An acceptable methodological evaluation method of real-time fluorescent RT-PCR targeting SARS-CoV-2

Background: it is necessary to evaluate real-time fluorescent reverse-transcription PCR (RT-PCR) methods to detect the nucleic acids of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: : considering lack of positive specimens in some particular locations in China, the specimens from healthy individuals were used to perform the methodology evaluations, in which the indexes were the differences of quantification cycle values (Δ C q) between human-derived internal reference control (IRC) genes of a specimen and quality control gene (QC). A series of experiments was conducted to evaluate various factors that might affect the results, such as types of virus transport media, methods of specimen pretreatment and template preparation, specimen vortex strength, specimen storage temperature, and duration. Results: : it was better to store specimens in normal saline (NS) transport medium, release more virus particles from swabs by vortex mixing, extract nucleic acids with centrifugation methods, and perform amplification assays timely. The above-mentioned options and optimum conditions were further confirmed using SARS-CoV-2 pseudoviruses and positive clinical specimens. Conclusions: : this study provides a solution for the accurate detection of SARS-CoV-2. Specifically, this study also indicates that the routine specimens from healthy individuals could be used to methodological evaluation of real-time fluorescent RT-PCR targeting SARS-CoV-2, of which the indexes were the Δ C q values.