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.

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 SARS-CoV2 in Clinical Samples: Target-specific Analysis of Qualitative Reverse Transcription–Polymerase Chain Reaction(RT-PCR) Diagnostic Kits

IJID Regions ◽  
2021 ◽  
Author(s):  
Ambica Rangaiah ◽  
Sathyanarayan Muthur Shankar ◽  
Shantala Gowdara Basawarajappa ◽  
Pritik A Shah ◽  
Aditya Chandrashekar ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Diagnostic Kits ◽  
Specific Analysis ◽  
Polymerase Chain

Biotypic characterisation of bovine viral diarrhoea virus (BVDV) using reverse transcription-polymerase chain reaction (RT-PCR) in clinical samples

2003 ◽  
Vol 51 (3) ◽  
pp. 425-431 ◽  
Author(s):  
T. Karaoğlu ◽  
I. Burgu ◽  
A. Özkul
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Bovine Viral Diarrhoea Virus ◽  
Clinical Samples ◽  
Bovine Viral Diarrhoea ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Molecular Differentiation ◽  
Diarrhoea Virus ◽  
Polymerase Chain

In this study, bovine viral diarrhoea virus (BVDV) was detected and biotypically characterised in clinical samples using reverse transcriptase-polymerase chain reaction (RT-PCR). The RT-PCR technique produced two different amplicons (402 and approx. 680 bp in size) in case of the presence of both biotypes (cp and ncp) in the sample. The mixture of the biotypes as detected by RT-PCR was verified by the immunoplaque assay (IPA). Purification of biotypes was carried out by native plaque isolation and subsequent RT-PCR revealed single products (402 or approx. 680 bp in size) in each clone. The results showed that RT-PCR can be used for accurate molecular differentiation between the BVDV biotypes.

1504: Molecular Diagnosis and Staging of Prostate Cancer Using Clusterin in Real Time Reverse Transcription Polymerase Chain Reaction (RT-PCR)

2006 ◽  
Vol 175 (4S) ◽  
pp. 485-486
Author(s):  
Sabarinath B. Nair ◽  
Christodoulos Pipinikas ◽  
Roger Kirby ◽  
Nick Carter ◽  
Christiane Fenske
Keyword(s):  
Prostate Cancer ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Molecular Diagnosis ◽  
Reverse Transcription ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

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.

Expression of hyaluronan synthase-3 in porcine oviducal epithelium during oestrus

2003 ◽  
Vol 15 (2) ◽  
pp. 99 ◽  
Author(s):  
Paisan Tienthai ◽  
Naoko Kimura ◽  
Paraskevi Heldin ◽  
Eimei Sato ◽  
Heriberto Rodriguez-Martinez
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Hyaluronan Synthase ◽  
Critical Periods ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Mean Values ◽  
Polymerase Chain ◽  
Sperm Reservoir ◽  
Oviduct Fluid

Hyaluronan (HA) has been related to fertilization and embryo development in the pig. Furthermore, HA is present in pig oviduct fluid and the lining epithelium, particularly of the pre-ovulatory sperm reservoir. Because the mechanisms that regulate HA synthesis have not yet been clarified, semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was conducted to assess the expression of mRNAs of two HA-synthesizing enzymes (has2 and has3) in the oviduct epithelium (uterotubal junction, isthmus, ampullary–isthmic junction and ampulla segments) of non-inseminated (control) and inseminated (treatment) sows at pre-, peri- and post-ovulatory oestrus. Only has3 mRNA was detected; it was present in all tubal segments of both control and treatment samples. The level of has3 expression did not vary significantly between non-inseminated and inseminated specimens, but there was a tendency (NS) for increased mean values during the peri- and post-ovulatory stages compared with pre-ovulation. It is concluded that has3 is expressed by the porcine endosalpinx epithelium and the levels of expression do not vary during the critical periods of sperm transport and fertilization, despite fluctuating levels of HA in the tubal fluid at corresponding periods.

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