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. © 2020 Wiley Periodicals LLC