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

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.

POCT Detection of 14 Respiratory Viruses Using Multiplex RT-PCR.

Over the past 6 years, acute respiratory infections have constituted an average of more than 70,000 cases in South Korea. It results in a high mortality rate in infants and the elderly with weak immunity. There are several types of respiratory viruses that invade the human respiratory tract and cause infectious disease. Reverse transcription PCR (RT-PCR) is mainly used for respiratory virus detection owing to its high sensitivity and reproducibility. In response, a multiplex real-time RT-PCR (rRT-PCR) assay was developed for the detection of influenza A and B viruses, parainfluenza viruses 1-4 (PIV1-4), human metapneumovirus, adenovirus, human rhinovirus, respiratory syncytial virus (RSV), and SARS-CoV-2. Detection ability of RT-PCR assay was confirmed by applying it to a portable device capable of point-of-care testing (POCT). Amplicons were synthesized using primer pairs and probe sets designed for each target virus, and a standard curve was constructed to confirm the limit of detection. An experiment using nasopharyngeal swab samples was conducted to understand the field applicability of the rRT-PCR assay. Detection was confirmed in most samples. This study demonstrated that rapid and accurate detection results can be obtained using the multiplex rRT-PCR based POC test, and that it is possible to detect 14 types of respiratory viruses that are generally difficult to distinguish at the same time, enabling timely treatment. Furthermore, we expect that the portable PCR device can significantly reduce the processing procedure of clinical samples before testing, which is the main disadvantage of common RT-PCR tests and can help reduce costs.