Flexible multiplex PCR to detect SARS‐CoV‐2, coronavirus OC43 and influenza A virus in nasopharyngeal swab samples

INTRODUCTION: Quantitative reverse transcription PCR (RT‐qPCR) is the leading tool to detect severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Given that it will almost certainly continue to coexist with other respiratory viruses in the coming years, our study aimed to design a multiplex PCR system not affected by supplier outages and with reduced cost compared to the existing commercially available kits. METHODS AND RESULTS: In this study, combinations of four primers/probe sets were used to construct a flexible RT‐qPCR assay which is capable of discriminating between SARS‐CoV‐2 and the seasonal human coronavirus HCoV‐OC43, or even influenza A virus. Additionally, the human RPP30 gene was used as an internal control. To demonstrate the robustness of the assay, it was applied to a collection of 150 clinical samples. The results showed 100% sensitivity and specificity compared to the automatized system used at the hospital and were better when indeterminate samples were analysed. CONCLUSIONS: This study provides an efficient method for the simultaneous detection of SARS‐CoV‐2, HCoV‐OC43 and influenza A virus, and its efficacy has been tested on clinical samples showing outstanding results. SIGNIFICANCE AND IMPACT OF THE STUDY: The multiplex RT‐qPCR design offers an accessible and economical alternative to commercial detection kits for hospitals and laboratories with limited economic resources or facing situations of supply shortage.