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.

Development and Validation of a Panel of One-Step Four-Plex qPCR/RT-qPCR Assays for Simultaneous Detection of SARS-CoV-2 and Other Pathogens Associated with Canine Infectious Respiratory Disease Complex

“…In view of our data, we have demonstrated that using our assays in a multiplex format did not affect the analytical parameters. Additionally, these parameters were similar to those from other assays recently developed in our laboratory for the detection of canine enteric viruses and feline respiratory pathogens [ 69 , 70 ]. In this study, two to three log 10 improvement of the LOD 95 was determined when compared to the previously published multiplex PCR assays for the detection of CAdV-2, CDV, CIV, and CPiV [ 38 ].…”

Variations in the persistence of 5′-end genomic and subgenomic SARS-CoV-2 RNAs in wastewater from aircraft, airports and wastewater treatment plants

“…This is consistent with previous work reporting that N1 and N2 were the sequence features of SARS-CoV-2 most likely to be recovered from wastewater [ 37 ]. We also found subgenomic Orf3a in abundance in one wastewater sample, a subgenomic RNA for which RT-qPCR assays [ 55 ] are also available, demonstrating the ability of wastewater sequencing to detect such RNA molecules. While looking at the mean coverage of the first 300 nucleotides on the 5′-end of the genome, we found that most samples directly collected from aircraft had this region covered (5 out of 6), while only 4 out of 10 samples had reads covering these regions in samples obtained from water tanks pooled from various sources.…”