Optimized qRT-PCR Approach for the Detection of Intra- and Extra-Cellular SARS-CoV-2 RNAs

Abstract

The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Meanwhile, increased demand for testing has led to a shortage of reagents and supplies and compromised the performance of diagnostic laboratories in many countries. Both the World Health Organization (WHO) and the Center for Disease Control and Prevention (CDC) recommend multi-step RT-PCR assays using multiple primer and probe pairs, which might complicate the interpretation of the test results, especially for borderline cases. In this study, we describe an alternative RT-PCR approach for the detection of SARS-CoV-2 RNA that can be used for the probe-based detection of clinical isolates in diagnostics as well as in research labs using a low-cost SYBR green method. For the evaluation, we used samples from patients with confirmed SARS-CoV-2 infections and performed RT-PCR assays along with successive dilutions of RNA standards to determine the limit of detection. We identified an M-gene binding primer and probe pair highly suitable for the quantitative detection of SARS-CoV-2 RNA for diagnostic and research purposes.

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“…RdRP-PCR was highly specific for SARS-CoV-2 and M-gene PCR being the most sensitive for SARS-CoV-2. These results are in line with recently published data by Toptan et al (2020) , where the efficiency of RT-qPCR primer and probes pairs to detect SARS-CoV-2 genes was compared. At least in this study, N-gene primers were less specific and were excluded due to limited linearity compared to other viral genes.…”

“…RNA was analysed by OneStep RT-qPCR using Luna Universal Probe One-Step RT-qPCR Kit (New England Biolabs) or LightCycler® Multiplex RNA Virus Master (Roche) and the CFX96 Real-Time System, with a C1000 Touch Thermal Cycler (Bio-Rad). Primer pairs for E-, N- and M-gene ( Toptan et al, 2020 ) specific PCRs were used in equimolar concentrations (0.4 μM each per reaction), while RdRP-primer pairs were used according to Corman et al (2020) with 0.6 μM and 0.8 μM for forward and reverse primers, respectively. The sequences of primers and probes are found in Table 3 .…”

“…Initial denaturation was allowed for 30 s at 95 °C followed by 45 cycles of denaturation for 5 s, extension for 30 s at 60 °C and final cool-down to 40 °C for 30 s. The PCR runs were analysed with Bio-Rad CFX Manager software version 3.1 (Bio-Rad Laboratories). For quantifications, standard curves using plasmid DNA (RdRP) or in vitro transcribed RNA (M-gene) were used as described previously ( Toptan et al, 2020 ). Depending on the RT-qPCR for M-gene or RdRP the detection limit was 200 copies per reaction.…”

“…To calculate from gene equivalents per reaction back to copies per mL, a PCR correction factor (cf) was determined with Luna Universal Probe One-Step RT-qPCR Kit (cf = 0.0037) and LightCycler® Multiplex RNA Virus Master (cf = 0.0015), respectively. To control PCR, viral RNA of SARS-CoV-2 (isolate SARS-CoV-2-FFM1) was used as a positive control ( Hoehl et al, 2020 ; Toptan et al, 2020 ) and water as a negative control. Furthermore, to verify specificity and sensitivity, RNA of SARS-CoV (isolate SARS-CoV-FFM1) ( Drosten et al, 2003 ) and HCoV-229E from cell culture supernatant was isolated and used in PCR.…”

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“…(v/v) L-glutamine (Sigma Aldrich, Germany). Both SARS-CoV-2/FFM1 and SARS-CoV-2/FFM7 were isolated as previously described (Hoehl et al, 2020;Toptan et al, 2020). The viral stocks were produced by passaging virus on Caco-2 cells at MOI 0.1.…”