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

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, and Influenza B viruses is essential for rapid differential diagnosis in patients with similar symptoms, especially during “ flu season” in the post-pandemic era.

So far, several multiplex methods have been approved for the simultaneous detection of SARS-CoV-2, Influenza A, and Influenza B .

However, due to the rapid mutation rate of the SARS-CoV-2 genome and the emergence of new variants, existing methods must be improved and updated.

Methods To identify a highly conserved region in the SARS-CoV-2 N-gene , a genomic survey was performed to increase the sensitivity and specificity of primer and probe sets targeting the SARS-CoV-2 genome.

The 95% LLOD (95% lower limits of detection) were calculated by probit analysis.

A total of 70 predetermined clinical samples using singleplex RT-qPCR assays, were included.

The clinical performance of the multiplex RT-qPCR assay was determined and compared with a commercial multiplex kit.

The Cohen’s kappa coefficient, P -value (McNemar’s test), Passing-Bablok regression, and Bland Altman agreement analysis were determined to monitor the agreement of the assays.

Results The novel SARS-CoV-2 primer and probe set designed in this assay was able to detect all variants of concern (VOCs) and variants of interest (VOIs) with high analytical and clinical performance.

The 95% LLOD for the multiplex RT-qPCR was 20 copies per reaction for the N gene of SARS-CoV-2 , 2 copies per reaction for M1 gene of Influenza A and NS1 gene of Influenza B .

The diagnostic sensitivity of the multiplex RT-qPCR was 94.4%, 93.7%, and 100% for the detection of SARS-CoV-2 , Influenza A , and Influenza B genomes, respectively.

Moreover, the specificity was identical (100%) in both assays.

According to the agreement analysis results, there was no statistical difference between our multiplex assay and the commercial kit.

Conclusions In this study, we developed a novel in-house made multiplex RT-qPCR assay, with high sensitivity, specificity, and reliability for the diagnosis of SARS-CoV-2 infection in clinical samples.

This is valuable during Influenza seasons when influenza co-circulates with SARS-CoV-2, as it saves costs, time, and thus specific and timely treatment of patients.