One-step quantitative RT-PCR assay with armored RNA controls for detection of SARS-CoV-2.

Abstract

Coronavirus disease 2019 (COVID‐19) has become pandemic since March 11, 2020. Thus, development and integration in clinics of fast and sensitive diagnostic tools are essential. The aim of the study is a development and evaluation of a one‐step quantitative reverse transcription‐polymerase chain reaction (RT‐qPCR) assay (COVID‐19 Amp) for severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) detection with an armored positive control and internal controls constructed from synthetic MS2‐phage‐based RNA particles. The COVID‐19 Amp assay limit of detection was 103 copies/ml, the analytical specificity was 100%. A total of 109 biological samples were examined using COVID‐19 Amp and World Health Organization (WHO)‐based assay. Discordance in nine samples was observed (negative by the WHO‐based assay) and discordant samples were retested as positive according to the results obtained from the Vector‐PCRrv‐2019‐nCoV‐RG assay. The developed COVID‐19 Amp assay has high sensitivity and specificity, includes virus particles‐based controls, provides the direct definition of the SARS‐CoV‐2 RdRp gene partial sequence, and is suitable for any hospital and laboratory equipped for RT‐qPCR.