotyping by conventional serological methods are costly, labour-intensive, and require significant technical expertise. We compared two different molecular methods to serotype pneumococci isolated from the nasopharynx of South African infants participating in a birth cohort study, the Drakenstein Child Health Study, in an area with high 13-valent pneumococcal conjugate vaccine (PCV13) coverage. Methods A real-time multiplex PCR (rmPCR) assay detecting 21 different serotypes/-groups and a sequetyping assay, based on the sequence of the wzh gene within the pneumococcal capsular locus, were compared. Forty pneumococcal control isolates, with serotypes determined by the Quellung reaction, were tested. In addition, 135 pneumococcal isolates obtained from the nasopharynx of healthy children were tested by both serotyping assays and confirmed by Quellung testing. Discordant results were further investigated by whole genome sequencing of four isolates. Results Of the 40 control isolates tested, 25 had a serotype covered by the rmPCR assay. These were all correctly serotyped/-grouped. Sequetyping PCR failed in 7/40 (18%) isolates. For the remaining isolates, sequetyping assigned the correct serotype/-group to 29/33 (88%) control isolates. Of the 132/135 (98%) nasopharyngeal pneumococcal isolates that could be typed, 69/132 (52%) and 112/132 (85%) were assigned the correct serotype/-group by rmPCR and sequetyping respectively. The serotypes of 63/132 (48%) isolates were not included in the rmPCR panel. All except three isolates (serotype 25A and 38) were theoretically amplified and differentiated into the correct serotype/-group with some strains giving ambigous results (serotype 13/20, 17F/33C, and 11A/D/1818F). Of the pneumococcal serotypes detected in this study, 69/91 (76%) were not included in the current PCV13. The most frequently

  13. The Use of NS1 Rapid Diagnostic Test and qRT-PCR to Complement IgM ELISA for Improved Dengue Diagnosis from Single Specimen.

    PubMed

    Teoh, Boon-Teong; Sam, Sing-Sin; Tan, Kim-Kee; Johari, Jefree; Abd-Jamil, Juraina; Hooi, Poh-Sim; AbuBakar, Sazaly

    2016-01-01

    Timely and accurate dengue diagnosis is important for differential diagnosis and immediate implementation of appropriate disease control measures. In this study, we compared the usefulness and applicability of NS1 RDT (NS1 Ag Strip) and qRT-PCR tests in complementing the IgM ELISA for dengue diagnosis on single serum specimen (n = 375). The NS1 Ag Strip and qRT-PCR showed a fair concordance (κ = 0.207, p = 0.001). While the NS1 Ag Strip showed higher positivity than qRT-PCR for acute (97.8% vs. 84.8%) and post-acute samples (94.8% vs. 71.8%) of primary infection, qRT-PCR showed higher positivity for acute (58.1% vs. 48.4%) and post-acute (50.0% vs.41.4%) samples in secondary infection. IgM ELISA showed higher positivity in samples from secondary dengue (74.2-94.8%) than in those from primary dengue (21.7-64.1%). More primary dengue samples showed positive with combined NS1 Ag Strip/IgM ELISA (99.0% vs. 92.8%) whereas more secondary samples showed positive with combined qRT-PCR/IgM ELISA (99.4% vs. 96.2%). Combined NS1 Ag Strip/IgM ELISA is a suitable combination tests for timely and accurate dengue diagnosis on single serum specimen. If complemented with qRT-PCR, combined NS1 Ag Strip/IgM ELISA would improve detection of secondary dengue samples. PMID:27278716

  14. Using Rapid Diagnostic Tests as a Source of Viral RNA for Dengue Serotyping by RT-PCR - A Novel Epidemiological Tool

    PubMed Central

    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

    2016-01-01

    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

  15. The Use of NS1 Rapid Diagnostic Test and qRT-PCR to Complement IgM ELISA for Improved Dengue Diagnosis from Single Specimen

    PubMed Central

    Teoh, Boon-Teong; Sam, Sing-Sin; Tan, Kim-Kee; Johari, Jefree; Abd-Jamil, Juraina; Hooi, Poh-Sim; AbuBakar, Sazaly

    2016-01-01

    Timely and accurate dengue diagnosis is important for differential diagnosis and immediate implementation of appropriate disease control measures. In this study, we compared the usefulness and applicability of NS1 RDT (NS1 Ag Strip) and qRT-PCR tests in complementing the IgM ELISA for dengue diagnosis on single serum specimen (n = 375). The NS1 Ag Strip and qRT-PCR showed a fair concordance (κ = 0.207, p = 0.001). While the NS1 Ag Strip showed higher positivity than qRT-PCR for acute (97.8% vs. 84.8%) and post-acute samples (94.8% vs. 71.8%) of primary infection, qRT-PCR showed higher positivity for acute (58.1% vs. 48.4%) and post-acute (50.0% vs.41.4%) samples in secondary infection. IgM ELISA showed higher positivity in samples from secondary dengue (74.2–94.8%) than in those from primary dengue (21.7–64.1%). More primary dengue samples showed positive with combined NS1 Ag Strip/IgM ELISA (99.0% vs. 92.8%) whereas more secondary samples showed positive with combined qRT-PCR/IgM ELISA (99.4% vs. 96.2%). Combined NS1 Ag Strip/IgM ELISA is a suitable combination tests for timely and accurate dengue diagnosis on single serum specimen. If complemented with qRT-PCR, combined NS1 Ag Strip/IgM ELISA would improve detection of secondary dengue samples. PMID:27278716

  16. Genotoxic mode of action predictions from a multiplexed flow cytometric assay and a machine learning approach.

    PubMed

    Bryce, Steven M; Bernacki, Derek T; Bemis, Jeffrey C; Dertinger, Stephen D

    2016-04-01

    Several endpoints associated with cellular responses to DNA damage as well as overt cytotoxicity were multiplexed into a miniaturized, "add and read" type flow cytometric assay. Reagents included a detergent to liberate nuclei, RNase and propidium iodide to serve as a pan-DNA dye, fluorescent antibodies against γH2AX, phospho-histone H3, and p53, and fluorescent microspheres for absolute nuclei counts. The assay was applied to TK6 cells and 67 diverse reference chemicals that served as a training set. Exposure was for 24 hrs in 96-well plates, and unless precipitation or foreknowledge about cytotoxicity suggested otherwise, the highest concentration was 1 mM. At 4- and 24-hrs aliquots were removed and added to microtiter plates containing the reagent mix. Following a brief incubation period robotic sampling facilitated walk-away data acquisition. Univariate analyses identified biomarkers and time points that were valuable for classifying agents into one of three groups: clastogenic, aneugenic, or non-genotoxic. These mode of action predictions were optimized with a forward-stepping process that considered Wald test p-values, receiver operator characteristic curves, and pseudo R(2) values, among others. A particularly high performing multinomial logistic regression model was comprised of four factors: 4 hr γH2AX and phospho-histone H3 values, and 24 hr p53 and polyploidy values. For the training set chemicals, the four-factor model resulted in 94% concordance with our a priori classifications. Cross validation occurred via a leave-one-out approach, and in this case 91% concordance was observed. A test set of 17 chemicals that were not used to construct the model were evaluated, some of which utilized a short-term treatment in the presence of a metabolic activation system, and in 16 cases mode of action was correctly predicted. These initial results are encouraging as they suggest a machine learning strategy can be used to rapidly and reliably predict new chemicals

  17. Multiplexed DNA repair assays for multiple lesions and multiple doses via transcription inhibition and transcriptional mutagenesis.

    PubMed

    Nagel, Zachary D; Margulies, Carrie M; Chaim, Isaac A; McRee, Siobhan K; Mazzucato, Patrizia; Ahmad, Anwaar; Abo, Ryan P; Butty, Vincent L; Forget, Anthony L; Samson, Leona D

    2014-05-01

    The capacity to repair different types of DNA damage varies among individuals, making them more or less susceptible to the detrimental health consequences of damage exposures. Current methods for measuring DNA repair capacity (DRC) are relatively labor intensive, often indirect, and usually limited to a single repair pathway. Here, we describe a fluorescence-based multiplex flow-cytometric host cell reactivation assay (FM-HCR) that measures the ability of human cells to repair plasmid reporters, each bearing a different type of DNA damage or different doses of the same type of DNA damage. FM-HCR simultaneously measures repair capacity in any four of the following pathways: nucleotide excision repair, mismatch repair, base excision repair, nonhomologous end joining, homologous recombination, and methylguanine methyltransferase. We show that FM-HCR can measure interindividual DRC differences in a panel of 24 cell lines derived from genetically diverse, apparently healthy individuals, and we show that FM-HCR may be used to identify inhibitors or enhancers of DRC. We further develop a next-generation sequencing-based HCR assay (HCR-Seq) that detects rare transcriptional mutagenesis events due to lesion bypass by RNA polymerase, providing an added dimension to DRC measurements. FM-HCR and HCR-Seq provide powerful tools for exploring relationships among global DRC, disease susceptibility, and optimal treatment. PMID:24757057

  18. Multiplexed DNA repair assays for multiple lesions and multiple doses via transcription inhibition and transcriptional mutagenesis

    PubMed Central

    Nagel, Zachary D.; Margulies, Carrie M.; Chaim, Isaac A.; McRee, Siobhan K.; Mazzucato, Patrizia; Ahmad, Anwaar; Abo, Ryan P.; Butty, Vincent L.; Forget, Anthony L.; Samson, Leona D.

    2014-01-01

    The capacity to repair different types of DNA damage varies among individuals, making them more or less susceptible to the detrimental health consequences of damage exposures. Current methods for measuring DNA repair capacity (DRC) are relatively labor intensive, often indirect, and usually limited to a single repair pathway. Here, we describe a fluorescence-based multiplex flow-cytometric host cell reactivation assay (FM-HCR) that measures the ability of human cells to repair plasmid reporters, each bearing a different type of DNA damage or different doses of the same type of DNA damage. FM-HCR simultaneously measures repair capacity in any four of the following pathways: nucleotide excision repair, mismatch repair, base excision repair, nonhomologous end joining, homologous recombination, and methylguanine methyltransferase. We show that FM-HCR can measure interindividual DRC differences in a panel of 24 cell lines derived from genetically diverse, apparently healthy individuals, and we show that FM-HCR may be used to identify inhibitors or enhancers of DRC. We further develop a next-generation sequencing-based HCR assay (HCR-Seq) that detects rare transcriptional mutagenesis events due to lesion bypass by RNA polymerase, providing an added dimension to DRC measurements. FM-HCR and HCR-Seq provide powerful tools for exploring relationships among global DRC, disease susceptibility, and optimal treatment. PMID:24757057

  19. Miniaturized, multiplexed readout of droplet-based microfluidic assays using time-domain modulation†

    PubMed Central

    Muluneh, Melaku; Kim, Bawul; Buchsbaum, Gershon

    2015-01-01

    Recent advances in microfluidics to generate and control picoliter emulsions of water in oil have enabled ultra-sensitive assays for small molecules, proteins, nucleic acids, and cells. Unfortunately, the conventional fluorescence detection used to measure the outcome of these droplet-based assays has not proven suited to match the time and space multiplexing capabilities of microfluidic systems. To address this challenge, we developed an in-flow fluorescence detection platform that enables multiple streams of droplets to be monitored using only a single photodetector and no lenses. The key innovation of our technology is the amplitude modulation of the signal from fluorescent droplets using distinct micro-patterned masks for each channel. By taking advantage of the high bandwidth of electronics, our technique enables the velocity-independent recovery of weak fluorescent signals (SNR ≪ 1) using only simple hardware, obviating the need for lasers, bulky detectors, and complex fluid control. We demonstrated a handheld-sized device that simultaneously monitors four independent channels with the capability to be scaled-up to more than sixteen, limited primarily by the droplet density. PMID:25311204

  20. Multiplex PCR assay specific for the multidrug-resistant strain W of Mycobacterium tuberculosis.

    PubMed

    Plikaytis, B B; Marden, J L; Crawford, J T; Woodley, C L; Butler, W R; Shinnick, T M

    1994-06-01

    In 1991, a multidrug-resistant strain of Mycobacterium tuberculosis was isolated from eight people with tuberculosis at a state correctional facility in New York. This strain, which is designated strain W (IS6110 restriction fragment length polymorphism type 212072), was resistant to isoniazid, rifampin, ethambutol, streptomycin, kanamycin, ethionamide, and rifabutin. Since that outbreak, the W strain has been associated with outbreaks in five hospitals in the New York City area and is a continuing public health problem in the area. To be able to identify this strain rapidly, we developed a multiplex PCR assay which targets a direct repeat of IS6110 with a 556-bp intervening sequence (NTF-1). The amplification generates two amplicons from strain W, which indicate the presence and orientation of the NTF-1 sequence between the direct repeat of IS6110, and a third amplicon, which serves as an internal PCR control. The assay was evaluated with 193 isolates of M. tuberculosis, and all 48 strain W isolates among those 193 isolates were correctly identified. PMID:7915723

  1. Evaluation of a multiplex PCR assay for simultaneous detection of bacterial and viral enteropathogens in stool samples of paediatric patients.

    PubMed

    Onori, Manuela; Coltella, Luana; Mancinelli, Livia; Argentieri, Marta; Menichella, Donato; Villani, Alberto; Grandin, Annalisa; Valentini, Diletta; Raponi, Massimiliano; Russo, Cristina

    2014-06-01

    We evaluated a multiplex PCR assay, the Seeplex Diarrhoea ACE detection, that simultaneously detects 15 enteric pathogens, including Salmonella spp., Shigella spp., Vibrio spp., toxin B producer Clostridium difficile, Campylobacter spp., Clostridium perfringens, Yersinia enterocolitica, Aeromonas spp., Escherichia coli O157:H7, verocytotoxin-producing Escherichia coli, adenovirus, Group A rotavirus, norovirus GI and GII, and astrovirus. We compared this assay with clinical methods routinely used in our laboratory, for detecting enteropathogens in stool samples collected from 245 paediatric patients with suspected infectious gastroenteritis. We recovered 61 bacterial pathogens and 121 enteric viruses with our laboratory assays, while we detected 78 bacteria and 167 viruses with the molecular assay. We calculated specificity and sensitivity for both methods after analysis of discordant results and demonstrated greater sensitivity for multiplex PCR than for our routine methods, with the exception of Salmonella spp. and toxigenic C. difficile detection. The multiplex PCR assay proved to be a reliable tool to directly detect the most common enteropathogens in stool samples but with some limitations. PMID:24656922

  2. Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae

    PubMed Central

    Teste, Marie-Ange; Duquenne, Manon; François, Jean M; Parrou, Jean-Luc

    2009-01-01

    Background Real-time RT-PCR is the recommended method for quantitative gene expression analysis. A compulsory step is the selection of good reference genes for normalization. A few genes often referred to as HouseKeeping Genes (HSK), such as ACT1, RDN18 or PDA1 are among the most commonly used, as their expression is assumed to remain unchanged over a wide range of conditions. Since this assumption is very unlikely, a geometric averaging of multiple, carefully selected internal control genes is now strongly recommended for normalization to avoid this problem of expression variation of single reference genes. The aim of this work was to search for a set of reference genes for reliable gene expression analysis in Saccharomyces cerevisiae. Results From public microarray datasets, we selected potential reference genes whose expression remained apparently invariable during long-term growth on glucose. Using the algorithm geNorm, ALG9, TAF10, TFC1 and UBC6 turned out to be genes whose expression remained stable, independent of the growth conditions and the strain backgrounds tested in this study. We then showed that the geometric averaging of any subset of three genes among the six most stable genes resulted in very similar normalized data, which contrasted with inconsistent results among various biological samples when the normalization was performed with ACT1. Normalization with multiple selected genes was therefore applied to transcriptional analysis of genes involved in glycogen metabolism. We determined an induction ratio of 100-fold for GPH1 and 20-fold for GSY2 between the exponential phase and the diauxic shift on glucose. There was no induction of these two genes at this transition phase on galactose, although in both cases, the kinetics of glycogen accumulation was similar. In contrast, SGA1 expression was independent of the carbon source and increased by 3-fold in stationary phase. Conclusion In this work, we provided a set of genes that are suitable reference

  3. Enzyme catalysis-electrophoresis titration for multiplex enzymatic assay via moving reaction boundary chip.

    PubMed

    Zhong, Ran; Xie, Haiyang; Kong, Fanzhi; Zhang, Qiang; Jahan, Sharmin; Xiao, Hua; Fan, Liuyin; Cao, Chengxi

    2016-09-21

    In this work, we developed the concept of enzyme catalysis-electrophoresis titration (EC-ET) under ideal conditions, the theory of EC-ET for multiplex enzymatic assay (MEA), and a related method based on a moving reaction boundary (MRB) chip with a collateral channel and cell phone imaging. As a proof of principle, the model enzymes horseradish peroxidase (HRP), laccase and myeloperoxidase (MPO) were chosen for the tests of the EC-ET model. The experiments revealed that the EC-ET model could be achieved via coupling EC with ET within a MRB chip; particularly the MEA analyses of catalysis rate, maximum rate, activity, Km and Kcat could be conducted via a single run of the EC-ET chip, systemically demonstrating the validity of the EC-ET theory. Moreover, the developed method had these merits: (i) two orders of magnitude higher sensitivity than a fluorescence microplate reader, (ii) simplicity and low cost, and (iii) fairly rapid (30 min incubation, 20 s imaging) analysis, fair stability (<5.0% RSD) and accuracy, thus validating the EC-ET method. Finally, the developed EC-ET method was used for the clinical assay of MPO activity in blood samples; the values of MPO activity detected via the EC-ET chip were in agreement with those obtained by a traditional fluorescence microplate reader, indicating the applicability of the EC-ET method. The work opens a window for the development of enzymatic research, enzyme assay, immunoassay, and point-of-care testing as well as titration, one of the oldest methods of analysis, based on a simple chip. PMID:27464600

  4. [Identification of Env-specific monoclonal antibodies from Chinese HIV-1 infected person by B cell activation and RT-PCR cloning].

    PubMed

    Wang, Hui-Min; Xu, Ke; Yu, Shuang-Qing; Ding, Lin-Lin; Luo, Hai-Yan; Flinko, Robin; Lewis, George K; Feng, Xia; Shao, Ji-Rong; Guan, Yong-Jun; Zeng, Yi

    2012-06-01

    To obtain protective human monoclonal antibody from HIV-1 infected person, we adapted a technology for isolating antigen specific monoclonal antibody from human memory B cells through in vitro B cell activation coupled with RT-PCT and expression cloning. Human B cells were purified by negative sorting from PBMCs of HIV-1 infected individuals and memory B cells were further enriched using anti-CD27 microbeads. Two hundred memory B cells per well were cultured in 96-well round-bottom plates Env-specific antibodies in supernatants were with feeder cells in medium containing EBV and CpG. screened by ELISA after 1-2 weeks' culture. Cells from positive wells of Env-specific antibody were harvested and total RNA was isolated. Human VH and Vkappa or Vlambda genes were amplified by RT-PCR and cloned into IgG1 and kappa or lambda expressing vectors. Functional VH and Vkappa or Vlambda were identified by cotransfecting 293T cells with individual heavy chain and light chain clones followed by analysis of culture supernatants by ELISA for Env-specific antibodies. Finally, corresponding mAb was produced by transient transfection of 293T cells with the identified VH and Vkappa/lambda pair and purified by protein A affinity chromatography. Purified monocolonal antibodies were used for HIV-1 specific antibody-dependent cell-mediated cytotoxicity (ADCC) and neutralizing activity assay. Four monocolonal Env-specific antibodies were isolated from one HIV-1 subtype B' infected individual. Two of them showed strong ADCC activity and one showed weak neutralizing activity against HIV-1. Its further studies on their application in therapeutic or prophylactic vaccines against HIV-1 should be grounded. PMID:22978159

  5. Quantification of human telomerase reverse transcriptase mRNA in testicular germ cell tumors by quantitative fluorescence real-time RT-PCR.

    PubMed

    Schrader, Mark; Burger, Angelika M; Müller, Markus; Krause, Hans; Straub, Bernd; Smith, Gilian L; Newlands, Eward S; Miller, Kurt

    2002-01-01

    Telomerase is a ribonucleoprotein enzyme which is endogenously expressed in germ, stem and tumor cells, but absent in benign somatic cells. The two major telomerase components are human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT). It has been shown that hTERT is rate-limiting for telomerase activity and that it plays a central role in human carcinogenesis. Here, we investigated the potential of hTERT and hTR gene expression as diagnostic markers in testicular germ cell tumors (TGCT). hTERT mRNA and hTR expression were quantified in 55 testicular germ cell tumors comprising 36 primary and 19 germ cell tumors from retroperitonal sides by fluorescence real-time RT-PCR using the LightCycler technology. Porphobilinogen deaminase (PBGD) was used as housekeeping gene and to enable relative quantification. For comparison to TGCTs, 38 benign testicular biopsies from patients with fertility disorders were assayed. hTERT expression was detected in all examined undifferentiated TGCTs and in the benign testicular tissue specimens with germ cell content (N(hTERT) 38-127). In contrast, mature teratomas from primary and post-chemotherapy masses, which are characterized by well-differentiated tissue components showed a nearly complete downregulation of hTERT expression (N(hTERT) 2-4, p<0.001). hTR levels however, were high in all tumors and independently of the presence of germ cells also in the benign tissue control group. hTERT mRNA is expressed in all undifferentiated TGCTs but repressed in mature teratomas. This suggests an inverse correlation between the differentiation status of germ cell tumors and hTERT expression. Thus, detection of hTERT expression in tumors histopathologically classified as mature teratomas enables a molecular-diagnostic confirmation and might aid decision making for treatment of patients presenting with this tumor subtype. PMID:12168080

  6. Rapid detection and characterization of Chikungunya virus by RT-PCR in febrile patients from Kerala, India.

    PubMed

    Joseph, Anu Yamuna; Babu, Vidhu Sankar; Dev, Sona S; Gopalakrishnapai, Jayashree; Harish, M; Rajesh, M D; Anisha, S; Mohankumar, C

    2008-08-01

    There has been a resurgence and prevalence of fever with symptoms of Chikungunya (CHIK) and increased death toll in Kerala, the southern-most state of India. The objective of this study was to develop a rapid detection method to determine the presence of CHIK- virus in the serum samples collected from febrile patients in Kerala, India. Serum specimens were analyzed for CHIK viral RNA by RT-PCR using primers specific for nsP1 and E1 genes. Five out of twenty clinical samples were positive for CHIK virus. The partial sequences of the E1 and nsP1 genes of the strain, IndKL01 were highly similar to the Reunion strains and the recently isolated Indian strains. A novel substitution, A148V, was detected in the E1 gene of the isolate, IndKL02. The detection procedure used in this study was simple, sensitive and rapid (less than 4 hr). This result suggests that CHIK viruses similar to the Reunion strains, which had resulted in high morbidity and mortality rates, may have caused the recent Chikungunya outbreak in India. The effect of the variant, E1-A148V, in the virulence and the rate of transmission of the virus deserves further investigation. PMID:18814485

  7. RT-PCR and sequence analysis of the full-length fusion protein of Canine Distemper Virus from domestic dogs.

    PubMed

    Romanutti, Carina; Gallo Calderón, Marina; Keller, Leticia; Mattion, Nora; La Torre, José

    2016-02-01

    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. PMID:26611227

  8. An RT-PCR primer pair for the detection of Pospiviroid and its application in surveying ornamental plants for viroids.

    PubMed

    Bostan, Hidayet; Nie, Xianzhou; Singh, Rudra P

    2004-03-15

    A primer pair for reverse transcription-polymerase chain reaction (RT-PCR), based on the conserved sequences of the members of genus Pospiviroid was designed to yield a fragment of about 200 base pairs (bp). Since pospiviroids infect a large number of plants species and a few members of the genus Pospiviroid have been already detected in some ornamental plants, the primer pair was evaluated for its efficacy using ornamental plants. The method of return-polyacrylamide gel electrophoresis (R-PAGE) was used to determine the general presence of viroids in the test samples. Efficacy of the primer pair for members of genus Pospiviroid was demonstrated by the detection of Potato spindle tuber viroid (PSTVd) and Tomato chlorotic dwarf viroid (TCDVd) in potato, Chrysanthemum stunt viroid and Iresine viroid in Verbena and Vinca species, and Citrus exocortis viroid in Impatiens species. Specificity of the primer pair became evident, where additional viroids were detected by R-PAGE in Coleus and Magilla species, but they were not amplified by the Pospiviroid primer. This primer pair would be of benefit in indexing ornamental plants in quarantine samples or in viroid-free certification schemes, irrespective of their actual identity. PMID:14738987

  9. Detection of rabies virus RNA isolated from several species of animals in Brazil by RT-PCR.

    PubMed

    Ito, M; Itou, T; Sakai, T; Santos, M F; Arai, Y T; Takasaki, T; Kurane, I; Ito, F H

    2001-12-01

    Brain samples from different animal species including humans: five vampire bats, 14 cattle, 12 dogs, 11 cats, two horses, one pig, one sheep and three humans collected from various geographical regions of Brazil were found to be positive for rabies by means of the fluorescent antibody test (FAT) and the mouse inoculation test (MIT). The brain samples were retested for rabies by means of the reverse transcription and polymerase chain reaction (RT-PCR) with 2 primer sets (P1/P2 and RHNI/RHNS3), which amplified full or partial regions on the nucleoprotein (N) gene of the rabies virus, respectively. Brain samples from five vampire bats, 13 cattle, one horse and one sheep failed to yield PCR products when the RHN1/RHNS3 primer pair was used, but all brain samples successfully yielded the products when the P1/P2 primer pair was used. These results suggest that Brazilian rabies virus isolates could be principally divided into two populations according to genetic difference. PMID:11789609

  10. RT-PCR for detecting five distinct Tospovirus species using degenerate primers and dsRNA template.

    PubMed

    Okuda, M; Hanada, K

    2001-08-01

    RT-PCR procedures for detection of multiple species of tospovirus from plant tissues were investigated. Downstream primers were designated from the 3' untranslated sequences of the S RNA. An upstream primer was designated from the degenerated sequences of the nucleocapsid protein. Approximately 450 bp DNA fragments were detected when Tomato spotted wilt virus (TSWV)- or Impatiens necrotic spot virus (INSV)- infected tissues were examined. Approximately 350 bp DNA fragments were detected when Watermelon silver mottle virus (WSMoV)- or Melon yellow spot virus (MYSV)-infected tissues were examined. Template RNA was extracted using CF 11 cellulose powder, and nonspecific amplification became unnoticeable when double-stranded RNA was used. The amplified fragments of WSMoV were differentiated from those of MYSV by AluI or TaqI digestion. The amplified fragments of TSWV were differentiated from those of INSV by DraI or HindIII digestion. An alstroemeria plant that was infected with an unidentified tospovirus was also examined, and a 350 bp fragment that was 97% identical to Iris yellow spot virus was detected. This method, therefore, detected at least five distinct Tospovirus species. PMID:11445145

  11. Vitamin D receptor alleles: Cloning and characterization of the VDR gene and RT-PCR of VDR cDNA

    SciTech Connect

    Javed, A.A.; Huang, Y.; Bombard, A.T.

    1994-09-01

    Vitamin D{sub 3} receptors (VDR) function as regulators through the action of the ligand 1{alpha}, 25-dihydroxy vitamin D{sub 3}. The receptor specifically finds its ligand and exerts it effect on the regulation of the expression of target genes. It has been shown that mutations in the VDR gene affect the function of the receptors and cause a corresponding disorder state. Recently, it has been reported that common allelic variations found normally in the Caucasian (Australian) population pose varying degrees of risk for osteoporosis. We present here the cloning of the VDR gene and RT-PCR of VDR cDNA. Studies are in progress to establish allele frequency in the Black, Hispanic and Caucasian populations to systematically study the influence of allele types and to develop a risk profile for osteoporosis. The present method for detection of various alleles is based on RFLP analysis. We are developing PCR-based methods for the rapid detection and typing of alleles.

  12. Development and Practical Use of RT-PCR for Seed-transmitted Prune dwarf virus in Quarantine

    PubMed Central

    Lee, Siwon; Shin, Yong-Gil

    2014-01-01

    Among imported plants, seeds are the items that have many latent pathogens and are difficult to inspect. Also, they are the import and export items whose market is expected to expand. The biggest problem with seeds is viruses. Prune dwarf virus (PDV) is the virus that is commonly inspected in Prunus cerasifera, P. persica, P. armeniaca, P. mandshurica, P. cerasus, P. avium or P. serotina seeds. In this study, two RT-PCR primer sets, which can promptly and specifically diagnose plant quarantine seed-transmitted PDV, were developed; and nested PCR primers, where products amplify 739 and 673 nucleotides (nt), and an nested PCR-product, 305 nt, can be obtained as these products are amplified again, were developed. Also, a modified-positive control plasmid was developed, where the restriction enzyme XhoI, which can identify the contamination of samples from the control, was inserted. The method developed in this study has detected PDV in 18 cases since 2007, and is expected to continuously contribute to the plant quarantine in Korea. PMID:25289000

  13. Identification and expression profiling of low oxygen regulated genes from Citrus flavedo tissues using RT-PCR differential display.

    PubMed

    Pasentsis, Konstantinos; Falara, Vasiliki; Pateraki, Irene; Gerasopoulos, Dimitrios; Kanellis, Angelos K

    2007-01-01

    The molecular basis for the adaptation of fruit tissues to low oxygen treatments remains largely unknown. RT-PCR differential display (DD) was employed to isolate anoxic and/or hypoxic genes whose expression responded to short, low-oxygen regimes. This approach led to the isolation, cloning, successful sequencing, and bioinformatic analysis of 98 transcripts from Citrus flavedo tissues that were differentially expressed in DD gels in response to 0, 0.5, 3, and 21% O(2) for 24 h. RNA blot analysis of 25 DD clones revealed that 11 genes were induced under hypoxia and/or anoxia, 11 exhibited constitutive expression and three transcripts were suppressed by low oxygen levels. Almost half of the DD cDNAs were either of unknown function or shared no apparent homology to any expressed sequences in the GenBank/EMBL databases. Six DD genes were similar to molecules of the following functions: C-compound and carbohydrate utilization, plant development, amino acid metabolism, and biosynthesis of brasinosteroids. Time-course and stress-related experiments of low O(2)-regulated genes indicated that these genes responded differently in terms of their earliness, band intensity, and their specificity to stresses, showing that some of them can be termed hypoxia- or anoxia-induced genes. PMID:17525081

  14. Development and Practical Use of RT-PCR for Seed-transmitted Prune dwarf virus in Quarantine.

    PubMed

    Lee, Siwon; Shin, Yong-Gil

    2014-06-01

    Among imported plants, seeds are the items that have many latent pathogens and are difficult to inspect. Also, they are the import and export items whose market is expected to expand. The biggest problem with seeds is viruses. Prune dwarf virus (PDV) is the virus that is commonly inspected in Prunus cerasifera, P. persica, P. armeniaca, P. mandshurica, P. cerasus, P. avium or P. serotina seeds. In this study, two RT-PCR primer sets, which can promptly and specifically diagnose plant quarantine seed-transmitted PDV, were developed; and nested PCR primers, where products amplify 739 and 673 nucleotides (nt), and an nested PCR-product, 305 nt, can be obtained as these products are amplified again, were developed. Also, a modified-positive control plasmid was developed, where the restriction enzyme XhoI, which can identify the contamination of samples from the control, was inserted. The method developed in this study has detected PDV in 18 cases since 2007, and is expected to continuously contribute to the plant quarantine in Korea. PMID:25289000

  15. Reference Gene Selection for qRT-PCR Analysis in the Sweetpotato Whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae)

    PubMed Central

    Li, Rumei; Xie, Wen; Wang, Shaoli; Wu, Qingjun; Yang, Nina; Yang, Xin; Pan, Huipeng; Zhou, Xiaomao; Bai, Lianyang; Xu, Baoyun; Zhou, Xuguo; Zhang, Youjun

    2013-01-01

    Background Accurate evaluation of gene expression requires normalization relative to the expression of reliable reference genes. Expression levels of “classical” reference genes can differ, however, across experimental conditions. Although quantitative real-time PCR (qRT-PCR) has been used extensively to decipher gene function in the sweetpotato whitefly Bemisia tabaci, a world-wide pest in many agricultural systems, the stability of its reference genes has rarely been validated. Results In this study, 15 candidate reference genes from B. tabaci were evaluated using two Excel-based algorithms geNorm and Normfinder under a diverse set of biotic and abiotic conditions. At least two reference genes were selected to normalize gene expressions in B. tabaci under experimental conditions. Specifically, for biotic conditions including host plant, acquisition of a plant virus, developmental stage, tissue (body region of the adult), and whitefly biotype, ribosomal protein L29 was the most stable reference gene. In contrast, the expression of elongation factor 1 alpha, peptidylprolyl isomerase A, NADH dehydrogenase, succinate dehydrogenase complex subunit A and heat shock protein 40 were consistently stable across various abiotic conditions including photoperiod, temperature, and insecticide susceptibility. Conclusion Our finding is the first step toward establishing a standardized quantitative real-time PCR procedure following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guideline in an agriculturally important insect pest, and provides a solid foundation for future RNA interference based functional study in B. tabaci. PMID:23308130

  16. Characterization of rabbit limbal epithelial side population cells using RNA sequencing and single-cell qRT-PCR.

    PubMed

    Kameishi, Sumako; Umemoto, Terumasa; Matsuzaki, Yu; Fujita, Masako; Okano, Teruo; Kato, Takashi; Yamato, Masayuki

    2016-05-01

    Corneal epithelial stem cells reside in the limbus, a transitional zone between the cornea and conjunctiva, and are essential for maintaining homeostasis in the corneal epithelium. Although our previous studies demonstrated that rabbit limbal epithelial side population (SP) cells exhibit stem cell-like phenotypes with Hoechst 33342 staining, the different characteristics and/or populations of these cells remain unclear. Therefore, in this study, we determined the gene expression profiles of limbal epithelial SP cells by RNA sequencing using not only present public databases but also contigs that were created by de novo transcriptome assembly as references for mapping. Our transcriptome data indicated that limbal epithelial SP cells exhibited a stem cell-like phenotype compared with non-SP cells. Importantly, gene ontology analysis following RNA sequencing demonstrated that limbal epithelial SP cells exhibited significantly enhanced expression of mesenchymal/endothelial cell markers rather than epithelial cell markers. Furthermore, single-cell quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that the limbal epithelial SP population consisted of at least two immature cell populations with endothelial- or mesenchymal-like phenotypes. Therefore, our present results may propose the presence of a novel population of corneal epithelial stem cells distinct from conventional epithelial stem cells. PMID:26546824

  17. Development of silicon photonic microring resonator biosensors for multiplexed cytokine assays and in vitro diagnostics

    NASA Astrophysics Data System (ADS)

    Luchansky, Matthew Sam

    In order to guide critical care therapies that are personalized to a patient's unique disease state, a diagnostic or theranostic medical device must quickly provide a detailed biomolecular understanding of disease onset and progression. This detailed molecular understanding of cellular processes and pathways requires the ability to measure multiple analytes in parallel. Though many traditional sensing technologies for biomarker analysis and fundamental biological studies (i.e. enzyme-linked immunosorbent assays, real-time polymerase chain reaction, etc.) rely on single-parameter measurements, it has become increasingly clear that the inherent complexity of many human illnesses and pathways necessitates quantitative and multiparameter analysis of biological samples. Currently used analytical methods are deficient in that they often provide either highly quantitative data for a single biomarker or qualitative data for many targets, but methods that simultaneously provide highly quantitative analysis of many targets have yet to be adequately developed. Fields such as medical diagnostics and cellular biology would benefit greatly from a technology that enables rapid, quantitative and reproducible assays for many targets within a single sample. In an effort to fill this unmet need, this doctoral dissertation describes the development of a clinically translational biosensing technology based on silicon photonics and developed in the chemistry research laboratory of Ryan C. Bailey. Silicon photonic microring resonators, a class of high-Q optical sensors, represent a promising platform for rapid, multiparameter in vitro measurements. The original device design utilizes 32-ring arrays for real-time biomolecular sensing without fluorescent labels, and these optical biosensors display great potential for more highly multiplexed (100s-1000s) measurements based on the impressive scalability of silicon device fabrication. Though this technology can be used to detect a variety of

  18. Detection and Typing of Human Papilloma Viruses by Nested Multiplex Polymerase Chain Reaction Assay in Cervical Cancer

    PubMed Central

    Jalal Kiani, Seyed; Shatizadeh Malekshahi, Somayeh; Yousefi Ghalejoogh, Zohreh; Ghavvami, Nastaran; Shafiei Jandaghi, Nazanin Zahra; Shahsiah, Reza; Jahanzad, Isa; Yavarian, Jila

    2015-01-01

    Background: Cervical cancer is the leading cause of death from cancer in under-developed countries. Human papilloma virus (HPV) 16 and 18 are the most prevalent types associated with carcinogenesis in the cervix. Conventional Polymerase Chain Reaction (PCR), type-specific and consensus primer-based PCR followed by sequencing, Restriction Fragment Length Polymorphism (RFLP) or hybridization by specific probes are common methods for HPV detection and typing. In addition, some researchers have developed a multiplex PCR for simultaneous detection and typing of different HPVs. Objectives: The aim of the present study was to investigate the prevalence of HPV infection and its types in cervical Squamous Cell Carcinoma (SCC) using the Nested Multiplex PCR (NMPCR) assay. Patients and Methods: Sixty-six samples with histologically confirmed SCC were evaluated. Total DNA was isolated by phenol–chloroform extraction and ethanol precipitation. Nested multiplex PCR was performed with first-round PCR by GP-E6/E7 consensus primers for amplification of the genomic DNA of all known mucosal HPV genotypes and second-round PCR by type-specific multiplex PCR primer cocktails. Results: Human papilloma virus infection was detected in 78.8% of samples, with the highest prevalence of HPV 16 (60.6%) while concurrent infections with two types was detected in 10.6%. Conclusions: The NMPCR assay is more convenient and easy for analysis of results, which is important for fast diagnosis and patient management, in a type-specific manner. PMID:26865940

  19. A novel multiplex PCR coupled with Luminex assay for the simultaneous detection of Cryptosporidium spp., Cryptosporidium parvum and Giardia duodenalis.

    PubMed

    Li, Wei; Zhang, Nan; Gong, Pengtao; Cao, Lili; Li, Jianhua; Su, Libo; Li, Shuhong; Diao, Yumei; Wu, Kang; Li, He; Zhang, Xichen

    2010-10-11

    Cryptosporidium parvum and Giardia duodenalis are the most frequently identified enteric parasites associated with diarrhea-causing disease outbreaks, and many non-parvum species of Cryptosporidium also can replicate and cause illness in mammals including humans. In this study, we describe a novel multiplex PCR coupled with Luminex assay for the identification of Cryptosporidium spp., C. parvum and G. duodenalis in a rapid manner. The multiplex PCR for the simultaneous detection of Cryptosporidium and Giardia was developed using three pairs of biotinylated primers which amplify 424, 223 and 267 bp products from the U1 small nuclear ribonucleoprotein (U1 snr) gene, 18S rRNA gene of Cryptosporidium and the beta-giardin gene of Giardia, respectively. The genus and species-specific capture probes linked to carboxylated Luminex microspheres hybridized to the multiplex PCR amplicons to enhance sensitivity and specificity. The conditions of multiplex PCR and Luminex hybridization reaction were optimized to enable the minimum detection limits of 5x10(-6), 5x10(-6), and 5x10(-6) ng DNAs (corresponding approximately to 0.1 oocyst/cyst). The Luminex approach proved to be 100% specific and accurate by testing a total of 240 fecal samples compared with microscopic examination of fecal smears and further modified acid-fast staining or iodine-staining observation. The established assay offers the potential for rapid detection of Cryptosporidium spp., C. parvum and G. duodenalis in fecal and environmental samples. PMID:20594647

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

    PubMed

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

    2010-06-01

    Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAIV) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds is often conducted in remote regions, but results are often delayed because of limited local analytical capabilities, difficulties with sample transportation and permitting, or problems keeping samples cold in the field. In response to these challenges, the performance of a portable real-time, reverse transcriptase-polymerase chain reaction (rRT-PCR) unit (RAPID((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. PMID:20206650

  1. A robust standard for absolute mRNA quantification of Saccharomyces cerevisiae by qRT-PCR using the universal RNA controls

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  2. Strand-specific real-time RT-PCR quantitation of Maize fine streak virus genomic and positive-sense RNAs using high temperature reverse transcription

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    USGS Publications Warehouse

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

    2010-01-01

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

  4. A bacterial community analysis using reverse transcription (RT) PCR which detects the bacteria with high activity in a wastewater treatment reactor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  5. Use of a custom RT-PCR array to analyze toxicity pathways at different life stages in Brown Norway Rat Brain following acute Toluene exposure.

    EPA Science Inventory

    To investigate the contribution of different life stages on response to toxicants, we utilized a custom designed RT-PCR array to examine the effects of acute exposure by oral gavage of the volatile organic solvent toluene (0.00, 0.65 or 1.0 glkg) in the brains of ma1e Brown Norwa...

  6. Strand-specific real-time RT-PCR quantitation of Maize fine streak virus genomic and positive-sense RNAs using high temperature reverse transcription

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  7. Analytical validation of a real-time RT-PCR test for Pan-American lineage H7 subtype avian influenza viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  8. Use of RT-PCR for diagnosis of infectious salmon anaemia virus (ISAV) in carrier sea trout Salmo trutta after experimental infection.

    PubMed

    Devold, M; Krossøy, B; Aspehaug, V; Nylund, A

    2000-02-24

    The emergence of infectious salmon anaemia virus (ISAV) in Canada and Scotland and frequent new outbreaks of the disease in Norway strongly suggest that there are natural reservoirs for the virus. The main host for the ISA virus is probably a fish occurring in the coastal area, most likely a salmonid fish. Since sea trout is an abundant species along the Norwegian coast, common in areas where ISA outbreaks occur, and possibly a life-long carrier of the ISA virus, a study was initiated to evaluate reverse transcriptase polymerase chain reaction (RT-PCR) for diagnosis of the virus in experimentally infected trout. Several tissues (kidney, spleen, heart and skin) were collected from the trout during a 135 d period. The following diagnostic methods for detection of the ISA virus were compared: cell culture (Atlantic Salmon Kidney, ASK cells), challenge of disease-free salmon with blood from the infected trout, and RT-PCR. The RT-PCR was the most sensitive of these methods. With the help of this technique it was possible to pick out positive individuals throughout the experimental period of 135 d. Challenge of disease-free salmon were more sensitive than cell culture (ASK cells). These 2 latter methods require use of the immunofluorescent antibody test (IFAT) or RT-PCR for verification of presence of ISA virus. PMID:10785858

  9. Multiplex enhancer-reporter assays uncover unsophisticated TP53 enhancer logic.

    PubMed

    Verfaillie, Annelien; Svetlichnyy, Dmitry; Imrichova, Hana; Davie, Kristofer; Fiers, Mark; Kalender Atak, Zeynep; Hulselmans, Gert; Christiaens, Valerie; Aerts, Stein

    2016-07-01

    Transcription factors regulate their target genes by binding to regulatory regions in the genome. Although the binding preferences of TP53 are known, it remains unclear what distinguishes functional enhancers from nonfunctional binding. In addition, the genome is scattered with recognition sequences that remain unoccupied. Using two complementary techniques of multiplex enhancer-reporter assays, we discovered that functional enhancers could be discriminated from nonfunctional binding events by the occurrence of a single TP53 canonical motif. By combining machine learning with a meta-analysis of TP53 ChIP-seq data sets, we identified a core set of more than 1000 responsive enhancers in the human genome. This TP53 cistrome is invariably used between cell types and experimental conditions, whereas differences among experiments can be attributed to indirect nonfunctional binding events. Our data suggest that TP53 enhancers represent a class of unsophisticated cell-autonomous enhancers containing a single TP53 binding site, distinct from complex developmental enhancers that integrate signals from multiple transcription factors. PMID:27197205

  10. Multiplex enhancer-reporter assays uncover unsophisticated TP53 enhancer logic

    PubMed Central

    Verfaillie, Annelien; Svetlichnyy, Dmitry; Imrichova, Hana; Davie, Kristofer; Fiers, Mark; Kalender Atak, Zeynep; Hulselmans, Gert; Christiaens, Valerie; Aerts, Stein

    2016-01-01

    Transcription factors regulate their target genes by binding to regulatory regions in the genome. Although the binding preferences of TP53 are known, it remains unclear what distinguishes functional enhancers from nonfunctional binding. In addition, the genome is scattered with recognition sequences that remain unoccupied. Using two complementary techniques of multiplex enhancer-reporter assays, we discovered that functional enhancers could be discriminated from nonfunctional binding events by the occurrence of a single TP53 canonical motif. By combining machine learning with a meta-analysis of TP53 ChIP-seq data sets, we identified a core set of more than 1000 responsive enhancers in the human genome. This TP53 cistrome is invariably used between cell types and experimental conditions, whereas differences among experiments can be attributed to indirect nonfunctional binding events. Our data suggest that TP53 enhancers represent a class of unsophisticated cell-autonomous enhancers containing a single TP53 binding site, distinct from complex developmental enhancers that integrate signals from multiple transcription factors. PMID:27197205

  11. Host factors associated with serologic inflammatory markers assessed using multiplex assays.

    PubMed

    McKay, Heather S; Bream, Jay H; Margolick, Joseph B; Martínez-Maza, Otoniel; Phair, John P; Rinaldo, Charles R; Abraham, Alison G; Jacobson, Lisa P

    2016-09-01

    Chronic systemic inflammation contributes to the development of adverse health conditions, yet the influence of fixed and modifiable risk factors on many serologic biomarkers of inflammation remains largely unknown. Serum concentrations of twenty-three biomarkers, including C-reactive protein (CRP), cytokines (CXCL11, CXCL8, CXCL10, CCL2, CCL13, CCL4, CCL17, CXCL13, IL-10, IL-12p70, IL-6, TNF-α, IL-2, IFN-γ, IL-1β, GM-CSF, BAFF), and soluble immune receptors (sCD14, sIL-2Rα, sCD27, sgp130, sTNF-R2) were measured longitudinally using multiplexed immunometric assays in 250 HIV-uninfected men followed in the Multicenter AIDS Cohort Study (1984-2009). Generalized gamma regression was used to determine the statistical significance of factors associated with each biomarker. After accounting for age, race, and education, and for analysis of multiple biomarkers, higher concentrations of specific individual biomarkers were significantly (P<0.002) associated with hypertension, obesity, hepatitis C infection, stimulant use, and diabetes and lower concentrations with hypercholesterolemia. These associations should be taken into account in epidemiological studies of these biomarkers, and may provide potential targets for disease prevention and treatment. PMID:27295613

  12. Early TBI-Induced Cytokine Alterations are Similarly Detected by Two Distinct Methods of Multiplex Assay

    PubMed Central

    Mukherjee, Sanjib; Katki, Khurshed; Arisi, Gabriel M.; Foresti, Maira L.; Shapiro, Lee A.

    2011-01-01

    Annually, more than a million persons experience traumatic brain injury (TBI) in the US and a substantial proportion of this population develop debilitating neurological disorders, such as, paralysis, cognitive deficits, and epilepsy. Despite the long-standing knowledge of the risks associated with TBI, no effective biomarkers or interventions exist. Recent evidence suggests a role for inflammatory modulators in TBI-induced neurological impairments. Current technological advances allow for the simultaneous analysis of the precise spatial and temporal expression patterns of numerous proteins in single samples which ultimately can lead to the development of novel treatments. Thus, the present study examined 23 different cytokines, including chemokines, in the ipsi and contralateral cerebral cortex of rats at 24 h after a fluid percussion injury (FPI). Furthermore, the estimation of cytokines were performed in a newly developed multiplex assay instrument, MAGPIX (Luminex Corp), and compared with an established instrument, Bio-Plex (Bio-Rad), in order to validate the newly developed instrument. The results show numerous inflammatory changes in the ipsi and contralateral side after FPI that were consistently reported by both technologies. PMID:21954376

  13. A digital microfluidic method for multiplexed cell-based apoptosis assays.

    PubMed

    Bogojevic, Dario; Chamberlain, M Dean; Barbulovic-Nad, Irena; Wheeler, Aaron R

    2012-02-01

    Digital microfluidics (DMF), a fluid-handling technique in which picolitre-microlitre droplets are manipulated electrostatically on an array of electrodes, has recently become popular for applications in chemistry and biology. DMF devices are reconfigurable, have no moving parts, and are compatible with conventional high-throughput screening infrastructure (e.g., multiwell plate readers). For these and other reasons, digital microfluidics has been touted as being a potentially useful new tool for applications in multiplexed screening. Here, we introduce the first digital microfluidic platform used to implement parallel-scale cell-based assays. A fluorogenic apoptosis assay for caspase-3 activity was chosen as a model system because of the popularity of apoptosis as a target for anti-cancer drug discovery research. Dose-response profiles of caspase-3 activity as a function of staurosporine concentration were generated using both the digital microfluidic method and conventional techniques (i.e., pipetting, aspiration, and 96-well plates.) As expected, the digital microfluidic method had a 33-fold reduction in reagent consumption relative to the conventional technique. Although both types of methods used the same detector (a benchtop multiwell plate reader), the data generated by the digital microfluidic method had lower detection limits and greater dynamic range because apoptotic cells were much less likely to de-laminate when exposed to droplet manipulation by DMF relative to pipetting/aspiration in multiwell plates. We propose that the techniques described here represent an important milestone in the development of digital microfluidics as a useful tool for parallel cell-based screening and other applications. PMID:22159547

  14. Development of a multiplex real-time PCR assay for identification of members of the Anopheles gambiae species complex.

    PubMed

    Bass, Chris; Williamson, Martin S; Field, Linda M

    2008-07-01

    Two high-throughput assays for the identification of members of the Anopheles gambiae sensu lato species complex have recently been reported. These methods, are based on TaqMan single nucleotide polymorphism (SNP) genotyping that enables rapid scoring of mosquito DNA samples in real-time PCR reactions. Unfortunately, both assays are restricted in the number of species that they can identify and a combination of the two assays may be required to identify all possible species in certain regions. To overcome this limitation, and thereby further increase throughput while reducing costs, we have developed a new multiplex real-time PCR assay for identifying members of the An. gambiae complex. The new method uses three probes labelled with fluorophores with distinct emission and excitation spectra, allowing simultaneous detection of the two main malaria vectors from the non-vector sibling species, and can be used on single mosquito legs from silica-dried specimens. A genotyping trial of over 450 specimens collected from 13 countries in sub-Saharan Africa showed the multiplex assay to be highly specific and sensitive and it compared well against the two previously reported TaqMan assays and standard allele-specific PCR. PMID:18490000

  15. Rapid detection and typing of pathogenic nonpneumophila Legionella spp. isolates using a multiplex real-time PCR assay.

    PubMed

    Benitez, Alvaro J; Winchell, Jonas M

    2016-04-01

    We developed a single tube multiplex real-time PCR assay that allows for the rapid detection and typing of 9 nonpneumophila Legionella spp. isolates that are clinically relevant. The multiplex assay is capable of simultaneously detecting and discriminating L. micdadei, L. bozemanii, L. dumoffii, L. longbeachae, L. feeleii, L. anisa, L. parisiensis, L. tucsonensis serogroup (sg) 1 and 3, and L. sainthelensis sg 1 and 2 isolates. Evaluation of the assay with nucleic acid from each of these species derived from both clinical and environmental isolates and typing strains demonstrated 100% sensitivity and 100% specificity when tested against 43 other Legionella spp. Typing of L. anisa, L. parisiensis, and L. tucsonensis sg 1 and 3 isolates was accomplished by developing a real-time PCR assay followed by high-resolution melt (HRM) analysis targeting the ssrA gene. Further typing of L. bozemanii, L. longbeachae, and L. feeleii isolates to the serogroup level was accomplished by developing a real-time PCR assay followed by HRM analysis targeting the mip gene. When used in conjunction with other currently available diagnostic tests, these assays may aid in rapidly identifying specific etiologies associated with Legionella outbreaks, clusters, sporadic cases, and potential environmental sources. PMID:26867966

  16. Multiplex PCR assay underreports true bloodstream infections with coagulase-negative staphylococci in hematological patients with febrile neutropenia.

    PubMed

    Reers, Yvonne; Idelevich, Evgeny A; Pätkau, Hanna; Sauerland, Maria Cristina; Tafelski, Sascha; Nachtigall, Irit; Berdel, Wolfgang E; Peters, Georg; Silling, Gerda; Becker, Karsten

    2016-08-01

    SeptiFast multiplex PCR assay was evaluated for detecting true bloodstream infections (BSIs) with coagulase-negative staphylococci (CoNS) in neutropenic hematological patients. Sensitivity for samples representing true CoNS-BSIs was 23.3% with an integrated cutoff and increased to 83.3% if the cutoff was neglected. Hence, the cutoff may prohibit timely targeted antimicrobial therapy. PMID:27220608

  17. Development of a suspension array assay in multiplex for simultaneous measurement of serum levels of four eosinophil granule proteins

    PubMed Central

    Makiya, Michelle A.; Herrick, Jesica A.; Khoury, Paneez; Prussin, Calman P.; Nutman, Thomas B.; Klion, Amy D.

    2014-01-01

    The concentrations of major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil derived neurotoxin (EDN) and eosinophil peroxidase (EPO) have been associated with eosinophilic disease severity. Whereas a variety of techniques have been used to measure individual eosinophil granule protein concentration, none of these methods efficiently measures MBP, ECP, EDN and EPO simultaneously. A multiplex suspension array system was developed to simultaneously measure the concentration of MBP, ECP, EDN and EPO in serum. The assay showed excellent inter- and intra-assay reliability, and serum levels of MBP, ECP and EDN from eosinophilic subjects analyzed by ELISA and multiplex were highly correlated (r = 0.8579; P < 0.0001, r = 0.6356; P = 0.0006 and r = 0.8600; P < 0.0001, respectively, Spearman rank correlation). Moreover, the multiplex assay required 500-fold less serum than a single ELISA to achieve comparable sensitivity. Absolute eosinophil count and eosinophil surface expression of the activation marker, CD69, were significantly correlated with concentrations of MBP, EDN and EPO, but not ECP, in serum from eosinophilic subjects. Furthermore, subjects with eosinophilic gastrointestinal disorder and normal peripheral absolute eosinophil counts (< 0.5 × 109/L) had significantly increased concentrations of MBP (P < 0.0001), ECP (P < 0.0001), EDN (P = 0.0001) and EPO (P < 0.0001) compared to normal donors. In summary, the eosinophil granule protein multiplex assay provides a rapid and reliable way to measure eosinophil granule protein levels and should prove useful in assessing patterns of degranulation in patients with eosinophilic disorders. PMID:24914990

  18. Environmental Detection of Genogroup I, II, and IV Noroviruses by Using a Generic Real-Time Reverse Transcription-PCR Assay

    PubMed Central

    Miura, Takayuki; Parnaudeau, Sylvain; Grodzki, Marco; Okabe, Satoshi; Atmar, Robert L.

    2013-01-01

    Norovirus is the most common agent implicated in food-borne outbreaks and is frequently detected in environmental samples. These viruses are highly diverse, and three genogroups (genogroup I [GI], GII, and GIV) infect humans. Being noncultivable viruses, real-time reverse transcription-PCR (RT-PCR) is the only sensitive method available for their detection in food or environmental samples. Selection of consensus sequences for the design of sensitive assays has been challenging due to sequence diversity and has led to the development of specific real-time RT-PCR assays for each genogroup. Thus, sample screening can require several replicates for amplification of each genogroup (without considering positive and negative controls or standard curves). This study reports the development of a generic assay that detects all three human norovirus genogroups on a qualitative basis using a one-step real-time RT-PCR assay. The generic assay achieved good specificity and sensitivity for all three genogroups, detected separately or in combination. At variance with multiplex assays, the choice of the same fluorescent dye for all three probes specific to each genogroup allows the levels of fluorescence to be added and may increase assay sensitivity when multiple strains from different genogroups are present. When it was applied to sewage sample extracts, this generic assay successfully detected norovirus in all samples found to be positive by the genogroup-specific RT-PCRs. The generic assay also identified all norovirus-positive samples among 157 archived nucleic acid shellfish extracts, including samples contaminated by all three genogroups. PMID:23956397

  19. Environmental detection of genogroup I, II, and IV noroviruses by using a generic real-time reverse transcription-PCR assay.

    PubMed

    Miura, Takayuki; Parnaudeau, Sylvain; Grodzki, Marco; Okabe, Satoshi; Atmar, Robert L; Le Guyader, Françoise S

    2013-11-01

    Norovirus is the most common agent implicated in food-borne outbreaks and is frequently detected in environmental samples. These viruses are highly diverse, and three genogroups (genogroup I [GI], GII, and GIV) infect humans. Being noncultivable viruses, real-time reverse transcription-PCR (RT-PCR) is the only sensitive method available for their detection in food or environmental samples. Selection of consensus sequences for the design of sensitive assays has been challenging due to sequence diversity and has led to the development of specific real-time RT-PCR assays for each genogroup. Thus, sample screening can require several replicates for amplification of each genogroup (without considering positive and negative controls or standard curves). This study reports the development of a generic assay that detects all three human norovirus genogroups on a qualitative basis using a one-step real-time RT-PCR assay. The generic assay achieved good specificity and sensitivity for all three genogroups, detected separately or in combination. At variance with multiplex assays, the choice of the same fluorescent dye for all three probes specific to each genogroup allows the levels of fluorescence to be added and may increase assay sensitivity when multiple strains from different genogroups are present. When it was applied to sewage sample extracts, this generic assay successfully detected norovirus in all samples found to be positive by the genogroup-specific RT-PCRs. The generic assay also identified all norovirus-positive samples among 157 archived nucleic acid shellfish extracts, including samples contaminated by all three genogroups. PMID:23956397

  20. A novel immunochromatographic assay based on a time-resolved chemiluminescence strategy for the multiplexed detection of ractopamine and clenbuterol.

    PubMed

    Wang, Wenwen; Su, Xiaoxiao; Ouyang, Hui; Wang, Lin; Fu, Zhifeng

    2016-04-21

    A novel multiplexed immunochromatographic assay (ICA) based on a time-resolved chemiluminescence (CL) strategy was developed for quantitative detection of β-agonists, by utilizing ractopamine (RAC) and clenbuterol (CLE) as the models. Different from conventional multiplexed ICA methods which usually require two or more test lines, this strategy was developed for detection of two β-agonists by using only one test line on the nitrocellulose membrane. In this study, horseradish peroxidase and alkaline phosphatase were used as the signal probes to label RAC antibody and CLE antibody, respectively. The two CL reactions with flash type and glow type kinetics characteristics were triggered simultaneously by injecting the coreactants, then the signals for RAC and CLE detections were recorded at 3 s and 300 s after coreactants injection, respectively. Owing to the utilization of CL detection, this protocol showed ideal sensitivity for quantitation. Under the optimal conditions, the detection limits for RAC and CLE were 0.17 ng mL(-1) and 0.067 ng mL(-1) (S/N = 3), respectively. The whole assay process can be accomplished within 20 min without complicated sample pretreatment. The proposed method was successfully applied for the detection of RAC and CLE in spiked swine urine. It opens up a new pathway for designing a low cost, time-efficiency and multiplexed strategy for rapid screening and field assay. PMID:27026603

  1. Differential display RT PCR of total RNA from human foreskin fibroblasts for investigation of androgen-dependent gene expression

    SciTech Connect

    Nitsche, E.M.; Moquin, A.; Adams, P.S.

    1996-05-03

    Male sexual differentiation is a process that involves androgen action via the androgen receptor. Defects in the androgen receptor, many resulting from point mutations in the androgen receptor gene, lead to varying degrees of impaired masculinization in chromosomally male individuals. To date no specific androgen regulated morphogens involved in this process have been identified and no marker genes are known that would help to predict further virilization in infants with partial androgen insensitivity. In the present study we first show data on androgen regulated gene expression investigated by differential display reverse transcription PCR (dd RT PCR) on total RNA from human neonatal genital skin fibroblasts cultured in the presence or absence of 100 nM testosterone. Using three different primer combinations, 54 cDNAs appeared to be regulated by androgens. Most of these sequences show the characteristics of expressed mRNAs but showed no homology to sequences in the database. However 15 clones with significant homology to previously cloned sequences were identified. Seven cDNAs appear to be induced by androgen withdrawal. Of these, five are similar to ETS (expression tagged sequences) from unknown genes; the other two show significant homology to the cDNAs of ubiquitin and human guanylate binding protein 2 (GBP-2). In addition, we have identified 8 cDNA clones which show homologies to other sequences in the database and appear to be upregulated in the presence of testosterone. Three differential expressed sequences show significant homology to the cDNAs of L-plastin and one to the cDNA of testican. This latter gene codes for a proteoglycan involved in cell social behavior and therefore of special interest in this context. The results of this study are of interest in further investigation of normal and disturbed androgen-dependent gene expression. 49 refs., 2 figs., 5 tabs.

  2. Selection of Stable Reference Genes for Quantitative RT-PCR Comparisons of Mouse Embryonic and Extra-Embryonic Stem Cells

    PubMed Central

    Veazey, Kylee J.; Golding, Michael C.

    2011-01-01

    Isolation and culture of both embryonic and tissue specific stem cells provide an enormous opportunity to study the molecular processes driving development. To gain insight into the initial events underpinning mammalian embryogenesis, pluripotent stem cells from each of the three distinct lineages present within the preimplantation blastocyst have been derived. Embryonic (ES), trophectoderm (TS) and extraembryonic endoderm (XEN) stem cells possess the developmental potential of their founding lineages and seemingly utilize distinct epigenetic modalities to program gene expression. However, the basis for these differing cellular identities and epigenetic properties remain poorly defined. Quantitative reverse transcription-polymerase chain reaction (qPCR) is a powerful and efficient means of rapidly comparing patterns of gene expression between different developmental stages and experimental conditions. However, careful, empirical selection of appropriate reference genes is essential to accurately measuring transcriptional differences. Here we report the quantitation and evaluation of fourteen commonly used references genes between ES, TS and XEN stem cells. These included: Actb, B2m, Hsp70, Gapdh, Gusb, H2afz, Hk2, Hprt, Pgk1, Ppia, Rn7sk, Sdha, Tbp and Ywhaz. Utilizing three independent statistical analysis, we identify Pgk1, Sdha and Tbp as the most stable reference genes between each of these stem cell types. Furthermore, we identify Sdha, Tbp and Ywhaz as well as Ywhaz, Pgk1 and Hk2 as the three most stable reference genes through the in vitro differentiation of embryonic and trophectoderm stem cells respectively. Understanding the transcriptional and epigenetic regulatory mechanisms controlling cellular identity within these distinct stem cell types provides essential insight into cellular processes controlling both embryogenesis and stem cell biology. Normalizing quantitative RT-PCR measurements using the geometric mean CT values obtained for the identified m

  3. Species-specific RT-PCR amplification of human enteroviruses: a tool for rapid species identification of uncharacterized enteroviruses.

    PubMed

    Oberste, M Steven; Maher, Kaija; Williams, Alford J; Dybdahl-Sissoko, Naomi; Brown, Betty A; Gookin, Michelle S; Peñaranda, Silvia; Mishrik, Nada; Uddin, Moyez; Pallansch, Mark A

    2006-01-01

    The 65 serotypes of human enteroviruses are classified into four species, Human enterovirus (HEV) A to D, based largely on phylogenetic relationships in multiple genome regions. The 3'-non-translated region of enteroviruses is highly conserved within a species but highly divergent between species. From this information, species-specific RT-PCR primers were developed that can be used to rapidly screen collections of enterovirus isolates to identify species of interest. The four primer pairs were 100 % specific when tested against enterovirus prototype strains and panels of isolates of known serotype (a total of 193 isolates). For evaluation in a typical application, the species-specific primers were used to screen 186 previously uncharacterized non-polio enterovirus isolates. The HEV-B primers amplified 68.3 % of isolates, while the HEV-A and HEV-C primers accounted for 9.7 and 11.3 % of isolates, respectively; no isolates were amplified with the HEV-D primers. Twelve isolates (6.5 %) were amplified by more than one primer set and eight isolates (4.3 %) were not amplified by any of the four primer pairs. Serotypes were identified by partial sequencing of the VP1 capsid gene, and in every case sequencing confirmed that the species-specific PCR result was correct; the isolates that were amplified by more than one species-specific primer pair were mixtures of two (11 isolates) or three (one isolate) species of viruses. The eight isolates that were not amplified by the species-specific primers comprised four new serotypes (EV76, EV89, EV90 and EV91) that appear to be unique members of HEV-A based on VP1, 3D and 3'-non-translated region sequences. PMID:16361424

  4. Quantification of mRNA in Salmonella sp. seeded soil and chicken manure using magnetic capture hybridization RT-PCR.

    PubMed

    Jacobsen, Carsten Suhr; Holben, William E

    2007-05-01

    Direct quantification of mRNA from Salmonella sp. seeded for 1 h to soil and chicken manure was accomplished using magnetic capture hybridization as a purification technique. This detection strategy targeted the invA gene present in Salmonella sp. After cell lysis, phenol/chloroform purification and isopropanol precipitation, the RNA extract was combined with the hybridization probe conjugated to paramagnetic beads. After hybridization, the captured nucleic acids were released by denaturation and purified of contaminating DNA using DNase. The resulting RNA was of high purity and there was no need for dilution of the samples prior to RT-PCR. The developed procedure was reproducibly used to quantify Salmonella sp. in high organic agricultural soil. The detection limit for mRNA using ordinary quantitative PCR (employing SYBRgreen-based detection) was 5 x 10(4)Salmonella sp. cells per gram of soil. Chicken manure amended into soil (1:4 w/w) did not reduce the ability to quantify Salmonella sp. mRNA in soil. Pasteurization (65 degrees C, 30 min) of chicken manure containing Salmonella sp. dramatically reduced the detection of invA mRNA (requiring 42 qPCR cycles for detection versus 26 cycles in unpasteurized manure), presumably due to degradation of the invA mRNA in Salmonella sp. cells killed by pasteurization. By contrast, DNA-based qPCR still detected Salmonella sp. in the pasteurized manure. Thus, in this case using samples seeded with fresh Salmonella sp. the mRNA-based detection appears to be superior to minimizing false-positive detection which was prevalent with DNA-based qPCR. PMID:17383760

  5. Factor VIII (F8) inversions in severe hemophilia A: Male germ cell origin and diagnosis with RT-PCR

    SciTech Connect

    Antonarakis, S.E. |; Rossiter, J.P.; Young, M.

    1994-09-01

    The Factor VIII (F8) gene, which is defective in hemophilia A, is located in the most telomeric megabase of Xq. Inversions due to intrachromosomal homologous recombination between mispaired copies of gene A located within intron 22 of the gene and about 500 kb telomeric to it account for nearly half of the cases of severe hemophilia A. We hypothesized that pairing of Xq with its homolog inhibits the inversion process, and that therefore the event originates predominantly in male germ cells. In all 21 informative cases in which the inversion originated in a maternal grandparent, DNA polymorphism analysis using markers within or very closely linked to F8, determined that it occurred in the male germline. In addition, all but one of 56 mothers of sporadic cases due to inversions were carriers. The data indicate that the F8 gene inversions leading to severe hemophilia A occur almost exclusively in male germ cells. The mean age of maternal grandfathers at the birth of their carrier daughters was 29.9 years (13 cases), i.e. not different from the mean paternal age in the general population, supporting the hypothesis that the inversions occur in meiosis. The inversions can be diagnosed by Southern blot analysis. For more rapid diagnosis we have used RT-PCR of RNA ectopically expressed in blood. Oligonucleotides were used to PCR amplify, after the initial RT reaction of RNA samples using random hexamers, either the normal transcript (F8 exons 21 to 24;312 bp product) or the novel abnormal transcript that is generated after the inversion. Both type 1 and 2 inversions can be recognized in affecteds and carriers by the presence of the diagnostic PcR product of 248 bp. Correct diagnoses were made in samples from 6 patients and 2 carriers with type 1 inversions, 2 patients and 2 carriers with type 2 inversions and 5 normal controls.

  6. Comprehensive Selection of Reference Genes for Gene Expression Normalization in Sugarcane by Real Time Quantitative RT-PCR

    PubMed Central

    Ling, Hui; Wu, Qibin; Guo, Jinlong; Xu, Liping; Que, Youxiong

    2014-01-01

    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

  7. Microarray multiplex assay for the simultaneous detection and discrimination of hepatitis B, hepatitis C, and human immunodeficiency type-1 viruses in human blood samples

    SciTech Connect

    Hsia, Chu Chieh . E-mail: chuchieh.hsia@fda.hhs.gov; Chizhikov, Vladimir E.; Yang, Amy X.; Selvapandiyan, Angamuthu; Hewlett, Indira; Duncan, Robert; Puri, Raj K.; Nakhasi, Hira L.; Kaplan, Gerardo G.

    2007-05-18

    Hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type-1 (HIV-1) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health worldwide. We developed a microarray multiplex assay for the simultaneous detection and discrimination of these three viruses. The microarray consists of 16 oligonucleotide probes, immobilized on a silylated glass slide. Amplicons from multiplex PCR were labeled with Cy-5 and hybridized to the microarray. The assay detected 1 International Unit (IU), 10 IU, 20 IU of HBV, HCV, and HIV-1, respectively, in a single multiplex reaction. The assay also detected and discriminated the presence of two or three of these viruses in a single sample. Our data represent a proof-of-concept for the possible use of highly sensitive multiplex microarray assay to screen and confirm the presence of these viruses in blood donors and patients.

  8. Evaluation of a Modular Multiplex-PCR Methicillin-Resistant Staphylococcus aureus Detection Assay Adapted for mecC Detection

    PubMed Central

    Larsen, Anders R.; Skov, Robert L.; Paterson, Gavin K.; Holmes, Mark A.; Sabat, Artur J.; Friedrich, Alexander W.; Köck, Robin; Peters, Georg; Kriegeskorte, André

    2013-01-01

    A mecC (mecALGA251)-adapted multiplex PCR-based methicillin-resistant Staphylococcus aureus (MRSA) detection assay was evaluated using an international, spa-typed Staphylococcus aureus collection comprising 51 mecC-positive MRSA, 240 mecA-positive MRSA, and 50 mecA- and mecC-negative methicillin-susceptible S. aureus (MSSA) isolates. The assay showed 100% sensitivity and specificity for S. aureus species identification as well as for mecA and mecC detection. PMID:23515551

  9. Development of silicon photonic microring resonator biosensors for multiplexed cytokine assays and in vitro diagnostics

    NASA Astrophysics Data System (ADS)

    Luchansky, Matthew Sam

    In order to guide critical care therapies that are personalized to a patient's unique disease state, a diagnostic or theranostic medical device must quickly provide a detailed biomolecular understanding of disease onset and progression. This detailed molecular understanding of cellular processes and pathways requires the ability to measure multiple analytes in parallel. Though many traditional sensing technologies for biomarker analysis and fundamental biological studies (i.e. enzyme-linked immunosorbent assays, real-time polymerase chain reaction, etc.) rely on single-parameter measurements, it has become increasingly clear that the inherent complexity of many human illnesses and pathways necessitates quantitative and multiparameter analysis of biological samples. Currently used analytical methods are deficient in that they often provide either highly quantitative data for a single biomarker or qualitative data for many targets, but methods that simultaneously provide highly quantitative analysis of many targets have yet to be adequately developed. Fields such as medical diagnostics and cellular biology would benefit greatly from a technology that enables rapid, quantitative and reproducible assays for many targets within a single sample. In an effort to fill this unmet need, this doctoral dissertation describes the development of a clinically translational biosensing technology based on silicon photonics and developed in the chemistry research laboratory of Ryan C. Bailey. Silicon photonic microring resonators, a class of high-Q optical sensors, represent a promising platform for rapid, multiparameter in vitro measurements. The original device design utilizes 32-ring arrays for real-time biomolecular sensing without fluorescent labels, and these optical biosensors display great potential for more highly multiplexed (100s-1000s) measurements based on the impressive scalability of silicon device fabrication. Though this technology can be used to detect a variety of

  10. Evaluation of Multiplex Tandem Real-Time PCR for Detection of Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis in Clinical Stool Samples ▿

    PubMed Central

    Stark, D.; Al-Qassab, S. E.; Barratt, J. L. N.; Stanley, K.; Roberts, T.; Marriott, D.; Harkness, J.; Ellis, J. T.

    2011-01-01

    The aim of this study was to describe the first development and evaluation of a multiplex tandem PCR (MT-PCR) assay for the detection and identification of 4 common pathogenic protozoan parasites, Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis, from human clinical samples. A total of 472 fecal samples submitted to the Department of Microbiology at St. Vincent's Hospital were included in the study. The MT-PCR assay was compared to four real-time PCR (RT-PCR) assays and microscopy by a traditional modified iron hematoxylin stain. The MT-PCR detected 28 G. intestinalis, 26 D. fragilis, 11 E. histolytica, and 9 Cryptosporidium sp. isolates. Detection and identification of the fecal protozoa by MT-PCR demonstrated 100% correlation with the RT-PCR results, and compared to RT-PCR, MT-PCR exhibited 100% sensitivity and specificity, while traditional microscopy of stained fixed fecal smears exhibited sensitivities and specificities of 56% and 100% for Cryptosporidium spp., 38% and 99% for D. fragilis, 47% and 97% for E. histolytica, and 50% and 100% for G. intestinalis. No cross-reactivity was detected in 100 stool samples containing various other bacterial, viral, and protozoan species. The MT-PCR assay was able to provide rapid, sensitive, and specific simultaneous detection and identification of the four most important diarrhea-causing protozoan parasites that infect humans. This study also highlights the lack of sensitivity demonstrated by microscopy, and thus, molecular methods such as MT-PCR must be considered the diagnostic methods of choice for enteric protozoan parasites. PMID:21048004

  11. Simultaneous Detection of Major Drug Resistance Mutations of HIV-1 Subtype B Viruses from Dried Blood Spot Specimens by Multiplex Allele-Specific Assay.

    PubMed

    Zhang, Guoqing; Cai, Fangping; de Rivera, Ivette Lorenzana; Zhou, Zhiyong; Zhang, Jing; Nkengasong, John; Gao, Feng; Yang, Chunfu

    2016-01-01

    A multiplex allele-specific (MAS) assay has been developed for the detection of HIV-1 subtype C drug resistance mutations (DRMs). We have optimized the MAS assay to determine subtype B DRMs in dried blood spots (DBS) collected from patients on antiretroviral therapy. The new assay accurately detected DRMs, including low-abundance mutations that were often missed by Sanger sequencing. PMID:26560533

  12. Simultaneous Detection of Major Drug Resistance Mutations of HIV-1 Subtype B Viruses from Dried Blood Spot Specimens by Multiplex Allele-Specific Assay

    PubMed Central

    Zhang, Guoqing; Cai, Fangping; de Rivera, Ivette Lorenzana; Zhou, Zhiyong; Zhang, Jing; Nkengasong, John

    2015-01-01

    A multiplex allele-specific (MAS) assay has been developed for the detection of HIV-1 subtype C drug resistance mutations (DRMs). We have optimized the MAS assay to determine subtype B DRMs in dried blood spots (DBS) collected from patients on antiretroviral therapy. The new assay accurately detected DRMs, including low-abundance mutations that were often missed by Sanger sequencing. PMID:26560533

  13. Highly Sensitive Multiplex Assay for Detection of Human Immunodeficiency Virus Type 1 and Hepatitis C Virus RNA

    PubMed Central

    Giachetti, C.; Linnen, J. M.; Kolk, D. P.; Dockter, J.; Gillotte-Taylor, K.; Park, M.; Ho-Sing-Loy, M.; McCormick, M. K.; Mimms, L. T.; McDonough, S. H.

    2002-01-01

    Various nucleic acid assays have been developed and implemented for diagnostics and therapeutic monitoring of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infections. The high-throughput, semiautomated assays described here were developed to provide a method suitable for screening plasma specimens for the presence of HIV-1 and HCV RNAs. Three assays were developed: a multiplex HIV-1/HCV assay for simultaneous detection of HIV-1 and HCV, and discriminatory assays for specific detection of HIV-1 and HCV. The assay systems utilize three proprietary technologies: (i) target capture-based sample preparation, (ii) transcription-mediated amplification (TMA), and (iii) hybridization protection assay (HPA). An internal control is incorporated into each reaction to control for every step of the assay and identify random false-negative reactions. The assays demonstrated a sensitivity of at least 100 copies/ml for each target, and they detected with similar sensitivity all major variants of HCV and HIV-1, including HIV-1 group O strains. Assay sensitivity for one virus was not affected by the presence of the other. The specificity of these TMA-driven assays was ≥99.5% in both normal donor specimens and plasma containing potentially interfering substances or other blood-borne pathogens. Statistical receiver operating characteristic plots of 1 − specificity versus sensitivity data determined very wide analyte cutoff values for each assay at the point at which the assay specificity and sensitivity were both ≥99.5%. The sensitivity, specificity, and throughput capability predict that these assays will be valuable for large-volume plasma screening, either in a blood bank setting or in other diagnostic applications. PMID:12089255

  14. Comparison of three multiplex PCR assays for the detection of respiratory viral infections: evaluation of xTAG respiratory virus panel fast assay, RespiFinder 19 assay and RespiFinder SMART 22 assay

    PubMed Central

    2012-01-01

    Background A broad spectrum of pathogens is causative for respiratory tract infections, but symptoms are mostly similar. Therefore, the identification of the causative viruses and bacteria is only feasible using multiplex PCR or several monoplex PCR tests in parallel. Methods The analytical sensitivity of three multiplex PCR assays, RespiFinder-19, RespiFinder-SMART-22 and xTAG-Respiratory-Virus-Panel-Fast-Assay (RVP), were compared to monoplex real-time PCR with quantified standardized control material. All assays include the most common respiratory pathogens. Results To compare the analytical sensitivity of the multiplex assays, samples were inoculated with 13 different quantified viruses in the range of 101 to 105 copies/ml. Concordant results were received for rhinovirus, whereas the RVP detected influenzavirus, RSV and hMPV more frequently in low concentrations. The RespiFinder-19 and the RespiFinder-SMART-22 showed a higher analytical sensitivity for adenoviruses and coronaviruses, whereas the RVP was incapable to detect adenovirus and coronavirus in concentrations of 104 copies/ml. The RespiFinder-19 and RespiFinder-SMART-22A did not detect influenzaviruses (104 copies/ml) and RSV (103 copies/ml). The detection of all 13 viruses in one sample was only achieved using monoplex PCR. To analyze possible competitive amplification reactions between the different viruses, samples were further inoculated with only 4 different viruses in one sample. Compared to the detection of 13 viruses in parallel, only a few differences were found. The incidence of respiratory viruses was compared in tracheal secretion (TS) samples (n = 100) of mechanically ventilated patients in winter (n = 50) and summer (n = 50). In winter, respiratory viruses were detected in 32 TS samples (64%) by RespiFinder-19, whereas the detection rate with RVP was only 22%. The most frequent viruses were adenovirus (32%) and PIV-2 (20%). Multiple infections were detected in 16 TS samples (32

  15. 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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  16. Rapid detection of Shigella and enteroinvasive Escherichia coli in produce enrichments by a conventional multiplex PCR assay.

    PubMed

    Binet, Rachel; Deer, Deanne M; Uhlfelder, Samantha J

    2014-06-01

    Faster detection of contaminated foods can prevent adulterated foods from being consumed and minimize the risk of an outbreak of foodborne illness. A sensitive molecular detection method is especially important for Shigella because ingestion of as few as 10 of these bacterial pathogens can cause disease. The objectives of this study were to compare the ability of four DNA extraction methods to detect Shigella in six types of produce, post-enrichment, and to evaluate a new and rapid conventional multiplex assay that targets the Shigella ipaH, virB and mxiC virulence genes. This assay can detect less than two Shigella cells in pure culture, even when the pathogen is mixed with background microflora, and it can also differentiate natural Shigella strains from a control strain and eliminate false positive results due to accidental laboratory contamination. The four DNA extraction methods (boiling, PrepMan Ultra [Applied Biosystems], InstaGene Matrix [Bio-Rad], DNeasy Tissue kit [Qiagen]) detected 1.6 × 10(3)Shigella CFU/ml post-enrichment, requiring ∼18 doublings to one cell in 25 g of produce pre-enrichment. Lower sensitivity was obtained, depending on produce type and extraction method. The InstaGene Matrix was the most consistent and sensitive and the multiplex assay accurately detected Shigella in less than 90 min, outperforming, to the best of our knowledge, molecular assays currently in place for this pathogen. PMID:24549197

  17. A novel multiplexing, polymerase chain reaction-based assay for the analysis of chromosome 18q status in colorectal cancer.

    PubMed

    Erill, Nadina; Colomer, Anna; Calvo, Miquel; Vidal, August; Román, Ruth; Verdú, Montse; Cordón-Cardó, Carlos; Puig, Xavier

    2005-10-01

    Chromosome 18q allelic loss has been reported to have prognostic significance in stage II colorectal carcinoma. We have developed a fluorescent multiplex polymerase chain reaction assay to analyze five microsatellite markers (D18S55, D18S58, D18S61, D18S64, and D18S69) for allelic loss at the long arm of chromosome 18. Amplicon detection and evaluation was accomplished by capillary electrophoresis using an ABI 310 genetic analyzer. Robustness of the assay when performed on DNA extracted from formalin-fixed, paraffin-embedded tissue sections was confirmed by analyzing its repeatability and reproducibility. Allelic loss was assessed in 61 stage II colorectal tumors and was detected in 58% (31 of 53) of tumors not showing instability. As part of the study, results of 207 previous polymerase chain reaction/polyacrylamide-based assays were re-evaluated by two independent observers to determine the degree of concordance of visual evaluation. In the case of stage II colorectal tumors, when electropherogram results were compared with those obtained from visual evaluation of the same markers after polyacrylamide gel electrophoresis, discrepancies between observers were detected in 16.4% of determinations. In conclusion, we have developed a robust and reliable assay for multiplexed loss of heterozygosity determination that improves assessment of chromosome 18q allelic loss in colorectal tumors processed as routine formalin-fixed, paraffin-embedded specimens. PMID:16237217

  18. Rapid and Multiplexed MicroRNA Diagnostic Assay Using Quantum Dot-Based Förster Resonance Energy Transfer.

    PubMed

    Qiu, Xue; Hildebrandt, Niko

    2015-08-25

    The detection of next generation microRNA (miRNA) biomarkers has become a highly important aspect for clinical diagnostics. We use multiplexed Förster resonance energy transfer (FRET) between a luminescent Tb complex and three different semiconductor quantum dots (QDs) to sensitively detect three different miRNAs from a single 150 μL sample with ca. 1 nM (subpicomol) detection limits. The rapid and amplification-free mix-and-measure assay format is based on careful design of miRNA base pairing and stacking to selectively detect different miRNAs with very strong sequence homologies. Clinical applicability is demonstrated by sensitive multiplexed quantification of three miRNAs at low (2 to 10 nM) and varying concentrations in samples that contained up to 10% serum. PMID:26192765

  19. Minimizing antibody cross-reactivity in multiplex detection of biomarkers in paper-based point-of-care assays.

    PubMed

    Dias, J T; Lama, L; Gantelius, J; Andersson-Svahn, H

    2016-04-21

    Highly multiplexed immunoassays could allow convenient screening of hundreds or thousands of protein biomarkers simultaneously in a clinical sample such as serum or plasma, potentially allowing improved diagnostic accuracy and clinical management of many conditions such as autoimmune disorders, infections, and several cancers. Currently, antibody microarray-based tests are limited in part due to cross reactivity from detection antibody reagents. Here we present a strategy that reduces the cross-reactivity between nanoparticle-bound reporter antibodies through the application of ultrasound energy. By this concept, it was possible to achieve a sensitivity 10(3)-fold (5 pg mL(-1)) lower than when no ultrasound was applied (50 ng mL(-1)) for the simultaneous detection of three different antigens. The detection limits and variability achieved with this technique rival those obtained with other types of multiplex sandwich assays. PMID:27030365

  20. [Deployment of a mobile RT-PCR laboratory molecular biology to deal with the A(H1N1) challenge in Kaboul].

    PubMed

    Maslin, J; Ducher, P; Fourel, D; Causse Le Dorze, P

    2010-11-01

    Since October 2009, the fear of swine flu spread in Afghanistan and severe cases were observed among NATO soldiers. Two patients were hospitalized in an Intensive Care Unit. To face this new challenge, the French Health Service decided the deployment of a mobile RT-PCR laboratory molecular biology in the Kabul International Military Hospital. We describe the implementation of the mobile RT-PCR laboratory for the diagnosis of A(H1N1). The analysis of the first nasopharyngeal samples confirmed the presence of this virus in Afghanistan. The peak of positive cases was observed in mid-November 2009, and some cluster cases were observed among units deployed on the field. PMID:20650585

  1. Minimizing antibody cross-reactivity in multiplex detection of biomarkers in paper-based point-of-care assays

    NASA Astrophysics Data System (ADS)

    Dias, J. T.; Lama, L.; Gantelius, J.; Andersson-Svahn, H.

    2016-04-01

    Highly multiplexed immunoassays could allow convenient screening of hundreds or thousands of protein biomarkers simultaneously in a clinical sample such as serum or plasma, potentially allowing improved diagnostic accuracy and clinical management of many conditions such as autoimmune disorders, infections, and several cancers. Currently, antibody microarray-based tests are limited in part due to cross reactivity from detection antibody reagents. Here we present a strategy that reduces the cross-reactivity between nanoparticle-bound reporter antibodies through the application of ultrasound energy. By this concept, it was possible to achieve a sensitivity 103-fold (5 pg mL-1) lower than when no ultrasound was applied (50 ng mL-1) for the simultaneous detection of three different antigens. The detection limits and variability achieved with this technique rival those obtained with other types of multiplex sandwich assays.Highly multiplexed immunoassays could allow convenient screening of hundreds or thousands of protein biomarkers simultaneously in a clinical sample such as serum or plasma, potentially allowing improved diagnostic accuracy and clinical management of many conditions such as autoimmune disorders, infections, and several cancers. Currently, antibody microarray-based tests are limited in part due to cross reactivity from detection antibody reagents. Here we present a strategy that reduces the cross-reactivity between nanoparticle-bound reporter antibodies through the application of ultrasound energy. By this concept, it was possible to achieve a sensitivity 103-fold (5 pg mL-1) lower than when no ultrasound was applied (50 ng mL-1) for the simultaneous detection of three different antigens. The detection limits and variability achieved with this technique rival those obtained with other types of multiplex sandwich assays. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr09207h

  2. Reference Gene Selection and Evaluation for Gene Expression Studies Using qRT-PCR in the White-Backed Planthopper, Sogatella furcifera (Hemiptera: Delphacidae).

    PubMed

    An, Xing-kui; Hou, Mao-lin; Liu, Yu-di

    2016-04-01

    The white-backed planthopper, Sogatella furcifera (Hemiptera, Delphacidae), is one of the most devastating rice pests. For a better control strategy, various genetic studies have been conducted using reverse-transcription quantitative real-time polymerase chain reaction (qRT-PCR). The appropriate application of qRT-PCR requires reliable endogenous controls; however, studies on this aspect of the white-backed planthopper are lacking. In the present study, nine commonly used reference genes, elongation factor 1-α (EF1-α), polyubiquitin (UB), ribosomal protein S18 (RPS18), actin 1 (ACT), α-1 tubulin (TUB), glyceraldehyde-3-phosphate (GAPDH), ribosomal protein L9 (RPL9), ribosomal protein L10 (RPL10), and 18S ribosomal RNA (18S), were evaluated by qRT-PCR for their expression stability under four different experimental conditions (different developmental stages, acquisition of Southern rice black-streaked dwarf virus (SRBSDV), different tissues, and different temperature stress). These results were analyzed using four software programs (geNorm, NormFinder, BestKeeper, and the delta Ct method) and a Web-based comprehensive tool RefFinder to compare and rank candidate reference genes. According to the results of RefFinder analysis, which integrates the abovementioned four software programs, TUB was ranked as the most suitable reference gene at different developmental stages and under different temperature stress, and GAPDH and RPL9 showed the highest stability for acquisition of SRBSDV and different tissues, respectively. These results will provide a solid foundation for future gene expression study on the white-backed planthopper, and also will give aids in establishing a standardized qRT-PCR procedure for other related insects. PMID:26612891

  3. Quantification of silkworm coactivator of MBF1 mRNA by SYBR Green I real-time RT-PCR reveals tissue- and stage-specific transcription levels.

    PubMed

    Li, Guang-li; Roy, Bhaskar; Li, Xing-hua; Yue, Wan-fu; Wu, Xiao-feng; Liu, Jian-mei; Zhang, Chuan-xi; Miao, Yun-gen

    2009-05-01

    Transcriptional coactivators play a crucial role in gene transcription and expression. Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator necessary for transcriptional activation caused by DNA-binding activators, such as FTZ-F1 and GCN4. Until now, very few studies have been reported in the silkworm. We selected the Bombyx mori because it is a model insect and acts as an economic animal for silk industry. In this study, we conducted the quantitative analysis of MBF1 mRNA in silkworm B. mori L. with actin (A3) as internal standard by means of SYBR Green I real-time RT-PCR method. The total RNA was extracted from the silk gland, epidermis, fat body, and midguts of the fifth instar B. mori larvae. The mRNA was reverse transcripted, and the cDNA fragments of MBF1 mRNA and actin gene were amplified by RT-PCR using specific primers. MBF1 mRNA expression in different tissues of silkworm B. mori L. was quantified using standardized SYBR Green I RT-PCR. The results suggested MBF1 gene was expressed in all investigated organs but highly expressed in the silk gland, showing its relation to biosynthesis of silk proteins. PMID:18612846

  4. Detection, quantitation and identification of enteroviruses from surface waters and sponge tissue from the Florida Keys using real-time RT-PCR

    USGS Publications Warehouse

    Donaldson, K.A.; Griffin, Dale W.; Paul, J.H.

    2002-01-01

    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.

  5. Genome-Wide Identification of New Reference Genes for qRT-PCR Normalization under High Temperature Stress in Rice Endosperm

    PubMed Central

    Dai, Ji-Song; Li, Yongqing; Zhu, Ying

    2015-01-01

    qRT-PCR is one of the most popular approaches to analyze specific gene expression level, and stably expressed reference genes are essential to obtain reliable results. However, many reference genes are only stable under certain circumstances and different reference genes might be required in different experiments. High temperature is a common stress that affects rice endosperm development and it has become a hot topic recently. Although study about reference genes at different developmental stages in rice has been reported, these genes may not be suitable to study high temperature mediated responses especially in endosperm. In our quest for proper reference genes to quantify gene expression in rice endosperm under high temperature, we studied 6 candidate genes selected from the transcriptome data and 11 housekeeping genes. All genes were analyzed with qRT-PCR and the expression stability was assessed with software geNorm and NormFinder. Fb15 and eIF-4a were identified as the two most stable genes in endosperm at different developmental stages, while high temperature treatment has a least effect on expression of Fb15 and UBQ5 in rice endosperm. Our results provide some good candidate reference genes for qRT-PCR normalization in rice endosperm under different temperatures. PMID:26555942

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

    PubMed

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

    2015-02-01

    A rapid and accurate method of detection and differentiation of virulent and avirulent Newcastle disease virus (NDV) pathotypes was developed. The NDV detection was carried out for different domestic avian field isolates and pigeon paramyxo virus-1 (25 field isolates and 9 vaccine strains) by using APMV-I "fusion" (F) gene Class II specific external primer A and B (535bp), internal primer C and D (238bp) based reverses transcriptase PCR (RT-PCR). The internal degenerative reverse primer D is specific for F gene cleavage position of virulent strain of NDV. The nested RT-PCR products of avirulent strains showed two bands (535bp and 424bp) while virulent strains showed four bands (535bp, 424bp, 349bp and 238bp) on agar gel electrophoresis. This is the first report regarding development and use of degenerate primer based nested RT-PCR for accurate detection and differentiation of NDV pathotypes by demonstrating multiple PCR band patterns. Being a rapid, simple, and economical test, the developed method could serve as a valuable alternate diagnostic tool for characterizing NDV isolates and carrying out molecular epidemiological surveillance studies for this important pathogen of poultry. PMID:25449112

  7. Development and validation of a multiplex conventional PCR assay for simultaneous detection and grouping of porcine bocaviruses.

    PubMed

    Zheng, Xiaowen; Liu, Gaopeng; Opriessnig, Tanja; Wang, Zining; Yang, Zongqi; Jiang, Yonghou

    2016-10-01

    Porcine bocavirus (PBoV), a newly described porcine parvovirus, has received attention because it can be commonly identified in clinically affected pigs including pigs with post-weaning multisystemic wasting syndrome (PWMS) and pigs with diarrhea. In recent years, novel PBoVs have been identified and were classified into three genogroups, but the ability to detect and classify these novel PBoVs is not comprehensive to date. In this study, a multiplex conventional PCR assay for simultaneous detection and grouping of PBoVs was developed by screening combinations of mixed primer pairs followed by optimization of the PCR conditions. This method exclusively amplifies targeted fragments of 531bp from the VP1 gene of PBoV G1, 291bp from the NP1 gene of PBoV G2, and 384bp from the NP1/VP1 gene of PBoV G3. The assay has a detection limit of 1.0×10(3)copies/μL for PBoV G1 4.5×10(3) for PBoV G2 and 3.8×10(3) for PBoV G3 based on testing mixed purified plasmid constructs containing the specific viral target fragments. The performance of the multiplex PCR assay was comparable to that of the single PCRs which used the same primer pairs. Using the newly established multiplex PCR assay, 227 field samples including faeces, serum and tissue samples from pigs were investigated. All three PBoV genogroups were detected in the clinical samples with a detection rate of 1.3%, 2.6% and 12.3%, respectively for PBoV G1, G2 and G3. Additionally, coinfections with two or more PBoV were detected in 1.7% of the samples investigated. These results indicate the multiplex PCR assay is specific, sensitive and rapid, and can be used for the detection and differentiation of single and multiple infections of the three PBoV genogroups in pigs. PMID:27448821

  8. Development of a multiplex PCR assay for identification of Klebsiella pneumoniae hypervirulent clones of capsular serotype K2.

    PubMed

    Bialek-Davenet, Suzanne; Criscuolo, Alexis; Ailloud, Florent; Passet, Virginie; Nicolas-Chanoine, Marie-Hélène; Decré, Dominique; Brisse, Sylvain

    2014-12-01

    Hypervirulent Klebsiella pneumoniae isolates of capsular serotype K2 (hvKP-K2) that cause community-acquired invasive infections represent several unrelated clones, which all belong to phylogenetic group KpI. These clones can be recognized using multilocus sequence typing and genomic analyses, but no rapid method currently exists to differentiate them. In this work, a multiplex PCR assay was developed to identify three hvKP-K2 groups: (i) sequence type (ST)86; (ii) ST380 and ST679 (i.e. clonal group 380); and (iii) ST65 and ST375. A specific genetic marker, Kp50233, allowing K. pneumoniae sensu stricto (corresponding to phylogroup KpI) to be distinguished from closely related species, was included in the assay. This PCR assay will be useful in better defining the epidemiology and clinical features of emerging virulent K. pneumoniae clones. PMID:25261063

  9. A two-tube multiplex reverse transcription PCR assay for simultaneous detection of viral and bacterial pathogens of infectious diarrhea.

    PubMed

    Wang, Ji; Xu, Ziqian; Niu, Peihua; Zhang, Chen; Zhang, Jingyun; Guan, Li; Kan, Biao; Duan, Zhaojun; Ma, Xuejun

    2014-01-01

    Diarrhea caused by viral and bacterial infections is a major health problem in developing countries. The purpose of this study is to develop a two-tube multiplex PCR assay using automatic electrophoresis for simultaneous detection of 13 diarrhea-causative viruses or bacteria, with an intended application in provincial Centers for Diseases Control and Prevention, China. The assay was designed to detect rotavirus A, norovirus genogroups GI and GII, human astrovirus, enteric adenoviruses, and human bocavirus (tube 1), and Salmonella, Vibrio parahaemolyticus, diarrheagenic Escherichia coli, Campylobacter jejuni, Shigella, Yersinia, and Vibrio cholera (tube 2). The analytical specificity was examined with positive controls for each pathogen. The analytical sensitivity was evaluated by performing the assay on serial tenfold dilutions of in vitro transcribed RNA, recombinant plasmids, or bacterial culture. A total of 122 stool samples were tested by this two-tube assay and the results were compared with those obtained from reference methods. The two-tube assay achieved a sensitivity of 20-200 copies for a single virus and 10(2)-10(3) CFU/mL for bacteria. The clinical performance demonstrated that the two-tube assay had comparable sensitivity and specificity to those of reference methods. In conclusion, the two-tube assay is a rapid, cost-effective, sensitive, specific, and high throughput method for the simultaneous detection of enteric bacteria and virus. PMID:24711998

  10. Rapid Focused Sequencing: A Multiplexed Assay for Simultaneous Detection and Strain Typing of Bacillus anthracis, Francisella tularensis, and Yersinia pestis

    PubMed Central

    Zolotova, Anna; Tan, Eugene; Selden, Richard F.

    2013-01-01

    Background The intentional release of Bacillus anthracis in the United States in 2001 has heightened concern about the use of pathogenic microorganisms in bioterrorism attacks. Many of the deadliest bacteria, including the Class A Select Agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis, are highly infectious via the pulmonary route when released in aerosolized form. Hence, rapid, sensitive, and reliable methods for detection of these biothreats and characterization of their potential impact on the exposed population are of critical importance to initiate and support rapid military, public health, and clinical responses. Methodology/Principal Findings We have developed microfluidic multiplexed PCR and sequencing assays based on the simultaneous interrogation of three pathogens per assay and ten loci per pathogen. Microfluidic separation of amplified fluorescently labeled fragments generated characteristic electrophoretic signatures for identification of each agent. The three sets of primers allowed significant strain typing and discrimination from non-pathogenic closely-related species and environmental background strains based on amplicon sizes alone. Furthermore, sequencing of the 10 amplicons per pathogen, termed “Rapid Focused Sequencing,” allowed an even greater degree of strain discrimination and, in some cases, can be used to determine virulence. Both amplification and sequencing assays were performed in microfluidic biochips developed for fast thermal cycling and requiring 7 µL per reaction. The 30-plex sequencing assay resulted in genotypic resolution of 84 representative strains belonging to each of the three biothreat species. Conclusions/Significance The microfluidic multiplexed assays allowed identification and strain differentiation of the biothreat agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis and clear discrimination from closely-related species and several environmental background strains. The

  11. Multiplex Real-Time PCR Assays that Measure the Abundance of Extremely Rare Mutations Associated with Cancer

    PubMed Central

    Vargas, Diana Y.; Kramer, Fred Russell; Tyagi, Sanjay; Marras, Salvatore A. E.

    2016-01-01

    We describe the use of “SuperSelective” primers that enable the detection and quantitation of somatic mutations whose presence relates to cancer diagnosis, prognosis, and therapy, in real-time PCR assays that can potentially analyze rare DNA fragments present in blood samples (liquid biopsies). The design of these deoxyribonucleotide primers incorporates both a relatively long “5' anchor sequence” that hybridizes strongly to target DNA fragments, and a very short, physically and functionally separate, “3' foot sequence” that is perfectly complementary to the mutant target sequence, but mismatches the wild-type sequence. As few as ten mutant fragments can reliably be detected in the presence of 1,000,000 wild-type fragments, even when the difference between the mutant and the wild type is only a single nucleotide polymorphism. Multiplex PCR assays employing a set of SuperSelective primers, and a corresponding set of differently colored molecular beacon probes, can be used in situations where the different mutations, though occurring in different cells, are located in the same codon. These non-symmetric real-time multiplex PCR assays contain limited concentrations of each SuperSelective primer, thereby enabling the simultaneous determination of each mutation’s abundance by comparing its threshold value to the threshold value of a reference gene present in the sample. PMID:27244445

  12. Multiplex Real-Time PCR Assays that Measure the Abundance of Extremely Rare Mutations Associated with Cancer.

    PubMed

    Vargas, Diana Y; Kramer, Fred Russell; Tyagi, Sanjay; Marras, Salvatore A E

    2016-01-01

    We describe the use of "SuperSelective" primers that enable the detection and quantitation of somatic mutations whose presence relates to cancer diagnosis, prognosis, and therapy, in real-time PCR assays that can potentially analyze rare DNA fragments present in blood samples (liquid biopsies). The design of these deoxyribonucleotide primers incorporates both a relatively long "5' anchor sequence" that hybridizes strongly to target DNA fragments, and a very short, physically and functionally separate, "3' foot sequence" that is perfectly complementary to the mutant target sequence, but mismatches the wild-type sequence. As few as ten mutant fragments can reliably be detected in the presence of 1,000,000 wild-type fragments, even when the difference between the mutant and the wild type is only a single nucleotide polymorphism. Multiplex PCR assays employing a set of SuperSelective primers, and a corresponding set of differently colored molecular beacon probes, can be used in situations where the different mutations, though occurring in different cells, are located in the same codon. These non-symmetric real-time multiplex PCR assays contain limited concentrations of each SuperSelective primer, thereby enabling the simultaneous determination of each mutation's abundance by comparing its threshold value to the threshold value of a reference gene present in the sample. PMID:27244445

  13. Combination of biobarcode assay with on-chip capillary electrophoresis for ultrasensitive and multiplex biological agent detection.

    PubMed

    Cho, Minkyung; Chung, Soyi; Jung, Jae Hwan; Rhie, Gi-eun; Jeon, Jun Ho; Seo, Tae Seok

    2014-11-15

    Early diagnosis of biological agents is of paramount importance to prevent the casualties and fatal disease in human during bioterrorism or biological warfare. In this study, we reported an efficient and sensitive multiplex biological agent detection method based on the DNA biobarcode assay and the micro-capillary electrophoresis (μCE) technology. Monoplex as well as multiplex pathogen identification was performed using five targets including Bacillus anthracis, Francisella tularensis, Yersinia pestis, Vaccinia virus and Botulinum toxin A. Through the DNA biobarcode assay process, the magnetic microparticle-pathogen-polystyrene microbead complexes were formed, and the FAM labeled single stranded barcode DNA could be released from the complexes upon denaturation. Different lengths of a barcode DNA were designed to designate each pathogen, so that the specific peak elution time in the capillary electrophoresis on a chip allows us to distinguish the target with high accuracy within 3 min. We improved the assignment accuracy of the peak in the electropherogram by adding two bracket ladders. Owing to the abundant amount of barcode DNAs, the presence of B. anthracis, F. tularensis, Y. pestis, Vaccinia virus was confirmed with a limit of detection of 50CFU/mL, while Botulinum toxin A was analyzed even at a concentration of 12.5 ag/mL. Multiple pathogen detection was also successfully conducted in a phosphate buffered saline (PBS) as well as a serum medium with background of other pathogens. Thus, our analytical platform based on the biobarcode assay and on-chip CE analysis provides rapid, sensitive, multiplex, and accurate biological agent identification. PMID:24878840

  14. Evaluation of two real-time polymerase chain reaction assays for porcine epidemic diarrhea virus (PEDV) to assess PEDV transmission in growing pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In April 2013 a porcine epidemic diarrhea virus (PEDV) epidemic began in the United States. As part of the response, real-time RT-PCR assays to detect PEDV were developed by several Veterinary Diagnostic Laboratories. This study evaluated RT-PCR PEDV assays that detect the N gene (gN) and S gene (gS...

  15. Multiplex analyte assays to characterize different dementias: brain inflammatory cytokines in poststroke and other dementias.

    PubMed

    Chen, Aiqing; Oakley, Arthur E; Monteiro, Maria; Tuomela, Katri; Allan, Louise M; Mukaetova-Ladinska, Elizabeta B; O'Brien, John T; Kalaria, Raj N

    2016-02-01

    Both the inflammatory potential and cognitive function decline during aging. The association between the repertoire of inflammatory biomarkers and cognitive decline is unclear. Inflammatory cytokines have been reported to be increased, decreased, or unchanged in the cerebrospinal fluid and sera of subjects with dementia. We assessed 112 postmortem brains from subjects diagnosed with poststroke dementia (PSD), vascular dementia, mixed dementia, and Alzheimer's disease (AD), comparing those to poststroke nondemented (PSND) subjects and age-matched controls. We analyzed 5 brain regions including the gray and white matter from the frontal and temporal lobes for a panel of cytokine and/or chemokine analytes using multiplex-array assays. Of the 37 analytes, 14 were under or near the detection limits, 7 were close to the lowest detection level, and 16 cytokines were within the linear range of the assay. We observed widely variable concentrations of C-reactive protein (CRP) and serum amyloid A at the high end (1-150 ng/mg protein), whereas several of the interleukins (IL, interferon-gamma and tumor necrosis factor) at the low end (1-10 pg/mg). There were also regional variations; most notable being high concentrations of some cytokines (e.g., CRP and angiogenesis panel) in the frontal white matter. Overall, we found decreased concentrations of several cytokines, including IL-1 beta (p = 0.000), IL-6 (p = 0.000), IL-7 (p = 0.000), IL-8 (p = 0.000), IL-16 (p = 0.001), interferon-inducible protein-10 (0.044), serum amyloid A (p = 0.011), and a trend in IL-1 alpha (p = 0.084) across all dementia groups compared to nondemented controls. IL-6 and IL-8 were significantly lower in dementia subjects than in nondemented subjects in every region. In particular, lower levels of IL-6 and IL-8 were notable in the PSD compared to PSND subjects. Because these 2 stroke groups had comparable degree of vascular pathology, the lower production of IL-6 and IL-8 in PSD reaffirms a

  16. Multiplex analyte assays to characterize different dementias: brain inflammatory cytokines in poststroke and other dementias

    PubMed Central

    Chen, Aiqing; Oakley, Arthur E.; Monteiro, Maria; Tuomela, Katri; Allan, Louise M.; Mukaetova-Ladinska, Elizabeta B.; O'Brien, John T.; Kalaria, Raj N.

    2016-01-01

    Both the inflammatory potential and cognitive function decline during aging. The association between the repertoire of inflammatory biomarkers and cognitive decline is unclear. Inflammatory cytokines have been reported to be increased, decreased, or unchanged in the cerebrospinal fluid and sera of subjects with dementia. We assessed 112 postmortem brains from subjects diagnosed with poststroke dementia (PSD), vascular dementia, mixed dementia, and Alzheimer's disease (AD), comparing those to poststroke nondemented (PSND) subjects and age-matched controls. We analyzed 5 brain regions including the gray and white matter from the frontal and temporal lobes for a panel of cytokine and/or chemokine analytes using multiplex-array assays. Of the 37 analytes, 14 were under or near the detection limits, 7 were close to the lowest detection level, and 16 cytokines were within the linear range of the assay. We observed widely variable concentrations of C-reactive protein (CRP) and serum amyloid A at the high end (1–150 ng/mg protein), whereas several of the interleukins (IL, interferon-gamma and tumor necrosis factor) at the low end (1–10 pg/mg). There were also regional variations; most notable being high concentrations of some cytokines (e.g., CRP and angiogenesis panel) in the frontal white matter. Overall, we found decreased concentrations of several cytokines, including IL-1 beta (p = 0.000), IL-6 (p = 0.000), IL-7 (p = 0.000), IL-8 (p = 0.000), IL-16 (p = 0.001), interferon-inducible protein–10 (0.044), serum amyloid A (p = 0.011), and a trend in IL-1 alpha (p = 0.084) across all dementia groups compared to nondemented controls. IL-6 and IL-8 were significantly lower in dementia subjects than in nondemented subjects in every region. In particular, lower levels of IL-6 and IL-8 were notable in the PSD compared to PSND subjects. Because these 2 stroke groups had comparable degree of vascular pathology, the lower production of IL-6 and IL-8 in PSD reaffirms a

  17. Evaluation of a multiplex PCR assay for detection of cytomegalovirus in stool samples from patients with ulcerative colitis

    PubMed Central

    Nahar, Saifun; Iraha, Atsushi; Hokama, Akira; Uehara, Ayako; Parrott, Gretchen; Ohira, Tetsuya; Kaida, Masatoshi; Kinjo, Tetsu; Kinjo, Takeshi; Hirata, Tetsuo; Kinjo, Nagisa; Fujita, Jiro

    2015-01-01

    AIM: To evaluate a multiplex PCR assay for the detection of bacterial and viral enteropathogens in stool samples from patients with ulcerative colitis (UC). METHODS: We prospectively analyzed 300 individuals, including immunocompetent patients, immunocompromised patients, and patients with UC. Stool samples were collected from the recto-sigmoid region of the colon by endoscopy. The samples were qualitatively analyzed for bacterial and viral enteropathogens with a multiplex PCR assay using a Seeplex® Kit. Additional clinical and laboratory data were collected from the medical records. RESULTS: A multiplex PCR assay detected 397 pathogens (191 bacteria and 206 viruses) in 215 samples (71.7%). The most frequently detected bacteria were Escherichia coli H7, 85 (28.3%); followed by Aeromonas spp., 43 (14.3%); and Clostridium perfringens, 36 (12.0%) samples. The most prevalent viruses were Epstein-Barr virus (EBV), 90 (30.0%); followed by human herpes virus-6 (HHV-6), 53 (17.7%); and cytomegalovirus (CMV), 37 (12.3%) samples. The prevalence rate of CMV infection was significantly higher in the immunocompromised group than in the immunocompetent group (P < 0.01). CMV infection was more common in patients with UC (26/71; 36.6%) than in the immunocompetent patients excluding UC (6/188; 3.2%) (P < 0.01). CMV infection was more prevalent in UC active patients (25/58; 43.1%) than in UC inactive patients (1/13; 7.7%) (P < 0.05). Among 4 groups which defined by the UC activity and immunosuppressive drugs, the prevalence rate of CMV infection was highest in the UC active patients with immunosuppressive drugs (19/34; 55.8%). Epstein-Barr virus (EBV) infection was more common in the immunocompromised patients excluding UC (18/41; 43.9%) than in the immunocompetent patients excluding UC (47/188; 25.0%) (P < 0.05). The simultaneous presence of CMV and EBV and/or HHV6 in UC active patients (14/58; 24.1%) was greater than in immunocompromised patients excluding UC (5/41; 12.2%) (P < 0

  18. Multiplex-polymerase chain reaction assay for the authentication of the mackerel Scomber colias in commercial canned products.

    PubMed

    Infante, Carlos; Manchado, Manuel

    2006-01-01

    A multiplex-polymerase chain reaction (PCR) system was developed for the authentication of the mackerel Scomber colias in commercial canned products. This novel method consists of an S. colias-specific fragment [159 base pairs (bp)] located in the nontranscribed spacer (NTS) sequence, and a Scomber genus-specific PCR product in the 5S rRNA gene (196-201 bp) as a positive amplification control. The system was assayed using 18 different canned products labeled as S. colias. A positive identification was made in all but one sample, revealing this methodology as a potential molecular tool for direct application in the authentication of S. colias canned products. PMID:16792069

  19. Blu-ray Technology-Based Quantitative Assays for Cardiac Markers: From Disc Activation to Multiplex Detection.

    PubMed

    Weng, Samuel; Li, Xiaochun; Niu, Michelle; Ge, Bixia; Yu, Hua-Zhong

    2016-07-01

    Acute myocardial infarction (AMI) is the leading cause of mortality and morbidity globally. To reduce the number of mortalities, reliable and rapid point-of-care (POC) diagnosis of AMI is extremely critical. We herein present a Blu-ray technology-based assay platform for multiplex cardiac biomarker detection; not only off-the-shelf Blu-ray discs (BDs) were adapted as substrates to prepare standard immunoassays and DNA aptamer/antibody hybrid assays for the three key cardiac marker proteins (myoglobin, troponin I, and C-creative protein) but also an unmodified optical drive was directly employed to read the assay results digitally. In particular, we have shown that all three cardiac markers can be quantitated in their respective physiological ranges of interest, and the detection limits achieved are comparable with conventional enzyme-linked immunosorbent assay (ELISA) kits. The Blu-ray assay platform was further validated by measuring real-world samples and establishing a linear correlation with the simultaneously obtained ELISA data. Without the need to modify either the hardware (Blu-ray discs and optical drives) or the software driver, this assay-on-a-BD technique promises to be a low-cost user-friendly quantitative tool for on-site chemical analysis and POC medical diagnosis. PMID:27268387

  20. Single-Step Multiplex PCR Assay for Determining 92 Pneumococcal Serotypes.

    PubMed

    Marimón, José M; Ercibengoa, María; Santacatterina, Erica; Alonso, Marta; Pérez-Trallero, Emilio

    2016-08-01

    For pneumococcal disease surveillance, simple and cost-effective methods capable of determining all serotypes are needed. Combining a single-tube multiplex PCR with fluorescently labeled primers followed by amplicon analysis using automated fluorescent capillary electrophoresis, each serotype of 92 reference isolates and 297 recently collected clinical isolates was successfully determined. PMID:27280423

  1. Performance of Multiplex Cytokine Assays in Serum and Saliva among Community-Dwelling Postmenopausal Women

    PubMed Central

    Browne, Richard W.; Kantarci, Alpdogan; LaMonte, Michael J.; Andrews, Christopher A.; Hovey, Kathleen M.; Falkner, Karen L.; Cekici, Ali; Stephens, Danielle; Genco, Robert J.; Scannapieco, Frank A.; Van Dyke, Thomas E.; Wactawski-Wende, Jean

    2013-01-01

    Multiplexing arrays increase the throughput and decrease sample requirements for studies employing multiple biomarkers. The goal of this project was to examine the performance of Multiplex arrays for measuring multiple protein biomarkers in saliva and serum. Specimens from the OsteoPerio ancillary study of the Women’s Health Initiative Observational Study were used. Participants required the presence of at least 6 teeth and were excluded based on active cancer and certain bone issues but were not selected on any specific condition. Quality control (QC) samples were created from pooled serum and saliva. Twenty protein markers were measured on five multiplexing array panels. Sample pretreatment conditions were optimized for each panel. Recovery, lower limit of quantification (LLOQ) and imprecision were determined for each analyte. Statistical adjustment at the plate level was used to reduce imprecision estimates and increase the number of usable observations. Sample pre-treatment improved recovery estimates for many analytes. The LLOQ for each analyte agreed with manufacturer specifications except for MMP-1 and MMP-2 which were significantly higher than reported. Following batch adjustment, 17 of 20 biomarkers in serum and 9 of 20 biomarkers in saliva demonstrated acceptable precision, defined as <20% coefficient of variation (<25% at LLOQ). The percentage of cohort samples having levels within the reportable range for each analyte varied from 10% to 100%. The ratio of levels in saliva to serum varied from 1∶100 to 28∶1. Correlations between saliva and serum were of moderate positive magnitude and significant for CRP, MMP-2, insulin, adiponectin, GM-CSF and IL-5. Multiplex arrays exhibit high levels of analytical imprecision, particularly at the batch level. Careful sample pre-treatment can enhance recovery and reduce imprecision. Following statistical adjustments to reduce batch effects, we identified biomarkers that are of acceptable quality in serum and to a

  2. Development of a set of multiplex standard polymerase chain reaction assays for the identification of infectious agents from aborted bovine clinical samples.

    PubMed

    Tramuta, Clara; Lacerenza, Daniela; Zoppi, Simona; Goria, Mariella; Dondo, Alessandro; Ferroglio, Ezio; Nebbia, Patrizia; Rosati, Sergio

    2011-07-01

    The current study describes the development of a set of 5 multiplex polymerase chain reaction (mPCR) assays for the simultaneous detection of abortive infection agents in bovine fetal tissues, including Brucella spp., Leptospira spp., and Campylobacter fetus (mPCR1); Hammondia heydorni, Neospora caninum, and Toxoplasma gondii (mPCR2); Coxiella burnetii and Chlamydophila psittaci (mPCR3); Mycoplasma bovis, Mycoplasma bovigenitalium, and Ureaplasma diversum (mPCR4); and Bovine viral diarrhea virus (BVDV) and Bovine herpesvirus-1 (BoHV-1; mPCR5). The protocol was tested on different tissue samples collected from 50 aborted bovine fetuses, and it showed that out of the 50 fetuses, 7 (14%, mPCR2) were PCR-positive for N. caninum, 4 (8%, mPCR5) were PCR-positive for BVDV, and 2 (4%, mPCR4) were PCR-positive for U. diversum. The results obtained by using each multiplex PCR were 100% concordant with those obtained by using the respective PCR assays targeting single genes on the same specimens. Moreover, all multiplex PCR assays on clinical samples were compared with reference methods, obtaining a perfect accordance in all samples and confirming the validity of the set of multiplex PCR assays. The proposed set of multiplex PCR assays is, therefore, suitable for the simultaneous detection of the main infectious agents responsible for bovine abortion. PMID:21908306

  3. Multiplex polymerase chain reaction-capillary gel electrophoresis: a promising tool for GMO screening--assay for simultaneous detection of five genetically modified cotton events and species.

    PubMed

    Nadal, Anna; Esteve, Teresa; Pla, Maria

    2009-01-01

    A multiplex polymerase chain reaction assay coupled to capillary gel electrophoresis for amplicon identification by size and color (multiplex PCR-CGE-SC) was developed for simultaneous detection of cotton species and 5 events of genetically modified (GM) cotton. Validated real-time-PCR reactions targeting Bollgard, Bollgard II, Roundup Ready, 3006-210-23, and 281-24-236 junction sequences, and the cotton reference gene acp1 were adapted to detect more than half of the European Union-approved individual or stacked GM cotton events in one reaction. The assay was fully specific (<1.7% of false classification rate), with limit of detection values of 0.1% for each event, which were also achieved with simulated mixtures at different relative percentages of targets. The assay was further combined with a second multiplex PCR-CGE-SC assay to allow simultaneous detection of 6 cotton and 5 maize targets (two endogenous genes and 9 GM events) in two multiplex PCRs and a single CGE, making the approach more economic. Besides allowing simultaneous detection of many targets with adequate specificity and sensitivity, the multiplex PCR-CGE-SC approach has high throughput and automation capabilities, while keeping a very simple protocol, e.g., amplification and labeling in one step. Thus, it is an easy and inexpensive tool for initial screening, to be complemented with quantitative assays if necessary. PMID:19610365

  4. Prototype of Multiplex Bead Assay for Quantification of Three Serum Biomarkers for In Vitro Diagnosis of Endometriosis.

    PubMed

    Signorile, Pietro Giulio; Baldi, Alfonso

    2016-12-01

    Endometriosis is a very common disease, affecting 10% of women in the reproductive age. To date, a significant delay between onset of the symptoms and definitive diagnosis is caused by the lack of a reliable non-invasive diagnostic test. Recently, the potential value as diagnostic markers for endometriosis of three proteins (Zn-alpha2-glycoprotein, serum albumin, and complement C3 precursor), has been showed. In this article, we have defined the experimental conditions for the development of a multiplex bead array assay for rapid and simultaneous quantification of these three biomarkers in the serum of patients with endometriosis. Finally, pivotal experiments on a small cohort of patients have confirmed the diagnostic value of this assay. J. Cell. Physiol. 231: 2622-2627, 2016. © 2016 Wiley Periodicals, Inc. PMID:27137486

  5. Point-of-care multiplexed assays of nucleic acids using microcapillary-based loop-mediated isothermal amplification.

    PubMed

    Zhang, Yi; Zhang, Lu; Sun, Jiashu; Liu, Yulei; Ma, Xingjie; Cui, Shangjin; Ma, Liying; Xi, Jianzhong Jeff; Jiang, Xingyu

    2014-07-15

    This report demonstrates a straightforward, robust, multiplexed and point-of-care microcapillary-based loop-mediated isothermal amplification (cLAMP) for assaying nucleic acids. This assay integrates capillaries (glass or plastic) to introduce and house sample/reagents, segments of water droplets to prevent contamination, pocket warmers to provide heat, and a hand-held flashlight for a visual readout of the fluorescent signal. The cLAMP system allows the simultaneous detection of two RNA targets of human immunodeficiency virus (HIV) from multiple plasma samples, and achieves a high sensitivity of two copies of standard plasmid. As few nucleic acid detection methods can be wholly independent of external power supply and equipment, our cLAMP holds great promise for point-of-care applications in resource-poor settings. PMID:24937125

  6. Real-time multiplex PCR assays for reliable detection of Clostridium perfringens toxin genes in animal isolates.

    PubMed

    Albini, S; Brodard, I; Jaussi, A; Wollschlaeger, N; Frey, J; Miserez, R; Abril, C

    2008-02-01

    Typing of Clostridium perfringens strains by PCR-based determination of toxin genes proved to be a reliable method for diagnosis of enterotoxaemia in various animal species. We report the establishment and validation of three real-time fluorogenic (TaqMan) multiplex PCRs for the detection of C. perfringens alpha-, beta-, beta2-, epsilon-, entero- and iota-toxin genes. The composition of the PCRs was chosen with regard to robustness of the assays and in order to increase sensitivity compared to the conventional simplex PCRs. The combination of probe dyes selected for the real-time assays (FAM/TAMRA, Cy-5/BHQ-2 and VIC/TAMRA) as well as the designation of the chromosome-borne alpha-toxin as internal positive control allowed the creation of highly specific and sensitive, as well as time and cost effective PCRs. One hundred and three strains of C. perfringens isolated in Switzerland derived from clinical or suspected cases of enterotoxaemia in 10 different animal species were tested. The toxin genotypes were in agreement in both the conventional PCRs and the newly designed multiplex PCRs. Furthermore, the real-time PCR carried out as simplex allows to quantitate the copy numbers of plasmid-borne toxin genes in relation to the chromosomally located alpha-toxin gene. PMID:17855025

  7. Multiplex Assay for Simultaneous Detection of Mycoplasma genitalium and Macrolide Resistance Using PlexZyme and PlexPrime Technology

    PubMed Central

    Tabrizi, Sepehr N.; Tan, Lit Y.; Walker, Samantha; Twin, Jimmy; Poljak, Marin; Bradshaw, Catriona S.; Fairley, Christopher K.; Bissessor, Melanie; Mokany, Elisa; Todd, Alison V.; Garland, Suzanne M.

    2016-01-01

    Mycoplasma genitalium is a cause of non-gonoccocal urethritis (NGU) in men and cervicitis and pelvic inflammatory disease in women. Recent international data also indicated that the first line treatment, 1 gram stat azithromycin therapy, for M. genitalium is becoming less effective, with the corresponding emergence of macrolide resistant strains. Increasing failure rates of azithromycin for M. genitalium has significant implications for the presumptive treatment of NGU and international clinical treatment guidelines. Assays able to predict macrolide resistance along with detection of M. genitalium will be useful to enable appropriate selection of antimicrobials to which the organism is susceptible and facilitate high levels of rapid cure. One such assay recently developed is the MG 23S assay, which employs novel PlexZyme™ and PlexPrime™ technology. It is a multiplex assay for detection of M. genitalium and 5 mutations associated with macrolide resistance. The assay was evaluated in 400 samples from 254 (186 males and 68 females) consecutively infected participants, undergoing tests of cure. Using the MG 23S assay, 83% (331/440) of samples were positive, with 56% of positives carrying a macrolide resistance mutation. Comparison of the MG 23S assay to a reference qPCR method for M. genitalium detection and high resolution melt analysis (HRMA) and sequencing for detection of macrolide resistance mutations, resulted in a sensitivity and specificity for M. genitalium detection and for macrolide resistance of 99.1/98.5% and 97.4/100%, respectively. The MG 23S assay provides a considerable advantage in clinical settings through combined diagnosis and detection of macrolide resistance. PMID:27271704

  8. Multiplex Assay for Simultaneous Detection of Mycoplasma genitalium and Macrolide Resistance Using PlexZyme and PlexPrime Technology.

    PubMed

    Tabrizi, Sepehr N; Tan, Lit Y; Walker, Samantha; Twin, Jimmy; Poljak, Marin; Bradshaw, Catriona S; Fairley, Christopher K; Bissessor, Melanie; Mokany, Elisa; Todd, Alison V; Garland, Suzanne M

    2016-01-01

    Mycoplasma genitalium is a cause of non-gonoccocal urethritis (NGU) in men and cervicitis and pelvic inflammatory disease in women. Recent international data also indicated that the first line treatment, 1 gram stat azithromycin therapy, for M. genitalium is becoming less effective, with the corresponding emergence of macrolide resistant strains. Increasing failure rates of azithromycin for M. genitalium has significant implications for the presumptive treatment of NGU and international clinical treatment guidelines. Assays able to predict macrolide resistance along with detection of M. genitalium will be useful to enable appropriate selection of antimicrobials to which the organism is susceptible and facilitate high levels of rapid cure. One such assay recently developed is the MG 23S assay, which employs novel PlexZyme™ and PlexPrime™ technology. It is a multiplex assay for detection of M. genitalium and 5 mutations associated with macrolide resistance. The assay was evaluated in 400 samples from 254 (186 males and 68 females) consecutively infected participants, undergoing tests of cure. Using the MG 23S assay, 83% (331/440) of samples were positive, with 56% of positives carrying a macrolide resistance mutation. Comparison of the MG 23S assay to a reference qPCR method for M. genitalium detection and high resolution melt analysis (HRMA) and sequencing for detection of macrolide resistance mutations, resulted in a sensitivity and specificity for M. genitalium detection and for macrolide resistance of 99.1/98.5% and 97.4/100%, respectively. The MG 23S assay provides a considerable advantage in clinical settings through combined diagnosis and detection of macrolide resistance. PMID:27271704

  9. Analytical validation of protein-based multiplex assays: a workshop report by the NCI-FDA interagency oncology task force on molecular diagnostics.

    PubMed

    Rodriguez, Henry; Tezak, Zivana; Mesri, Mehdi; Carr, Steven A; Liebler, Daniel C; Fisher, Susan J; Tempst, Paul; Hiltke, Tara; Kessler, Larry G; Kinsinger, Christopher R; Philip, Reena; Ransohoff, David F; Skates, Steven J; Regnier, Fred E; Anderson, N Leigh; Mansfield, Elizabeth

    2010-02-01

    Clinical proteomics has the potential to enable the early detection of cancer through the development of multiplex assays that can inform clinical decisions. However, there has been some uncertainty among translational researchers and developers as to the specific analytical measurement criteria needed to validate protein-based multiplex assays. To begin to address the causes of this uncertainty, a day-long workshop titled "Interagency Oncology Task Force Molecular Diagnostics Workshop" was held in which members of the proteomics and regulatory communities discussed many of the analytical evaluation issues that the field should address in development of protein-based multiplex assays for clinical use. This meeting report explores the issues raised at the workshop and details the recommendations that came out of the day's discussions, such as a workshop summary discussing the analytical evaluation issues that specific proteomic technologies should address when seeking US Food and Drug Administration approval. PMID:20007859

  10. A multiplex RT-PCR test for the differential identification of turkey astrovirus type-1, turkey astrovirus type-2, chicken astrovirus, avian nephritis virus and avian rotavirus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Viral enteric diseases cause substantial economic loss to the US poultry industry because they lead to decreased weight gain, increased morbidity, increased mortality, and increased production costs from poor feed conversions and increased use of therapeutic anti-microbial treatments. Astroviruses ...

  11. Identification of normalization factors for quantitative real-time RT-PCR analysis of gene expression in Pacific abalone Haliotis discus hannai

    NASA Astrophysics Data System (ADS)

    Qiu, Reng; Sun, Boguang; Fang, Shasha; Sun, Li; Liu, Xiao

    2013-03-01

    Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without validation. To identify appropriate reference genes for qRT-PCR in Pacific abalone Haliotis discus hannai, we examined the transcription stability of six housekeeping genes in abalone tissues in the presence and absence of bacterial infection. For this purpose, abalone were infected with the bacterial pathogen Vibrio anguillarum for 12 h and 48 h. The mRNA levels of the housekeeping genes in five tissues (digestive glands, foot muscle, gill, hemocyte, and mantle) were determined by qRT-PCR. The PCR data was subsequently analyzed with the geNorm and NormFinder algorithms. The results show that in the absence of bacterial infection, elongation factor-1-alpha and beta-actin were the most stably expressed genes in all tissues, and thus are suitable as cross-tissue type normalization factors. However, we did not identify any universal reference genes post infection because the most stable genes varied between tissue types. Furthermore, for most tissues, the optimal reference genes identified by both algorithms at 12 h and 48 h post-infection differed. These results indicate that bacterial infection induced significant changes in the expression of abalone housekeeping genes in a manner that is dependent on tissue type and duration of infection. As a result, different normalization factors must be used for different tissues at different infection points.

  12. Concordance between RT-PCR-based detection of respiratory viruses from nasal swabs collected for viral testing and nasopharyngeal swabs collected for bacterial testing

    PubMed Central

    Grijalva, Carlos G.; Griffin, Marie R.; Edwards, Kathryn M.; Johnson, Monika; Gil, Ana I.; Verastegui, Héctor; Lanata, Claudio F.; Williams, John V.

    2014-01-01

    Background Epidemiologic studies of respiratory infections frequently rely on separate sample collections for the detection of bacteria and viruses. The requirement for two specimens presents cost, logistical, and acceptability challenges. Objectives To determine the agreement in detection of respiratory viruses using RT-PCR between two different types of samples collected on the same day: nasal swabs preserved in viral transport medium (NS) and nasopharyngeal swabs preserved in skim milk-tryptone-glucose-glycerol [STGG] medium (NP), the current standard for pneumococcal colonization studies. Study design Paired NS and NP samples were collected between May 2009 and September 2011 as part of the RESPIRA-PERU study, a large prospective cohort of Andean children <3 years of age. NS samples used polyester swabs and viral transport medium whereas NP samples used rayon wire-handled swabs and STGG medium. Samples were tested for influenza, human metapneumovirus (MPV), respiratory syncytial virus (RSV), human rhinovirus (HRV), parainfluenza virus 3 (PIV3) and adenovirus (ADV) using real-time RT-PCR. We calculated the agreement, and compared cycle thresholds (CT) between NP and NS samples. Results Among 226 paired NP-NS samples, we observed very high agreement with a Kappa statistic ranging from 0.71 for ADV to 0.97 for MPV. CT values were similar for both strategies. Conclusions NP samples preserved in STGG provide a simple and reliable strategy for identification of both pneumococcus and respiratory viruses. This single specimen collection strategy could be used for epidemiologic studies, especially in resource-limited settings. Furthermore, archived NP-STGG specimens from previous studies could be reliably tested by RT-PCR for viruses. PMID:24875136

  13. RT-PCR and statistical analyses of adeABC expression in clinical isolates of Acinetobacter calcoaceticus-Acinetobacter baumannii complex.

    PubMed

    Ruzin, Alexey; Immermann, Frederick W; Bradford, Patricia A

    2010-06-01

    The relationship between expression of adeABC and minimal inhibitory concentration (MIC) of tigecycline was investigated by RT-PCR and statistical analyses in a population of 106 clinical isolates (MIC range, 0.0313-16 microg/ml) of Acinetobacter calcoaceticus-Acinetobacter baumannii complex. There was a statistically significant linear relationship (p < 0.0001) between log-transformed expression values and log-transformed MIC values, indicating that overexpression of AdeABC efflux pump is a prevalent mechanism for decreased susceptibility to tigecycline in A. calcoaceticus-A. baumannii complex. PMID:20438348

  14. Selection and validation of reference genes for qRT-PCR expression analysis of candidate genes involved in olfactory communication in the butterfly Bicyclus anynana.

    PubMed

    Arun, Alok; Baumlé, Véronique; Amelot, Gaël; Nieberding, Caroline M

    2015-01-01

    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

  15. Selection and Validation of Reference Genes for qRT-PCR Expression Analysis of Candidate Genes Involved in Olfactory Communication in the Butterfly Bicyclus anynana

    PubMed Central

    Arun, Alok; Baumlé, Véronique; Amelot, Gaël; Nieberding, Caroline M.

    2015-01-01

    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

  16. Genomic quantitative real-time PCR proves residual disease positivity in more than 30% samples with negative mRNA-based qRT-PCR in Chronic Myeloid Leukemia

    PubMed Central

    Pagani, Ilaria S.; Spinelli, Orietta; Mattarucchi, Elia; Pirrone, Cristina; Pigni, Diana; Amelotti, Elisabetta; Lilliu, Silvia; Boroni, Chiara; Intermesoli, Tamara; Giussani, Ursula; Caimi, Luigi; Bolda, Federica; Baffelli, Renata; Candi, Eleonora; Pasquali, Francesco; Lo Curto, Francesco; Lanfranchi, Arnalda; Porta, Fulvio; Rambaldi, Alessandro; Porta, Giovanni

    2014-01-01

    Imatinib mesylate (IM) is the first line therapy against Chronic Myeloid Leukemia, effectively prolonging overall survival. Because discontinuation of treatment is associated with relapse, IM is required indefinitely to maintain operational cure. To assess minimal residual disease, cytogenetic analysis is insensitive in a high background of normal lymphocytes. The qRT-PCR provides highly sensitive detection of BCR-ABL1 transcripts, but mRNA levels are not directly related to the number of leukemic cells, and undetectable results are difficult to interpret. We developed a sensitive approach to detect the number of leukemic cells by a genomic DNA (gDNA) Q-PCR assay based on the break-point sequence, with a formula to calculate the number of Ph-positive cells. We monitored 8 CML patients treated with IM for more than 8 years. We tested each samples by patient specific gDNA Q-PCR in parallel by the conventional techniques. In all samples positive for chimeric transcripts we showed corresponding chimeric gDNA by Q-PCR, and in 32.8% (42/128) of samples with undetectable levels of mRNA we detected the persistence of leukemic cells. The gDNA Q-PCR assay could be a new diagnostic tool used in parallel to conventional techniques to support the clinician's decision to vary or to STOP IM therapy. PMID:25594053

  17. Rapid Simultaneous Detection of Enterovirus and Parechovirus RNAs in Clinical Samples by One-Step Real-Time Reverse Transcription-PCR Assay

    PubMed Central

    Bennett, Susan; Harvala, Heli; Witteveldt, Jeroen; McWilliam Leitch, E. Carol; McLeish, Nigel; Templeton, Kate; Gunson, Rory; Carman, William F.; Simmonds, Peter

    2011-01-01

    Enteroviruses (EVs) are recognized as the major etiological agent in meningitis in children and young adults. The use of molecular techniques, such as PCR, has substantially improved the sensitivity of enterovirus detection compared to that of virus culture methods. PCR-based methods also can detect a much wider range of EV variants, including those within species A, as well as human parechoviruses (HPeVs) that often grow poorly in vitro and which previously have been underdiagnosed by traditional methods. To exploit these developments, we developed a real-time one-step reverse transcription-PCR (RT-PCR) for the rapid and sensitive detection of EV and HPeV in clinical specimens. Two commercially available RT-PCR kits were used (method I, Platinum one-step kit; method II, Express qPCR one-step kit) with primers and probes targeting the EV and HPeV 5′-untranslated regions (5′UTR). Amplification dynamics (threshold cycle [CT]values and efficiencies) of absolutely quantified full-length RNA transcripts representative of EV species A to D and HPeV were similar, demonstrating the effectiveness of both assays across the range of currently described human EV and HPeV variants. Probit analysis of multiple endpoint replicates demonstrated comparable sensitivities of the assays for EV and HPeV (method I, approximately 10 copies per reaction for both targets; method II, 20 copies per reaction). CT values were highly reproducible on repeat testing of positive controls within assays and between assay runs. Considering the sample turnaround time of less than 3 h, the multiplexed one-step RT-PCR method provides rapid diagnostic testing for EV and HPeV in cases of suspected central nervous system infections in a clinically relevant time frame. PMID:21593263

  18. A novel multiplex pyrosequencing assay for genotyping functionally relevant CTLA-4 polymorphisms: potential applications in autoimmunity and cancer.

    PubMed

    Banelli, Barbara; Morabito, Anna; Laurent, Stefania; Piccioli, Patrizia; Dozin, Beatrice; Ghio, Massimo; Ascierto, Paolo Antonio; Monteghirfo, Stefano; Marasco, Antonella; Ottaviano, Vincenzo; Queirolo, Paola; Romani, Massimo; Pistillo, Maria Pia

    2014-08-01

    CTLA-4 expression/function can be affected by single nucleotide polymorphisms (SNPs) of CTLA-4 gene, which have been widely associated with susceptibility or progression to autoimmune diseases and cancer development. In this study, we analyzed six CTLA-4 SNPs (-1661A>G, -1577G>A, -658C>T, -319C>T, +49A>G, CT60G>A) in 197 DNA samples from 43 B-lymphoblastoid cell lines (B-LCLs), 40 systemic sclerosis (SSc) patients, 14 pre-analyzed melanoma patients and 100 Italian healthy subjects. Genotyping of -1661A>G, -1577G>A, -658C>T and CT60G>A was performed by newly developed multiplex pyrosequencing (PSQ) assays, whereas -319C>T and +49A>G by T-ARMS PCR and direct sequencing. Genotype/allele frequency were analyzed using χ(2) or Fisher exact test. Our study provides the first multiplex PSQ method that allows simultaneous genotyping of two CTLA-4 SNP pairs (i.e. -1661A>G/-658C>T and -1577G>A/CT60G>A) by two multiplex PSQ reactions. Herein, we show the CTLA-4 genotype distribution in the B-LCLs providing the first and best characterized cell line panel typed for functionally relevant CTLA-4 SNPs. We also report the significant association of the -1661A/G genotype, -1661 & -319 AC-GT dipl