RT-RC-PCR: a novel and highly scalable next-generation sequencing method for simultaneous detection of SARS-COV-2 and typing variants of concern
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Abstract
We describe a novel assay method: reverse-transcription reverse-complement polymerase chain reaction (RT-RC-PCR), which rationalises reverse transcription and NGS library preparation into a single closed tube reaction. By simplifying the analytical process and cross-contamination risks, RT-RC-PCR presents disruptive scalability and economy while using NGS and LIMS infrastructure widely available across health service, institutional and commercial laboratories.
We present a validation of RT-RC-PCR for the qualitative detection of SARS-CoV-2 RNA by NGS. The limit of detection is comparable to real-time RT-PCR, and no obvious difference in sensitivity was detected between extracted nasopharyngeal swab (NPS) RNA and native saliva samples.
The end point measurement of RT-RC-PCR is NGS of amplified sequences within the SARS-CoV-2 genome; we demonstrated its capacity to detect different variants using amplicons containing delH69-V70 and N501Y, both of which emerged in the UK Variant of Concern B.1.1.7 in 2020.
In summary, RT-RC-PCR has potential to facilitate accurate mass testing at disruptive scale and cost, with concurrent detection of variants of concern.
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SciScore for 10.1101/2021.03.02.21252704: (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 Diagnostic test evaluation parameters were calculated using Medcalc (https://www.medcalc.org/calc/diagnostic_test.php). Medcalcsuggested: (MedCalc, RRID:SCR_015044)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 …SciScore for 10.1101/2021.03.02.21252704: (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 Diagnostic test evaluation parameters were calculated using Medcalc (https://www.medcalc.org/calc/diagnostic_test.php). Medcalcsuggested: (MedCalc, RRID:SCR_015044)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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