Endpoint PCR Detection of Sars-CoV-2 RNA
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Abstract
Quantitative real-time PCR methods have been used to perform approximately 278 million tests for COVID-19 up to mid-July 2020. Real-time PCR involves a rate limiting step where the samples are measured in situ during each PCR amplification cycle. This creates a bottleneck limiting scalability and as a consequence reducing access to inexpensive reliable testing at national and international scales. We investigated endpoint PCR for the qualitative detection of SARS-CoV-2 sequences on synthetic RNA standards and hospital patient samples. The endpoint PCR detection limit is constrained only by the stochastics of low copy numbers and reliably detected single copies of synthetic RNA standards. On a set of 30 patient samples, endpoint PCR found one additional positive sample and was able to confirm an indeterminate sample as negative. These results were found using 4 μl reagent and 1 μl of sample representing an 80% reduction in required RNA extract input and PCR reagent volumes relative to the NHS protocol (20 μl reagent and 5 μl sample). These results indicate that endpoint PCR should be the method of choice for large scale testing programmes. Based on the experience from ultra-high throughput genotyping efforts a single workflow using 384-well plates has similar PCR capacity (250 Million) to that required for all testing done worldwide during the first 7 month of the pandemic.
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SciScore for 10.1101/2020.07.21.20158337: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources The ensuing Promega extracts underwent PCR by either GeneFinder COVID-19 PLUS RealAmp Kit or the Viasure SARS-CoV-2 Real Time PCR Detection Kit. GeneFindersuggested: (GENEFINDER, RRID:SCR_009190)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not …SciScore for 10.1101/2020.07.21.20158337: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources The ensuing Promega extracts underwent PCR by either GeneFinder COVID-19 PLUS RealAmp Kit or the Viasure SARS-CoV-2 Real Time PCR Detection Kit. GeneFindersuggested: (GENEFINDER, RRID:SCR_009190)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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SciScore for 10.1101/2020.07.21.20158337: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources Cost and performance comparisons have appeared in the literature and industrial scale PCR cost has been well below US$1/data point for several years and costs as low as US$0.064/data point documented. Costsuggested: (COST, SCR_014098)Hospital real-time PCRs: Both the GeneFinder RealAmp PCR and the Viasure SARS-CoV-2 Real Time PCR assays used 5 μl of RNA eluate added to 20μl of respective assay’s detection … SciScore for 10.1101/2020.07.21.20158337: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources Cost and performance comparisons have appeared in the literature and industrial scale PCR cost has been well below US$1/data point for several years and costs as low as US$0.064/data point documented. Costsuggested: (COST, SCR_014098)Hospital real-time PCRs: Both the GeneFinder RealAmp PCR and the Viasure SARS-CoV-2 Real Time PCR assays used 5 μl of RNA eluate added to 20μl of respective assay’s detection mastermix which then underwent PCR reaction in a 25 μl reaction (96-well plate). GeneFindersuggested: (GENEFINDER, SCR_009190)The Abbott RealTime SARS-C0V-2 assay utilised 15 μl of RNA eluate from Abbott M2000 extraction added to 90μl of assay detection mastermix prior to real-time PCR in the Abbott M2000 analyser. Abbottsuggested: (Abbott, SCR_010477)Data from additional tools added to each annotation on a weekly basis.
About SciScore
SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.
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