Development of a Novel, Genome Subtraction-Derived, SARS-CoV-2-Specific COVID-19-nsp2 Real-Time RT-PCR Assay and Its Evaluation Using Clinical Specimens
SARS-CoV-2, COVID-19, Nsp2, Real-time RT-PCR, genome subtraction, GolayMetaMiner
The pandemic novel coronavirus infection, Coronavirus Disease 2019 (COVID-19), has affected at least 190 countries or territories, with 465,915 confirmed cases and 21,031 deaths. In a containment-based strategy, rapid, sensitive and specific testing is important in epidemiological control and clinical management. Using 96 SARS-CoV-2 and 104 non-SARS-CoV-2 coronavirus genomes and our in-house program, GolayMetaMiner, four specific regions longer than 50 nucleotides in the SARS-CoV-2 genome were identified. Primers were designed to target the longest and previously untargeted nsp2 region and optimized as a probe-free real-time reverse transcription-polymerase chain reaction (RT-PCR) assay. The new COVID-19-nsp2 assay had a limit of detection (LOD) of 1.8 TCID50/mL and did not amplify other human-pathogenic coronaviruses and respiratory viruses. Assay reproducibility in terms of cycle threshold (Cp) values was satisfactory, with the total imprecision (% CV) values well below 5%. Evaluation of the new assay using 59 clinical specimens from 14 confirmed cases showed 100% concordance with our previously developed COVID-19-RdRp/Hel reference assay. A rapid, sensitive, SARS-CoV-2-specific real-time RT-PCR assay, COVID-19-nsp2, was developed.
Cyril Chik-Yan Yip, Chi-Chun Ho, Jasper Fuk-Woo Chan, Kelvin Kai-Wang To, Helen Shuk-Ying Chan, Sally Cheuk-Ying Wong, Kit-Hang Leung, Agnes Yim-Fong Fung, Anthony Chin-Ki Ng, Zijiao Zou, Anthony Raymond Tam, Tom Wai-Hin Chung, Kwok-Hung Chan, Ivan Fan-Ngai Hung, Vincent Chi-Chung Cheng, Owen Tak-yin Tsang, Stephen Kwok-Wing Tsui, Kwok-yung Yuen
International Journal of Molecular Sciences