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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 Apr 08; 21(7)IJ

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.

Authors+Show Affiliations

Department of Microbiology, Queen Mary Hospital, HKSAR, Hong Kong, China.Genomics and Bioinformatics Programme, The Chinese University of Hong Kong, HKSAR, Hong Kong, China.State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, HKSAR, Hong Kong, China. Department of Microbiology, The University of Hong Kong, HKSAR, Hong Kong, China. Department of Clinical Microbiology and Infection, The University of Hong Kong-Shenzhen Hospital, Shenzhen 518053, China. Carol Yu Centre for Infection, Li Ka Shing Faculty of Medicine, The University of Hong Kong, HKSAR, Hong Kong, China.State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, HKSAR, Hong Kong, China. Department of Microbiology, The University of Hong Kong, HKSAR, Hong Kong, China. Department of Clinical Microbiology and Infection, The University of Hong Kong-Shenzhen Hospital, Shenzhen 518053, China. Carol Yu Centre for Infection, Li Ka Shing Faculty of Medicine, The University of Hong Kong, HKSAR, Hong Kong, China.Department of Medicine, Queen Elizabeth Hospital, HKSAR, Hong Kong, China.Department of Pathology, Queen Elizabeth Hospital, HKSAR, Hong Kong, China.Department of Microbiology, The University of Hong Kong, HKSAR, Hong Kong, China.Department of Microbiology, The University of Hong Kong, HKSAR, Hong Kong, China.Department of Microbiology, The University of Hong Kong, HKSAR, Hong Kong, China.Department of Microbiology, The University of Hong Kong, HKSAR, Hong Kong, China.Department of Medicine, Queen Mary Hospital, HKSAR, Hong Kong, China.Department of Microbiology, Queen Mary Hospital, HKSAR, Hong Kong, China.State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, HKSAR, Hong Kong, China. Department of Microbiology, The University of Hong Kong, HKSAR, Hong Kong, China. Carol Yu Centre for Infection, Li Ka Shing Faculty of Medicine, The University of Hong Kong, HKSAR, Hong Kong, China.Department of Medicine, The University of Hong Kong, HKSAR, Hong Kong, China.Department of Microbiology, Queen Mary Hospital, HKSAR, Hong Kong, China.Department of Medicine and Geriatrics, Princess Margaret Hospital, HKSAR, Hong Kong, China.School of Biomedical Sciences, The Chinese University of Hong Kong, HKSAR, Hong Kong, China.State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, HKSAR, Hong Kong, China. Department of Microbiology, The University of Hong Kong, HKSAR, Hong Kong, China. Department of Clinical Microbiology and Infection, The University of Hong Kong-Shenzhen Hospital, Shenzhen 518053, China. Carol Yu Centre for Infection, Li Ka Shing Faculty of Medicine, The University of Hong Kong, HKSAR, Hong Kong, China.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

32276333

Citation

Yip, Cyril Chik-Yan, et al. "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." International Journal of Molecular Sciences, vol. 21, no. 7, 2020.
Yip CC, Ho CC, Chan JF, et al. 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(7).
Yip, C. C., Ho, C. C., Chan, J. F., To, K. K., Chan, H. S., Wong, S. C., Leung, K. H., Fung, A. Y., Ng, A. C., Zou, Z., Tam, A. R., Chung, T. W., Chan, K. H., Hung, I. F., Cheng, V. C., Tsang, O. T., Tsui, S. K. W., & Yuen, K. Y. (2020). 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. International Journal of Molecular Sciences, 21(7). https://doi.org/10.3390/ijms21072574
Yip CC, et al. 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 Apr 8;21(7) PubMed PMID: 32276333.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Yip,Cyril Chik-Yan, AU - Ho,Chi-Chun, AU - Chan,Jasper Fuk-Woo, AU - To,Kelvin Kai-Wang, AU - Chan,Helen Shuk-Ying, AU - Wong,Sally Cheuk-Ying, AU - Leung,Kit-Hang, AU - Fung,Agnes Yim-Fong, AU - Ng,Anthony Chin-Ki, AU - Zou,Zijiao, AU - Tam,Anthony Raymond, AU - Chung,Tom Wai-Hin, AU - Chan,Kwok-Hung, AU - Hung,Ivan Fan-Ngai, AU - Cheng,Vincent Chi-Chung, AU - Tsang,Owen Tak-Yin, AU - Tsui,Stephen Kwok Wing, AU - Yuen,Kwok-Yung, Y1 - 2020/04/08/ PY - 2020/03/27/received PY - 2020/04/04/revised PY - 2020/04/06/accepted PY - 2020/4/12/entrez PY - 2020/4/12/pubmed PY - 2020/4/24/medline KW - COVID-19 KW - COVID-19-nsp2 assay KW - GolayMetaMiner KW - SARS-CoV-2 KW - clinical evaluation KW - genome subtraction KW - nsp2 KW - real-time RT-PCR KW - sensitivity KW - specificity JF - International journal of molecular sciences JO - Int J Mol Sci VL - 21 IS - 7 N2 - 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. SN - 1422-0067 UR - https://www.unboundmedicine.com/medline/citation/32276333/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_ L2 - https://www.mdpi.com/resolver?pii=ijms21072574 DB - PRIME DP - Unbound Medicine ER -