Automated multiplex nucleic acid tests for rapid detection of SARS-CoV-2, influenza A and B infection with direct reverse-transcription quantitative PCR (dirRT-qPCR) assay in a centrifugal microfluidic platform

Abstract

The coronavirus disease 2019 (COVID-19) pandemic, caused by the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, has posed a threat to public health worldwide. Also, influenza virus has caused a large number of deaths annually. Since co-infection of SARS-CoV-2 and influenza virus, which share similar symptoms, hampers current treatment efficiency, multiple simultaneous detection of these viruses is needed to provide the right treatment for patients. We developed a microfluidic disc-direct RT-qPCR (dirRT-qPCR) assay for rapid multiplex detection of SARS-CoV-2, influenza A and B viral infection in pharyngeal swab samples in an automated manner. Choices of the DNA polymerase, concentrations of dTPs and MgCl₂ were characterized to optimize the assay. A detection limit of 2 × 10¹ copies per reaction was found in all three viral RNAs with as little as 2 μL of swab samples. The accuracy of our assay was evaluated with 2127 clinical swab samples of infection with these three viruses and healthy controls, and it possessed a consistency rate of 100, 99.54 and 99.25% in SARS-CoV-2, influenza A and B detection in comparison to standard RT-qPCR. The reported scheme of our assay is capable of screening other viral infections for up to 16 targets simultaneously. The whole diagnosis could be completed in 1.5 hours after simple sample loading by a non-technical expert. This constitutes an enabling strategy for large-scale point-of-care screening of multiple viral infections, which ultimately lead to a pathway for resolving the critical issue of early diagnosis for the prevention and control of viral outbreaks.