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
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Abstract
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.
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SciScore for 10.1101/2021.04.10.21254091: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Consent: 2.2 Ethical statement: The samples generated and disseminated as part of Projects A and B were approved under a waiver of consent by the University of Washington institutional review board (IRB; STUDY00000408).
IRB: 2.2 Ethical statement: The samples generated and disseminated as part of Projects A and B were approved under a waiver of consent by the University of Washington institutional review board (IRB; STUDY00000408).Randomization not detected. Blinding To confirm that all reagents arrived safely and that every laboratory could perform the direct RT-PCR method, each laboratory tested a set of eight nucleic acid samples purified from patients with … SciScore for 10.1101/2021.04.10.21254091: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Consent: 2.2 Ethical statement: The samples generated and disseminated as part of Projects A and B were approved under a waiver of consent by the University of Washington institutional review board (IRB; STUDY00000408).
IRB: 2.2 Ethical statement: The samples generated and disseminated as part of Projects A and B were approved under a waiver of consent by the University of Washington institutional review board (IRB; STUDY00000408).Randomization not detected. Blinding To confirm that all reagents arrived safely and that every laboratory could perform the direct RT-PCR method, each laboratory tested a set of eight nucleic acid samples purified from patients with COVID-19 and supplied by UWVL, which included six blinded samples (three positive and three negative), one sample identified positive, and one sample identified negative to serve as controls, plus a laboratory-supplied no-template water control. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
No key resources detected.
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: We detected the following sentences addressing limitations in the study:Although the reproducibility of the method has been reported in single-laboratory studies previously, this study is the first to demonstrate that a globally diverse set of laboratories operating with different equipment, clinical sample collection and handling conditions, resource limitations, and operating practices can successfully implement the method. As described above, when centrally disseminated pooled samples were evaluated with a common Master Mix and primers/probes (Projects A and B), the Propagate partner laboratory results were >99.5% concordant (all negatives and all but one positive correctly identified and strong agreement on Ct values). This result demonstrates the robustness of the methodology. Although all Propagate partner laboratories had prior experience with standard RNA extraction RT-PCR analysis of SARS-CoV-2 test samples, they were able to adopt and implement the direct method on Project A and B samples with only a minimum of instruction (a brief written protocol and a few minutes of discussions via web meeting), showing that the method is easily transferable. Due to halts and delays in air shipments, the partner laboratories in Malawi and Nigeria were unable to receive or analyze the Project A/B sample kits. While this was unfortunate, it is emblematic of the challenges that the African continent (among others) continues to face in receiving needed laboratory supplies and the importance of resource-sensitive methods development efforts such as these....
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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