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.

Misidentification of the SARS-CoV-2 Mu variant using commercial mutation screening assays.

Detection of mutations by multiplex real-time RT-PCR is a widely used method for the screening of SARS-CoV-2 variants, but this method has several limitations. We describe three cases in which a Mu strain containing the mutation K417N was initially misclassified as the Beta variant. We recommend the detection of P681H to distinguish between these two variants. Our experience highlights the importance of keeping track of new variants and mutations in order to adapt the current workflows.