Development of a robust TaqMan probe-based one-step multiplex RT-qPCR for Simultaneous detection of SARS-CoV-2 and influenza A/B viruses

Abstract

Abstract Background: During the coronavirus disease 2019 (COVID-19) pandemic, the simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A/B viruses are essential in the rapid differential diagnosis in patients with similar symptoms. So far, several multiplex methods were published for simultaneous detection of SARS-CoV-2 and Flu A/B. However, due to the rapid mutation rate of the SARS-CoV-2 genome and the emergence of new variants, making it necessary to improve and update existing methods. Methods: To identify a highly conserved region on SARS-CoV-2 N-gene, variant genome survey was performed to increase the sensitivity and specificity of primers and probe annealing to the SARS-CoV-2 genome. The analytical and diagnostic sensitivity and specificity of our multiplex RT-qPCR assay were determined; and the kappa agreement was calculated to highlight the agreement using the Chi-square test by SPSS 22.0. Results: The new set of SARS-CoV-2 primer-probes designed in the present assay was able to detect all variants of concern (VOCs) and variants of interest (VOIs) with high analytical specificity among other respiratory viruses. The analytical sensitivity by our multiplex assay was ≤ 30 copies of the plasmid per reaction. A total of 70 clinical samples were analyzed using our multiplex assay and compared with multiplex commercial kit. The sensitivity of our multiplex RT-qPCR assay and commercial kit were 95.4% and 93.1%, respectively. Also, the specificity was the same (100%) in both methods. Kappa agreement was measured at 0.97 and 0.90 for our multiplex assay and commercial kit respectively. Conclusions: A novel and robust TaqMan probe-based one-step multiplex RT-qPCR assay, with high analytical and clinical performance and agreement was developed and validated in this study. It will be valuable in influenza seasons when influenza expected co-circulate with SARS-CoV-2, due to saving costs, time, and consequently, specific and timely treatment of patients.