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.

Keywords: COVID-19; SARS-CoV-2; coronavirus; qRT-PCR detection; test protocol.

Figure 1

The WHO E-gene PCR for the pre-screening for SARS-CoV-2 using in vitro diagnostic (IVD)-certified test kits (Roche) produces unspecific, template-independent side-products. (a) Detection of SARS-CoV-2 false-positive samples (Pat. 20–23) tested with primers targeting the E-gene. (b) Confirmatory PCR using SARS-CoV-2 RdRP-specific primer and probe. RFU, relative fluorescence units; pos, positive control SARS-CoV-2 positive Pat.1.

Figure 2

Characteristics of M-gene based qRT-PCR. (a) Representative amplification curves of WHO RdRP- and (b) SARS-CoV-2 M-gene-specific qRT-PCR using plasmid standard copy numbers. (c) Standard curves of both genes determined with plasmid templates. The Cq was plotted against the log starting quantity of the indicated plasmid DNA template. (d) Representative amplification curves and (e) standard curves of SARS-CoV-2 M-gene-specific qRT-PCR using in vitro-transcribed RNA templates. Log starting quantity (copies/reaction) is indicated and plotted against the Cq. (f) Representative amplification curves for SARS-CoV-2 M- and RdRP-gene-specific qRT-PCR using in vitro-transcribed RNA templates harboring both target sequences. (g) Distinct M-gene amplicons visualized in a 2% agarose gel. The RNA copy numbers per reaction are indicated on the top. RFU, relative fluorescence units; bp, base pairs; Cq, quantification cycle; NTC, no template control (H₂O); E, amplification efficiency.

Figure 3

SARS-CoV-2 RNA detection in patient samples detection using M- versus RdRP-specific primers. (a) Comparison of M- and RdRP-specific primers and probes measured with the indicated one step qRT-PCR kits. (b) Mean values of the Cq difference between RdRP- and M-gene PCR. Cq, quantification cycle. Only samples positive in all assays are shown (n = 6).

Figure 4

Detection of intracellular SARS-CoV-2 RNAs. (a) Vero cells were infected with SARS-CoV-2 isolate FFM1 (MT358638) with high and low MOI, and 24 h post infection, the RNA was subjected to M- and RdRP-gene-specific one-step probe qRT-PCR analysis (Luna, NEB). Intracellular copy numbers (b) and representative amplification curves (c) of SARS-CoV-2 isolate FFM1-infected Caco-2 cells determined with the M-gene primer pairs and probe. Total cellular RNA including viral RNA was harvested at the indicated time points and subjected to M-gene specific qRT-PCR analysis.

Figure 5

SARS-CoV-2 sequences are highly conserved in terms of the M-gene. (a) SARS-CoV-2 isolates FFM1–7 described in this study were mapped to the NC_045512 (SARS-CoV-2) reference genome using the Geneious Prime software. Single nucleotide polymorphisms and their corresponding positions are indicated as colored boxes. Amino acid substitutions are shown above red boxes. Silent mutations are indicated in yellow boxes. (b) Phylogenetic tree of SARS-CoV-2 isolates, inferred by using the Maximum Likelihood method and Tamura–Nei model using the MEGA-X software. The tree is drawn to scale, with branch lengths representing the number of substitutions per site.