Gargle-Direct: Extraction-Free Detection of SARS-CoV-2 using Real-time PCR (RT-qPCR) of Saline Gargle Rinse Samples
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Abstract
Background
Saline mouth rinse/gargle samples have recently been shown to be a suitable option for swab-independent self-collection for SARS-CoV-2 diagnosis. We sought to evaluate a simplified process for direct reverse transcriptase PCR (RT-qPCR) testing of this novel sample type and to compare performance with routine RT-qPCR using automated nucleic acid extraction.
Methods
Clinical saline mouth rinse/gargle samples were subjected to automated nucleic acid extraction (“standard method”), followed by RT-qPCR using three assays including the FDA authorized US-CDC’s N1/N2 assay, which was the reference standard for determining sensitivity/specificity. For extraction-free workflow, an aliquot of each gargle sample underwent viral heat inactivation at 65 °C for 20 minutes followed by RT-qPCR testing, without an intermediate extraction step. An in-house validated RT-qPCR lab developed test (LDT), targeting the SARS-CoV-2’s S/ORF8 genes (SORP triplex assay) and the N1/N2 US-CDC assay was used to evaluate the extraction-free protocol. To improve the analytical sensitivity, we developed a single-tube hemi-nested (STHN) version of the SORP triplex assay.
Results
A total of 38 SARS-CoV-2 positive and 75 negative saline mouth rinse/gargle samples were included in this evaluation. A 100% concordance in detection rate was obtained between the standard method and the extraction-free approach for the SORP assay. An average increase of +2.63 to +5.74 of the cycle threshold (C T ) values was observed for both the SORP and N1/N2 assay when extraction-free was compared between the standard method. The average ΔC T [ΔC T =C T(Direct PCR) -C T(Extracted RNA) ], for each of the gene targets were: S (ΔC T = +4.24), ORF8 (ΔC T =+2.63), N1 (ΔC T =+2.74) and N2 (ΔC T =+5.74). The ΔC T for the STHN SORP assay was +1.51 and −2.05 for the S and ORF8 targets respectively, when extracted method was compared to the standard method.
Conclusion
Our Gargle-Direct SARS-CoV-2 method is operationally simple, minimizes pre-analytical sample processing and is potentially implementable by most molecular diagnostic laboratories. The empirical demonstration of single-tube hemi-nested RT-qPCR, to specifically address and alleviate the widely-acknowledged problem of reduced analytical sensitivity of detection of extraction-free templates, should help diagnostic laboratories in choosing Gargle-Direct protocol for high-throughput testing.
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SciScore for 10.1101/2020.10.09.20203430: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Approval was obtained from the BC Children’s and Women’s Hospital Research Ethics Board for this study (H20-02538). Randomization not detected. Blinding not detected. 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:Limitations of this study include the relatively small number of positive specimens although, we have included more than …
SciScore for 10.1101/2020.10.09.20203430: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Approval was obtained from the BC Children’s and Women’s Hospital Research Ethics Board for this study (H20-02538). Randomization not detected. Blinding not detected. 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:Limitations of this study include the relatively small number of positive specimens although, we have included more than the 30 positive and negative clinical samples recommended by the FDA for evaluation of new molecular assays as part of the Emergency Use Authorization authority. Future studies should include larger numbers of prospectively tested specimens, especially those with low viral loads (CT>35), to confirm these findings and assess the operational feasibility of implementing larger scale robotic Gargle-Direct testing of saline mouth rinse/gargle specimens. For example, adjusting the protocol such that heat inactivation occurs in the primary collection tube, would likely be feasible and significantly simplify the workflow. In summary, we describe a sensitive and specific diagnostic method for SARS-CoV-2 that is operationally simple, bypasses multiple supply chain bottlenecks, utilizes a self-collected swab independent sample type, is appropriate for large scale testing, is cost effective, and can be readily adopted by other laboratories. This STHNRT-qPCR could also be applied in other SARS-CoV-2 testing scenarios where a loss of sensitivity is routinely expected e.g. SARS-CoV-2 testing by sample pooling. This would facilitate large scale SARS-CoV-2 testing, a crucial tool for the control the COVID-19 pandemic.
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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