Objective: To develop a novel quadruplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay for the diagnosis of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, differential diagnosis and detection of co-infections.
Methods: A one-step quadruplex rRT-PCR assay was developed for simultaneous detection and differentiation of SARS-CoV-2 ORF1ab and N genes, influenza A virus (hIAV) and influenza B virus (hIBV).
Results: The quadruplex rRT-PCR assay had good sensitivity and specificity. Correlation coefficients and amplification efficiencies of all singleplex and quadruplex rRT-PCR reactions were within acceptable ranges. The 95% lower limits of detection for plasmid standards and positive nucleic acid extracts of the quadruplex rRT-PCR assay were 57.38-95.11 copies/μL and 114.65-154.25 copies/μL, respectively. Excellent results were attained for inter- and intra-assay reproducibility. Among these clinical samples, only four samples showed results inconsistent with the singleplex rRT-PCR assays.
Conclusions: To the authors' knowledge, this is the first study to report a quadruplex rRT-PCR assay for the detection of two SARS-CoV-2 genes, hIAV and hIBV with perfect clinical performance.
Keywords: Co-infection; Differentiation; Influenza A virus; Influenza B virus; Quadruplex; SARS-CoV-2.
Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.