Rapid, Sensitive Bluetongue Virus Serogroup and Serotype Detection Using Polymerase Chain Reaction

1995 ◽  
Author(s):  
Bennie Osburn ◽  
Marius Ianconescu ◽  
Geoffrey Akita ◽  
Rozalia Kaufman
Keyword(s):  
Cell Culture ◽  
Polymerase Chain Reaction ◽  
Bluetongue Virus ◽  
Animal Health ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Diagnostic Pcr ◽  
Polymerase Chain ◽  
The U.S

The objectives of this proposal were to enhance animal health by 1) development of a BTV serogroup diagnostic assay using polymerase chain reaction (PCR) and 2) development of a BTV serotype specific diagnostic PCR assay. A PCR assay for diagnosis of bluetongue virus (BTV) serogroup from clinical samples meeting the criteria of objective 1 was developed. This PCR assay is more sensitive than virus isolation and has been adopted by both the U.S. and Israeli collaborating laboratories of this project, as well as at least one other U.S. laboratory for routine diagnosis of BTV infection in ruminants. The basic BTV PCR protocol has also become an essential tool in BTV molecular research in both collaborating laboratories. During development of the BTV serotype specific PCR we had the opportunity to investigate a nationwide outbreak of abortions and fatal disease in dogs in the U.S. purportedly due to BTV infection via a BTV contaminated canine vaccine. The BTV serogroup PCR was integral in confirming BTV in tissues from affected dogs and in lots of the suspect vaccine. This led to the first published report of BTV infection in dogs. We discovered that BTV can produce silent persistent infection in canine cell culture. This indicated a need for more stringent screening of biologics for occult BTV infection. A novel mixed cell culture method was developed to identify occult BTV and other occult viral infection cell cultures. Serotype specific primers for PCR detection of all U.S. BTV serotypes and two Israel serotypes (BTV-2 and 10) have been evaluated and are available. A subsequent collaboration would logically include sequencing of the L2 genes of Israel BTV-4, 6 and 16, allowing incorporation of these Israel BTV serotypes into a multiplex PCR assay.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

scholarly journals Evaluation of Amplification Targets for the Specific Detection ofBordetella pertussisUsing Real-Time Polymerase Chain Reaction

2014 ◽  
Vol 25 (4) ◽  
pp. 217-221 ◽  
Author(s):  
Mohammad Rubayet Hasan ◽  
Rusung Tan ◽  
Ghada N Al-Rawahi ◽  
Eva Thomas ◽  
Peter Tilley
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Target Genes ◽  
Reference Panel ◽  
Superior Performance ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain

BACKGROUND:Bordetella pertussisinfections continue to be a major public health challenge in Canada. Polymerase chain reaction (PCR) assays to detectB pertussisare typically based on the multicopy insertion sequence IS481, which offers high sensitivity but lacks species specificity.METHODS: A novelB pertussisreal-time PCR assay based on the porin gene was tested in parallel with several previously published assays that target genes such as IS481,ptx-promoter, pertactin and a putative thialase. The assays were evaluated using a reference panel of common respiratory bacteria including differentBordetellaspecies and 107 clinical nasopharyngeal specimens. Discrepant results were confirmed by sequencing the PCR products.RESULTS: Analytical sensitivity was highest for the assay targeting the IS481element; however, the assay lacked specificity forB pertussisin the reference panel and in the clinical samples. False-positive results were also observed with assays targeting theptx-promoter and pertactin genes. A PCR assay based on the thialase gene was highly specific but failed to detect all reference strains ofB pertussis. However, a novel assay targeting the porin gene demonstrated high specificity forB pertussisboth in the reference panel and in clinical samples and, based on sequence-confirmed results, correctly predicted allB pertussis-positive cases in clinical samples. According to Probit regression analysis, the 95% detection limit of the new assay was 4 colony forming units/reaction.CONCLUSION: A novel porin assay forB pertussisdemonstrated superior performance and may be useful for improved molecular detection ofB pertussisin clinical specimens.

scholarly journals Systematic Review and Meta-analysis of Real-time Polymerase Chain Reaction assay for the detection of COVID-19 from clinical samples in low-and middle-income countries: Protocol

2021 ◽  
Author(s):  
Emmanuel Oladipo Babafemi
Keyword(s):  
Systematic Review ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Meta Analysis ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Middle Income ◽  
Polymerase Chain

Abstract Background: COVID-19 has spread globally since its discovery in Hubei province, China in December 2019 and became pandemic in 2020. COVID-19 is a new betacoronavirus and a variant of severe acute respiratory syndrome coronavirus 2 (SARA- CoV-2). Rapid, accurate and reliable diagnosis of COVID-19 will prevent the spread and allow for appropriate management. The main objective of this systematic review is to identify, appraise and summarise the published evidence on the diagnostic performance and effectiveness of SARS-CoV-2 virus in the diagnosis of current or previous COVID-19 using real-time polymerase chain reaction (RT-PCR) assay in low-and middle-income countries (LMICs). Methods: We will search MEDLINE/PubMed, EMBASE, BIOSIS, LILACS, Cochrane Infectious Diseases Group Specialised Register (CIDG SR), Global Health, and CINAHL for published studies for the diagnosis of COVID-19 using real-time polymerase chain reaction assay in LMICs There will be no restriction regarding the language, date of publication, and publication status. We will include retrospective, cross-sectional and cohort observational studies will be included in the review. Selection of studies, data extraction and management, assessment of risk of bias, and quality of evidence will be performed by two independent reviewers (EB and BC). A third researcher (GM) will be consulted in case of discrepancies. Depending on the availability and quality of the data, a meta-analysis will be performed. Otherwise, findings will be qualitatively reported. Discussion: To our knowledge, this is the first systematic review and meta-analysis to assess the uptake of RT-PCR assay for SARS-CoV-2 detection from clinical samples in human in LMICs. This review will make available evidence on the uptake, accuracy, approach, and interpretation of results of this assay in the context of COVID-19 diagnosis which will meet an urgent need, considering the diagnostic challenges of RT-PCR assay for COVID-19 diagnosis in humans. Systematic review registration: PROSPERO CRD42021271894

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30–60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method: In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The LoD (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI): 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

DETECTION OF RESPIRATORY SYNCITIAL VIRUS (RSV) IN LOWER ACUTE RESPIRATORY INFECTIONS BY NESTED REVERSE TRANSCRIPTION- POLYMERASE CHAIN REACTION

2011 ◽  
pp. 11-15
Author(s):  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Respiratory Infections ◽  
Clinical Samples ◽  
Acute Respiratory Infections ◽  
Rt Pcr ◽  
Minimal Level ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain

Objective: To develop and apply a nested reverse transcription- polymerase chain reaction (nested RT-PCR) for detection of RSV in lower acute respiratory infections. Materials and methods: A nested reverse transcription- polymerase chain reaction was used to amplify a sequence of the F gene in the RSV genomic RNA, optimized and compared the sensitivity and specificity of this assay with the control samples and then applied this procedure for diagnosing RSV from clinical samples. Results: This nested RT-PCR assay amplified the specific target fragment of RSV RNA and did not amplify any sequence of genomes of the tested common viruses and bacteria causing respiratory infections. The minimal level of detection of this procedure was 102 copies/ml. Results for detection of RSV on 109 samples of throat swabs or nasopharyngeal swabs from children with lower respiratory infections showed that twenty seven patients were positive with RSV ( 24.8%), among which six out of 30 (20%) were with bronchitis, seven out of 26 ( 27%) were with bronchiolitis and fourteen out of 53 (26.4%) were with pneumonia. Conclusion: This nested RT-PCR was found to be useful and reliable for detection of RSV in respiratory infections.

scholarly journals Detection of Bluetongue Virus Serogroup by Polymerase Chain Reaction

1992 ◽  
Vol 4 (4) ◽  
pp. 400-405 ◽  
Author(s):  
Geoffrey Y. Akita ◽  
Jarasvech Chinsangaram ◽  
Bennie I. Osburn ◽  
Marius Ianconescu ◽  
Rozalia Kaufman
Keyword(s):  
Polymerase Chain Reaction ◽  
Bluetongue Virus ◽  
Wide Spectrum ◽  
Genome Segment ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Field Isolates ◽  
Viral Particles ◽  
Polymerase Chain

To facilitate detection of active bluetongue virus (BTV) infection, a polymerase chain reaction (PCR) protocol was developed. The BTV reverse transcriptase PCR (RT-PCR) is a 1-tube reaction and involves chemical denaturation of the double-stranded viral RNA target, a complementary DNA (cDNA) synthesis step, and PCR amplification of the cDNA. BTV RT-PCR using primers derived from highly conserved genome segment 10 results in a 251–base pair (bp) product. BTV RNA from all USA prototype serotypes 2, 10, 11, 13, and 17; a wide spectrum of USA BTV field isolates including serotypes 10, 11, 13, and 17; and a spectrum of Israeli field isolates including serotypes 2, 4, 6, 10, and 16 were detected by BTV RT-PCR. With agarose gels, the 251–bp product was detected from as little as 100 fg-1 pg of BTV RNA, which is equivalent to 5 × 103-5 × 104 viral particles or 5 × 102-5 × 103 infectious units. With dot blot hybridization, specific PCR product was detected from as little as 1 fg of BTV RNA, which is equivalent to 50 viral particles, or 5 infectious units. This level of sensitivity is comparable to that of virus isolation. The BTV RT-PCR using primers derived from genome segment 10 can detect a wide spectrum of USA and Israeli BTV serotypes and has potential for detection of infection by the BTV serogroup. Application of this BTV PCR to clinical samples is in progress.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30%-60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. The LoD (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI): 363.23-1145.69) for ORF1ab and 528.1 (95% CI: 347.7-1248.7) for N, 401.8 (95% CI: 284.8-938.3) for ORF1ab and 336.8 (95% CI: 244.6-792.5) for N, and 194.74 (95% CI: 139.7-430.9) for ORF1ab and 189.1 (95% CI: 130.9-433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

scholarly journals Detection of Bluetongue Virus in Clinical Samples by Polymerase Chain Reaction

1993 ◽  
Vol 5 (2) ◽  
pp. 154-158 ◽  
Author(s):  
Geoffrey Y. Akita ◽  
John Glenn ◽  
Anthony E. Castro ◽  
Bennie I. Osburn
Keyword(s):  
Polymerase Chain Reaction ◽  
Bluetongue Virus ◽  
Vero Cells ◽  
Clinical Samples ◽  
Great Promise ◽  
Chain Reaction ◽  
Blood Samples ◽  
Polymerase Chain ◽  
Chicken Eggs ◽  
Embryonated Chicken

A previously described bluetongue virus (BTV) serogroup polymerase chain reaction (PCR) assay was applied to clinical samples. The sensitivity of the BTV serogroup PCR was increased by the use of non-radioactive chemiluminescent hybridization. Unfractionated whole blood samples from rams experimentally inoculated with cell culture-adapted BTV- 11 UC-8 were analyzed by virus isolation (VI) on Vero cells and PCR. VI and PCR were in agreement, with the exception of 3 blood samples that were VI negative and PCR positive. In semen spiked with BTV- 11 UC-8, PCR detected as little as 1.6 × 102 plaque-forming units of BTV/ml of semen. BTV in the spleen of a sheep submitted for necropsy for suspect BTV infection was detected by both PCR and VI in embryonated chicken eggs. BTV PCR with nonradioactive chemiluminescent hybridization resulted in a level of sensitivity comparable to that of VI and likly more sensitive than VI on Vero cells for blood. This BTV PCR has great promise for rapid, sensitive, and specific detection of active BTV infection in a variety of clinical samples.

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