Simultaneous detection and differentiation of SARS-CoV-2, influenza A virus and influenza B virus by one-step quadruplex real-time RT-PCR in patients with clinical manifestations

15 Dec, 2020


In order to develop a novel method based on the quadruplex assay which is a valuable diagnostic tool for the diagnosis of SARS-CoV-2 infection, differential diagnosis and detection of coinfections. 
A one-step quadruplex real-time reverse transcription-PCR (rRT-PCR) assay was developed for simultaneous detection and differenation of SARS-CoV-2 ORF1ab and N genes, influenza A virus (hIAV) and influenza B virus (hIBV).
The results showed that the quadruplex assay presents good sensitivity and specificity. Correlation coefficients (R2) and amplification efficiencies (E) of all singleplex and multiplex rRT-PCR reactions are within the acceptable range. The 95% LLoDs of plasmid standards and positive nucleic acid extracts of quadruplex assay were in the range [57.38–95.11] copies genomic/µl and [114.65–154.25] copies genomic/µl, respectively. Excellent results were attained in the inter-assay and intra-assay reproducibility evaluation. Among these clinical samples, only 4 samples showed results inconsistent with the singleplex rRT-PCR assay. 
This study is the first report to establish a quadruple rRT-PCR assay for the detection of two SARS-CoV-2 genes, hIAV and hIBV, owing to perfect clinical performance.

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