• J. Clin. Microbiol. · Apr 2020

    Improved Molecular Diagnosis of COVID-19 by the Novel, Highly Sensitive and Specific COVID-19-RdRp/Hel Real-Time Reverse Transcription-PCR Assay Validated In Vitro and with Clinical Specimens.

    • Jasper Fuk-Woo Chan, Cyril Chik-Yan Yip, Kelvin Kai-Wang To, Tommy Hing-Cheung Tang, Sally Cheuk-Ying Wong, Kit-Hang Leung, Agnes Yim-Fong Fung, Anthony Chin-Ki Ng, Zijiao Zou, Hoi-Wah Tsoi, Garnet Kwan-Yue Choi, Anthony Raymond Tam, Vincent Chi-Chung Cheng, Kwok-Hung Chan, Owen Tak-Yin Tsang, and Kwok-Yung Yuen.
    • State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, China.
    • J. Clin. Microbiol. 2020 Apr 23; 58 (5).

    AbstractOn 31 December 2019, the World Health Organization was informed of a cluster of cases of pneumonia of unknown etiology in Wuhan, China. Subsequent investigations identified a novel coronavirus, now named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), from the affected patients. Highly sensitive and specific laboratory diagnostics are important for controlling the rapidly evolving SARS-CoV-2-associated coronavirus disease 2019 (COVID-19) epidemic. In this study, we developed and compared the performance of three novel real-time reverse transcription-PCR (RT-PCR) assays targeting the RNA-dependent RNA polymerase (RdRp)/helicase (Hel), spike (S), and nucleocapsid (N) genes of SARS-CoV-2 with that of the reported RdRp-P2 assay, which is used in >30 European laboratories. Among the three novel assays, the COVID-19-RdRp/Hel assay had the lowest limit of detection in vitro (1.8 50% tissue culture infective doses [TCID50]/ml with genomic RNA and 11.2 RNA copies/reaction with in vitro RNA transcripts). Among 273 specimens from 15 patients with laboratory-confirmed COVID-19 in Hong Kong, 77 (28.2%) were positive by both the COVID-19-RdRp/Hel and RdRp-P2 assays. The COVID-19-RdRp/Hel assay was positive for an additional 42 RdRp-P2-negative specimens (119/273 [43.6%] versus 77/273 [28.2%]; P < 0.001), including 29/120 (24.2%) respiratory tract specimens and 13/153 (8.5%) non-respiratory tract specimens. The mean viral load of these specimens was 3.21 × 104 RNA copies/ml (range, 2.21 × 102 to 4.71 × 105 RNA copies/ml). The COVID-19-RdRp/Hel assay did not cross-react with other human-pathogenic coronaviruses and respiratory pathogens in cell culture and clinical specimens, whereas the RdRp-P2 assay cross-reacted with SARS-CoV in cell culture. The highly sensitive and specific COVID-19-RdRp/Hel assay may help to improve the laboratory diagnosis of COVID-19.Copyright © 2020 Chan et al.

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