Semi-Automated and Miniaturized SARS-CoV-2 Detection using TaqPath COVID-19 Multiplex Real-Time RT-PCR Assay v1

Abstract

The purpose of this procedure is to describe the setup of a semi-automated and miniaturized workflow for SARS-CoV-2 detection using the TaqPath COVID-19 Multiplex Real-Time RT-PCR Assay. This procedure applies to specimens received for SARS-CoV-2 testing in a clinical setting. Samples are collected, processed, and RNA is extracted as described elsewhere, using the MagMax Viral Pathogen (MVPII) 96 Kit for SARS-CoV-2 Detection protocol or the Omega Bio-tek Mag-Bind Viral DNA/RNA 96 Kit for SARS-CoV-2 Detection protocol. The TaqPath COVID-19 Multiplex Real-Time RT-PCR Assay is a multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) test intended for the qualitative detection of nucleic acid from SARS-CoV-2, the virus that causes Coronavirus Disease 2019 (COVID-19). The procedure has been modified to accommodate reactions as opposed to the standard reactions, significantly reducing per-reaction cost. This is achieved by using the SPT LabTech Mosquito HV and the SPT LabTech Mosquito X1 liquid handling robots. Purified viral ribonucleic acid (RNA) is reverse-transcribed into cDNA and amplified using the TaqPath™ COVID-19 Combo Kit and the QuantStudio 7 Pro 384 Real-Time PCR Systems. In the process, the probe oligos anneal to four (4) specific target sequences located between four (4) unique forward and reverse primers for the following genes: ORF1ab, N Gene, S Gene, and the MS2 bacteriophage RNA extraction positive control. During the extension phase of the qPCR cycle, the 5’ nuclease activity of Taq polymerase degrades the probe, causing the reporter dye to separate from the quencher dye, generating a fluorescent signal. With each qPCR cycle, additional reporter dye molecules are cleaved from their respective probes, increasing the fluorescence intensity. Fluorescence intensity is monitored during each PCR cycle by the QuantStudio 7 Pro 384 Real-Time PCR System.