A two-pronged approach for rapid and high-throughput SARS-CoV-2 nucleic acid testing
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Abstract
Improved molecular screening and diagnostic tools are needed to substantially increase SARS-CoV-2 testing capacity and throughput while reducing the time to receive test results. Here we developed multiplex reverse transcriptase polymerase chain reaction (m-RT-PCR) for detection of SARS-CoV-2 using rapid DNA electrophoresis and alternatively using multiplex viral sequencing (mVseq). For RNA specimens extracted from nasopharyngeal (NP) swabs in viral transport media (VTM), our assays achieved a sensitivity for SARS-CoV-2 detection corresponding to cycle threshold (Ct) of 37.2 based on testing of these specimens using quantitative reverse transcription PCR (RT-qPCR). For NP swab-VTM specimens without prior RNA extraction, sensitivity was reduced to Ct of 31.6, which was due to lower concentration of SARS-CoV-2 genome copies in VTM compared to RNA-extracted samples. Assay turnaround time was 60 minutes using rapid gel electrophoresis, 90 minutes using Agilent Bioanalyzer, and 24-48 hours using Illumina sequencing, the latter of which required a second PCR to produce a sequence-ready library using m-RT-PCR products as the template. Our assays can be employed for high-throughput sequencing-based detection of SARS-CoV-2 directly from a clinical specimen without RNA isolation, while ease-of-use and low cost of the electrophoresis-based readout enables screening, particularly in resource-constrained settings.
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SciScore for 10.1101/2020.12.04.20234450: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Study population: The Yale Institutional Review Board determined this study to be not human subjects research (IRB Protocol #2000029556). 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 Volumes of 5 μl or 1 μl of the m-RT-PCR product was run directly on a 1.5% agarose gel or the Agilent Bioanalyzer, respectively (Figure 2). Agilent Bioanalyzersuggested: NoneFollowing quantification, a small aliquot of the library (18 μl of 1.3 nM final concentration) was run on Illumina NovaSeq 6000 or MiSeq using paired-end 150 bp sequencing. MiSeqsug…SciScore for 10.1101/2020.12.04.20234450: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Study population: The Yale Institutional Review Board determined this study to be not human subjects research (IRB Protocol #2000029556). 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 Volumes of 5 μl or 1 μl of the m-RT-PCR product was run directly on a 1.5% agarose gel or the Agilent Bioanalyzer, respectively (Figure 2). Agilent Bioanalyzersuggested: NoneFollowing quantification, a small aliquot of the library (18 μl of 1.3 nM final concentration) was run on Illumina NovaSeq 6000 or MiSeq using paired-end 150 bp sequencing. MiSeqsuggested: (A5-miseq, RRID:SCR_012148)The paired-end reads were aligned using BWA MEM to a reference containing the SARS-CoV-2 genome (NC_045512.2) and RPP30 gene (NM_001104546.1). BWAsuggested: (BWA, RRID:SCR_010910)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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