Duplex One-Step RT-qPCR Assays for Simultaneous Detection of Genomic and Subgenomic RNAs of SARS-CoV-2 Variants

Abstract

A hallmark of severe acute respiratory syndrome virus (SARS-CoV-2) replication is the discontinuous transcription of open reading frames (ORFs) encoding structural virus proteins. Real-time reverse transcription PCR (RT-qPCR) assays in previous publications used either single or multiplex assays for SARS-CoV-2 genomic RNA detection and a singleplex approach for subgenomic RNA detection. Although multiplex approaches often target multiple genomic RNA segments, an assay that concurrently detects genomic and subgenomic targets has been lacking. To bridge this gap, we developed two duplex one-step RT-qPCR assays that detect SARS-CoV-2 genomic ORF1a and either subgenomic spike or subgenomic ORF3a RNAs. All primers and probes for our assays were designed to bind to variants of SARS-CoV-2. In this study, our assays successfully detected SARS-CoV-2 Washington strain and delta variant isolates at various time points during the course of live virus infection in vitro. The ability to quantify subgenomic SARS-CoV-2 RNA is important, as it may indicate the presence of active replication, particularly in samples collected longitudinally. Furthermore, specific detection of genomic and subgenomic RNAs simultaneously in a single reaction increases assay efficiency, potentially leading to expedited lucidity about viral replication and pathogenesis of any variant of SARS-CoV-2.

Keywords: RNA; SARS-CoV-2; duplex RT-qPCR; genomic; subgenomic; variant.

Figure 1

Singleplex RT-qPCR assays for the detection of SARS-CoV-2 RNA using synthetic gene fragments. Singleplex RT-qPCR assays were performed on serially diluted gene fragment standards to optimize detection of SARS-CoV-2 genomic ORF1a (gORF1a; (A)), subgenomic S (sgS; (B)) and subgenomic ORF3a (sgORF3a; (C)). Experiments were performed thrice, with three replicates for each reaction. RT-qPCR = real-time reverse transcription PCR; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2.

Figure 2

Duplex RT-qPCR assays specifically detect SARS-CoV-2 genomic and subgenomic nucleic acids. Duplex RT-qPCR assays were performed on serially diluted synthetic gene fragment mixtures using (gORF1a + sgS; (A)) or (gORF1a + sgORF3a; (B)). Experiments were performed thrice, with three replicates for each reaction. Mean Ct values for duplex assays for RNA from SARS-CoV-2-exposed Vero cells and positive controls (gORF1a + sgS/gORF1a + sgORF3a) gene fragments (C,D). Error bars depict standards deviations. Gel images show amplification products after RT-qPCR in (C,D). RT-qPCR = real-time reverse transcription PCR; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2.

Figure 3

Schematic of SARS-CoV-2 genome organization and transcription. SARS-CoV-2 genomic RNA (gORF1a and gORF1b) and subgenomic RNA (sgRNA) structure (comprising S, spike; E, envelope; M, membrane; and N, nucleocapsid) with 5′ leader sequence and transcriptional regulatory sequences (TRS-L and TRS-B) (A). The negative-sense and positive-sense sgRNA—sgRNA (−) and sgRNA (+), respectively—arising from discontinuous transcription and the primer and probe binding regions for gORF1a, sgS, and sgORF3a gene fragments are illustrated (B). SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2.

Figure 4

Singleplex RT-qPCR assays specifically detect SARS-CoV-2 genomic or subgenomic nucleic acids. The amplification product after RT-qPCR for genomic ORF1a (gORF1a; (A)), subgenomic spike (sgS; (B)), and subgenomic ORF3a (sgORF3a; (C)). SARS-CoV-2 RNA from supernatants of infected Vero cells (orange) and positive control gene fragment (green), negative non-template control (NTC; blue). The genomic S (gS; (B)), genomic ORF3a (gORF3a; (C)) were used as negative control gene fragments (gold). Amplification curves for the respective product seen on the gel for target gORF (A), sgS (B), and sgORF3a (C) are shown. The experiment was performed thrice, and each reaction was run in triplicate. RT-qPCR = real-time reverse transcription PCR; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2.

Figure 5

Longitudinal detection of SARS-CoV-2 genomic and subgenomic RNAs in Vero E6 cells expressing TMPRSS2. SARS-CoV-2 genomic and subgenomic RNAs were detected longitudinally using the gORF1a + sgS duplex RT-qPCR assay (A,B) and the gORF1a + sgORF3a duplex RT-qPCR assay (C,D) in TMPRSS2 Vero E6 cells that had been exposed to SARS-CoV-2. Experiments were performed thrice in 6-well plates, and duplex assays were performed in triplicates for each well. SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2; RT-qPCR = real-time reverse transcription PCR; TMPRSS2 = human transmembrane serine protease 2.

Figure 6

SARS-CoV-2 Washington strain and delta variant titers correlate with genomic or subgenomic RNA copy numbers. Plaque assays were performed for supernatants collected from human Vero E6 cells expressing TMPRSS2 exposed to SARS-CoV-2 (MOI = 0.01) at various time points (A). Experiments were performed thrice, and plaque assays were performed in duplicate for each infection. Comparison of SARS-CoV-2 genomic and subgenomic RNA copies in TMPRSS2 Vero E6 cells at 20, 24, 30, and 36 h after exposure to SARS-CoV-2 Washington strain or delta variant. Statistical significance is represented as *** (p = 0.001) on the scatter plot (B). Average SARS-CoV-2 titers from three independent experiments during which infected TMPRSS2 Vero E6 supernatants were collected for plaque assays at 20, 24, 30, and 36 h post-exposure (C). Statistical significance, established with an unpaired two-tailed t-test, is presented as ** (p < 0.05) and *** (p < 0.001). Error bars represent standard deviations. SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2; RT-qPCR = real-time reverse transcription PCR; TMPRSS2 = human transmembrane serine protease 2.