Establishment of a Rapid Typing Method for Coronavirus Disease 2019 Mutant Strains Based on PARMS Technology.

ABSTRACT

Purpose: This study aims to establish a competitive allele-specific PCR based on penta-primer amplification refractory mutation system (PARMS) technology to detect the key mutation sites of variant of concern (VOC) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus for rapid typing. Methods: Competitive allele-specific primers and universal primers were designed for the key gene mutation sites N501Y, E484K, L452R, and K417N of SARS-CoV-2 VOCs, respectively.Using the principle of allele-specific polymerase chain reaction and fluorescence energy resonance transfer, different VOCs can be differentiated. Results: Using reverse transcribed cDNA of different VOCs as specimens, through double-blind detection, different VOC types can be effectively identified, with an accuracy rate of 100%. Through the identification and detection of different VOCs, effective differentiation can be achieved. Conclusions: The system has high specificity and sensitivity, with a detection limit of 1.28 copies/reaction.PARMS technology is fast, efficient, and low-cost. It is used for the identification and detection of the popular SARS-CoV-2 VOCs, which is helpful for the rapid and accurate prevention and control of COVID-19 epidemic.