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.
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.
['Yip|Cyril Chik-Yan|CC|', 'Ho|Chi-Chun|CC|', 'Chan|Jasper Fuk-Woo|JF|0000-0001-6336-6657', 'To|Kelvin Kai-Wang|KK|', 'Chan|Helen Shuk-Ying|HS|', 'Wong|Sally Cheuk-Ying|SC|', 'Leung|Kit-Hang|KH|', 'Fung|Agnes Yim-Fong|AY|', 'Ng|Anthony Chin-Ki|AC|', 'Zou|Zijiao|Z|', 'Tam|Anthony Raymond|AR|', 'Chung|Tom Wai-Hin|TW|', 'Chan|Kwok-Hung|KH|', 'Hung|Ivan Fan-Ngai|IF|', 'Cheng|Vincent Chi-Chung|VC|', 'Tsang|Owen Tak-Yin|OT|', 'Tsui|Stephen Kwok Wing|SKW|', 'Yuen|Kwok-Yung|KY|']
D000073640:Betacoronavirus D000086382:COVID-19 D000086742:COVID-19 Testing D000086663:COVID-19 Vaccines D019411:Clinical Laboratory Techniques D018352:Coronavirus Infections D016679:Genome, Viral D006801:Humans D058873:Pandemics D011024:Pneumonia, Viral D012367:RNA, Viral D060888:Real-Time Polymerase Chain Reaction D015203:Reproducibility of Results D020133:Reverse Transcriptase Polymerase Chain Reaction D000086402:SARS-CoV-2 D012680:Sensitivity and Specificity
real-time RT-PCR COVID-19 sensitivity specificity SARS-CoV-2 GolayMetaMiner nsp2 COVID-19-nsp2 assay clinical evaluation genome subtraction