Simultaneous detection of SARS-CoV-2 and identification of spike D614G mutation using point-of-care real-time polymerase chain reaction

Highlights

• Rapid identification of SARS-CoV-2 is critical.

• RT-PCR, for virus detection, is conducted in centralized laboratories, delays TAT.

• We developed a POCT platform to simultaneously detect SARS-CoV-2 and its mutants.

• It showed high detection sensitivity and accuracy.

• We present a rapid detection method for continuously appearing virus mutations.

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with high mortality and infectivity rates in humans since its emergence. Analysis using high-accuracy real-time polymerase chain reaction (PCR) is recommended for the detection of general respiratory viruses including SARS-CoV-2, but it takes a long time (e.g. ~ 6 h); moreover, on-site diagnosis is difficult owing to the need for skilled technicians and advanced laboratory facilities. Currently, the importance of point-of-care testing (POCT) is being emphasized for the rapid detection of SARS-CoV-2. Here, we developed a multiplex real-time reverse transcription PCR (rRT-PCR) analysis that not only detects SARS-CoV-2 but also D614G strains with higher contagiousness than wild types among SARS-CoV-2 mutants using probe-based rRT-PCR. Moreover, this method was applied to portable PCR equipment capable of POCT to confirm high detection sensitivity and specificity. Multiple assays were possible with fluorescence labeling of individual probes. Furthermore, using a microfluidic chip-based point-of-care testing rRT-PCR platform, detection time was reduced by more than half compared with the commonly used detection system. This demonstrates that our assay has 100% of high sensitivity and specificity and could thus aid in the rapid and simple screening of SARS-CoV-2 carrying the mutation. We present a rapid detection method for mutations in SARS-CoV-2.