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Open AccessArticle

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

1
Department of Microbiology, Queen Mary Hospital, HKSAR, Hong Kong, China
2
Genomics and Bioinformatics Programme, The Chinese University of Hong Kong, HKSAR, Hong Kong, China
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State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, HKSAR, Hong Kong, China
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Department of Microbiology, The University of Hong Kong, HKSAR, Hong Kong, China
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Department of Clinical Microbiology and Infection, The University of Hong Kong-Shenzhen Hospital, Shenzhen 518053, China
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Carol Yu Centre for Infection, Li Ka Shing Faculty of Medicine, The University of Hong Kong, HKSAR, Hong Kong, China
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Department of Medicine, Queen Elizabeth Hospital, HKSAR, Hong Kong, China
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Department of Pathology, Queen Elizabeth Hospital, HKSAR, Hong Kong, China
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Department of Medicine, Queen Mary Hospital, HKSAR, Hong Kong, China
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Department of Medicine, The University of Hong Kong, HKSAR, Hong Kong, China
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Department of Medicine and Geriatrics, Princess Margaret Hospital, HKSAR, Hong Kong, China
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School of Biomedical Sciences, The Chinese University of Hong Kong, HKSAR, Hong Kong, China
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Int. J. Mol. Sci. 2020, 21(7), 2574; https://doi.org/10.3390/ijms21072574
Received: 27 March 2020 / Revised: 4 April 2020 / Accepted: 6 April 2020 / Published: 8 April 2020
(This article belongs to the Section Molecular Pathology, Diagnostics, and Therapeutics)
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. View Full-Text
Keywords: SARS-CoV-2; COVID-19; nsp2; real-time RT-PCR; genome subtraction; GolayMetaMiner; sensitivity; specificity; clinical evaluation; COVID-19-nsp2 assay SARS-CoV-2; COVID-19; nsp2; real-time RT-PCR; genome subtraction; GolayMetaMiner; sensitivity; specificity; clinical evaluation; COVID-19-nsp2 assay
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Yip, C. .-Y.; Ho, C.-C.; Chan, J. .-W.; To, K. .-W.; Chan, H. .-Y.; Wong, S. .-Y.; Leung, K.-H.; Fung, A. .-F.; Ng, A. .-K.; Zou, Z.; Tam, A.R.; Chung, T. .-H.; Chan, K.-H.; Hung, I. .-N.; Cheng, V. .-C.; Tsang, O. .-Y.; Tsui, S.K.W.; Yuen, K.-Y. 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. Int. J. Mol. Sci. 2020, 21, 2574.

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