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BackgroundSaline 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. MethodsClinical 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-CDCs 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 {degrees}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-2s 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. ResultsA 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 (CT) values was observed for both the SORP and N1/N2 assay when extraction-free was compared between the standard method. The average {Delta}CT [{Delta}CT=CT(Direct PCR)-CT(Extracted RNA)], for each of the gene targets were: S ({Delta}CT= +4.24), ORF8 ({Delta}CT=+2.63), N1 ({Delta}CT=+2.74) and N2 ({Delta}CT=+5.74). The {Delta}CT 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. ConclusionOur 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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