Development and validation of a reverse transcription quantitative polymerase chain reaction for tilapia lake virus detection in clinical samples and experimentally challenged fish

2017 ◽  
Vol 41 (2) ◽  
pp. 255-261 ◽  
Author(s):  
P Tattiyapong ◽  
K Sirikanchana ◽  
W Surachetpong
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Quantitative Polymerase Chain Reaction ◽  
Virus Detection ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Development And Validation

scholarly journals An international, interlaboratory ring trial confirms the feasibility of an extraction-less “direct” RT-qPCR method for reliable detection of SARS-CoV-2 RNA in clinical samples

PLoS ONE ◽  
2022 ◽  
Vol 17 (1) ◽  
pp. e0261853
Author(s):  
Margaret G. Mills ◽  
Emily Bruce ◽  
Meei-Li Huang ◽  
Jessica W. Crothers ◽  
Ollivier Hyrien ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Correct Identification ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Ring Trial ◽  
Feasible Option ◽  
Polymerase Chain

Reverse transcription–quantitative polymerase chain reaction (RT-qPCR) is used worldwide to test and trace the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). “Extraction-less” or “direct” real time–reverse transcription polymerase chain reaction (RT-PCR) is a transparent and accessible qualitative method for SARS-CoV-2 detection from nasopharyngeal or oral pharyngeal samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged 10 global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international interlaboratory ring trial. Participating laboratories were provided a common protocol, common reagents, aliquots of identical pooled clinical samples, and purified nucleic acids and used their existing in-house equipment. We observed 100% concordance across laboratories in the correct identification of all positive and negative samples, with highly similar cycle threshold values. The test also performed well when applied to locally collected patient nasopharyngeal samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that direct RT-PCR assay methods can be clearly translated across sites utilizing readily available equipment and expertise and are thus a feasible option for more efficient COVID-19 coronavirus disease testing as demanded by the continuing pandemic.

scholarly journals Screening of Additive Formulations Enables Off-Chip Drop Reverse Transcription Quantitative Polymerase Chain Reaction of Single Influenza A Virus Genomes

2021 ◽  
Vol 93 (10) ◽  
pp. 4365-4373
Author(s):  
Emma Kate Loveday ◽  
Geoffrey K. Zath ◽  
Dimitri A. Bikos ◽  
Zackary J. Jay ◽  
Connie B. Chang
Keyword(s):  
Polymerase Chain Reaction ◽  
Influenza A Virus ◽  
Reverse Transcription ◽  
Influenza A ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Virus Genomes

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.

Comparison of reverse transcription–quantitative polymerase chain reaction methods and platforms for single cell gene expression analysis

2012 ◽  
Vol 427 (2) ◽  
pp. 178-186 ◽  
Author(s):  
Bridget C. Fox ◽  
Alison S. Devonshire ◽  
Marc-Olivier Baradez ◽  
Damian Marshall ◽  
Carole A. Foy
Keyword(s):  
Gene Expression ◽  
Polymerase Chain Reaction ◽  
Single Cell ◽  
Reverse Transcription ◽  
Gene Expression Analysis ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Cell Gene Expression ◽  
Polymerase Chain ◽  
Cell Gene

Quantitative Real-Time Polymerase Chain Reaction Detection of BK Virus Using Labeled Primers

2010 ◽  
Vol 134 (3) ◽  
pp. 444-448 ◽  
Author(s):  
Zhengming Gu ◽  
Jianmin Pan ◽  
Matthew J. Bankowski ◽  
Randall T. Hayden
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Polymerase Chain Reaction ◽  
Bk Virus ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Chain Reaction ◽  
Pcr Method ◽  
Polymerase Chain

Abstract Context.—BK virus infections among immunocompromised patients are associated with disease of the kidney or urinary bladder. High viral loads, determined by quantitative polymerase chain reaction (PCR), have been correlated with clinical disease. Objective.—To develop and evaluate a novel method for real-time PCR detection and quantification of BK virus using labeled primers. Design.—Patient specimens (n = 54) included 17 plasma, 12 whole blood, and 25 urine samples. DNA was extracted using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Indianapolis, Indiana); sample eluate was PCR-amplified using the labeled primer PCR method. Results were compared with those of a user-developed quantitative real-time PCR method (fluorescence resonance energy transfer probe hybridization). Results.—Labeled primer PCR detected less than 10 copies per reaction and showed quantitative linearity from 101 to 107 copies per reaction. Analytical specificity of labeled primer PCR was 100%. With clinical samples, labeled primer PCR demonstrated a trend toward improved sensitivity compared with the reference method. Quantitative assay comparison showed an R2 value of 0.96 between the 2 assays. Conclusions.—Real-time PCR using labeled primers is highly sensitive and specific for the quantitative detection of BK virus from a variety of clinical specimens. These data demonstrate the applicability of labeled primer PCR for quantitative viral detection and offer a simplified method that removes the need for separate oligonucleotide probes.

Considerations in the development and validation of real-time quantitative polymerase chain reaction and its application in regulated bioanalysis to characterize the cellular kinetics of CAR-T products in clinical studies

Bioanalysis ◽  
2020 ◽  
Author(s):  
Tong-yuan Yang ◽  
Rajitha Doddareddy
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Method Development ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Cellular Kinetics ◽  
Car T ◽  
Polymerase Chain ◽  
Development And Validation ◽  
Kinetics Of

Real-time quantitative polymerase chain reaction (qPCR) has become the standard method for monitoring cellular kinetics of CAR-T therapies with measurement of the CAR transgene copy numbers in peripheral blood mononuclear cells isolated from patients receiving the treatment. Unlike other biophysical and immunological methodologies for bioanalytical characterization of conventional small molecule drugs or protein biologics, there is no relevant regulatory guidance to date on the method development and validation for quantitative qPCR assays employed during clinical development of CAR-T products. This paper will provide an overview and considerations in the development and validation of a qPCR assay from sample extraction to assay parameters and its implementation in regulated bioanalysis for CAR-T or other types of cell therapies.

Expression of DNA repair genes in oral squamous cell carcinoma using reverse transcription-quantitative polymerase chain reaction

2020 ◽  
Vol 130 (3) ◽  
pp. 298-305
Author(s):  
Mônica Ghislaine Oliveira Alves ◽  
Natália da Silva Miguel ◽  
Camila Cristina Panisello Ferreira ◽  
Elis Ribeiro Alvarenga ◽  
Bruna Manzanares Tonon ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Squamous Cell Carcinoma ◽  
Dna Repair ◽  
Oral Squamous Cell Carcinoma ◽  
Reverse Transcription ◽  
Quantitative Polymerase Chain Reaction ◽  
Dna Repair Genes ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Repair Genes
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