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
Cyril Chik‐Yan Yip, Chi-Chun Ho, Jasper Fuk‐Woo Chan, Kelvin Kai‐Wang To, Helen Shuk‐Ying Chan, Sally C. Y. 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
Abstract
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.