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. 2020 Apr 23;58(5):e00310-20.
doi: 10.1128/JCM.00310-20. Print 2020 Apr 23.

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 #  1   2   3   4   5 , Cyril Chik-Yan Yip #  6 , Kelvin Kai-Wang To  1   2   3   4 , Tommy Hing-Cheung Tang  7 , Sally Cheuk-Ying Wong  8 , Kit-Hang Leung  3 , Agnes Yim-Fong Fung  3 , Anthony Chin-Ki Ng  3 , Zijiao Zou  3 , Hoi-Wah Tsoi  3 , Garnet Kwan-Yue Choi  6 , Anthony Raymond Tam  9 , Vincent Chi-Chung Cheng  6 , Kwok-Hung Chan  1   3   4 , Owen Tak-Yin Tsang  10 , Kwok-Yung Yuen  11   3   4   5
Affiliations

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 et al. J Clin Microbiol. .

Abstract

On 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.

Keywords: COVID-19; PCR; SARS; Wuhan; coronavirus; diagnostic; emerging; virus.

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Figures

FIG 1
FIG 1
The number of clinical specimens that were positive for SARS-CoV-2 RNA by the COVID-19-RdRp/Hel assay or RdRp-P2 assay on different days after symptom onset from nasopharyngeal aspirates/swabs and/or throat swabs (A), saliva specimens (B), sputum specimens (C), plasma specimens (D), and feces or rectal swabs (E).
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