Improved molecular diagnosis of COVID-19 by the novel, highly sensitive and specific COVID-19-RdRp/Hel real-time reverse transcription-polymerase chain reaction assay validated in vitro and with clinical specimens.
Identifieur interne : 000321 ( Main/Exploration ); précédent : 000320; suivant : 000322Improved molecular diagnosis of COVID-19 by the novel, highly sensitive and specific COVID-19-RdRp/Hel real-time reverse transcription-polymerase chain reaction assay validated in vitro and with clinical specimens.
Auteurs : Jasper Fuk-Woo Chan [République populaire de Chine] ; Cyril Chik-Yan Yip [République populaire de Chine] ; Kelvin Kai-Wang To [République populaire de Chine] ; Tommy Hing-Cheung Tang [République populaire de Chine] ; Sally Cheuk-Ying Wong [République populaire de Chine] ; Kit-Hang Leung [République populaire de Chine] ; Agnes Yim-Fong Fung [République populaire de Chine] ; Anthony Chin-Ki Ng [République populaire de Chine] ; Zijiao Zou [République populaire de Chine] ; Hoi-Wah Tsoi [République populaire de Chine] ; Garnet Kwan-Yue Choi [République populaire de Chine] ; Anthony Raymond Tam [République populaire de Chine] ; Vincent Chi-Chung Cheng [République populaire de Chine] ; Kwok-Hung Chan [République populaire de Chine] ; Owen Tak-Yin Tsang [République populaire de Chine] ; Kwok-Yung Yuen [République populaire de Chine]Source :
- Journal of clinical microbiology [ 1098-660X ] ; 2020.
Abstract
On 31st 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 as 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 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 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 RdRd-P2-negative specimens [119/273 (43.6%) vs 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.
DOI: 10.1128/JCM.00310-20
PubMed: 32132196
Affiliations:
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sortKey="Yip, Cyril Chik Yan" sort="Yip, Cyril Chik Yan" uniqKey="Yip C" first="Cyril Chik-Yan" last="Yip">Cyril Chik-Yan Yip</name><affiliation wicri:level="1"><nlm:affiliation>Department of Microbiology, Queen Mary Hospital, Pokfulam, Hong Kong Special Administrative Region, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Department of Microbiology, Queen Mary Hospital, Pokfulam, Hong Kong Special Administrative Region</wicri:regionArea><wicri:noRegion>Hong Kong Special Administrative Region</wicri:noRegion></affiliation></author><author><name sortKey="To, Kelvin Kai Wang" sort="To, Kelvin Kai Wang" uniqKey="To K" first="Kelvin Kai-Wang" last="To">Kelvin Kai-Wang To</name><affiliation wicri:level="1"><nlm:affiliation>State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, China.</nlm:affiliation><country xml:lang="fr">République populaire de 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Chin-Ki Ng</name><affiliation wicri:level="1"><nlm:affiliation>Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region</wicri:regionArea><wicri:noRegion>Hong Kong Special Administrative Region</wicri:noRegion></affiliation></author><author><name sortKey="Zou, Zijiao" sort="Zou, Zijiao" uniqKey="Zou Z" first="Zijiao" last="Zou">Zijiao Zou</name><affiliation wicri:level="1"><nlm:affiliation>Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region</wicri:regionArea><wicri:noRegion>Hong Kong Special Administrative Region</wicri:noRegion></affiliation></author><author><name sortKey="Tsoi, Hoi Wah" sort="Tsoi, Hoi Wah" uniqKey="Tsoi H" first="Hoi-Wah" last="Tsoi">Hoi-Wah Tsoi</name><affiliation wicri:level="1"><nlm:affiliation>Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region</wicri:regionArea><wicri:noRegion>Hong Kong Special Administrative Region</wicri:noRegion></affiliation></author><author><name sortKey="Choi, Garnet Kwan Yue" sort="Choi, Garnet Kwan Yue" uniqKey="Choi G" first="Garnet 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Jasper Fuk Woo" sort="Chan, Jasper Fuk Woo" uniqKey="Chan J" first="Jasper Fuk-Woo" last="Chan">Jasper Fuk-Woo Chan</name><affiliation wicri:level="1"><nlm:affiliation>State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region</wicri:regionArea><wicri:noRegion>Hong Kong Special Administrative Region</wicri:noRegion></affiliation></author><author><name sortKey="Yip, Cyril Chik Yan" sort="Yip, Cyril Chik Yan" uniqKey="Yip C" first="Cyril Chik-Yan" last="Yip">Cyril Chik-Yan Yip</name><affiliation wicri:level="1"><nlm:affiliation>Department of Microbiology, Queen Mary Hospital, Pokfulam, Hong Kong Special Administrative Region, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Department of Microbiology, Queen Mary Hospital, Pokfulam, Hong Kong Special Administrative Region</wicri:regionArea><wicri:noRegion>Hong Kong Special Administrative Region</wicri:noRegion></affiliation></author><author><name sortKey="To, Kelvin Kai Wang" sort="To, Kelvin Kai Wang" uniqKey="To K" first="Kelvin Kai-Wang" last="To">Kelvin Kai-Wang To</name><affiliation wicri:level="1"><nlm:affiliation>State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region</wicri:regionArea><wicri:noRegion>Hong Kong Special Administrative Region</wicri:noRegion></affiliation></author><author><name sortKey="Tang, Tommy Hing Cheung" sort="Tang, Tommy Hing Cheung" uniqKey="Tang T" first="Tommy Hing-Cheung" last="Tang">Tommy Hing-Cheung Tang</name><affiliation wicri:level="1"><nlm:affiliation>Department of Medicine, Queen Elizabeth Hospital, Hong Kong Special Administrative Region, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Department of Medicine, Queen Elizabeth Hospital, Hong Kong Special Administrative Region</wicri:regionArea><wicri:noRegion>Hong Kong Special Administrative Region</wicri:noRegion></affiliation></author><author><name sortKey="Wong, Sally Cheuk Ying" sort="Wong, Sally Cheuk Ying" uniqKey="Wong S" first="Sally Cheuk-Ying" last="Wong">Sally Cheuk-Ying Wong</name><affiliation wicri:level="1"><nlm:affiliation>Department of Pathology, Queen Elizabeth Hospital, Hong Kong Special Administrative Region, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Department of Pathology, Queen Elizabeth Hospital, Hong Kong Special Administrative Region</wicri:regionArea><wicri:noRegion>Hong Kong Special Administrative Region</wicri:noRegion></affiliation></author><author><name sortKey="Leung, Kit Hang" sort="Leung, Kit Hang" uniqKey="Leung K" first="Kit-Hang" last="Leung">Kit-Hang Leung</name><affiliation wicri:level="1"><nlm:affiliation>Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region</wicri:regionArea><wicri:noRegion>Hong Kong Special Administrative Region</wicri:noRegion></affiliation></author><author><name sortKey="Fung, Agnes Yim Fong" sort="Fung, Agnes Yim Fong" uniqKey="Fung A" first="Agnes Yim-Fong" last="Fung">Agnes Yim-Fong Fung</name><affiliation wicri:level="1"><nlm:affiliation>Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region</wicri:regionArea><wicri:noRegion>Hong Kong Special Administrative Region</wicri:noRegion></affiliation></author><author><name sortKey="Ng, Anthony Chin Ki" sort="Ng, Anthony Chin Ki" uniqKey="Ng A" first="Anthony Chin-Ki" last="Ng">Anthony Chin-Ki Ng</name><affiliation wicri:level="1"><nlm:affiliation>Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region</wicri:regionArea><wicri:noRegion>Hong Kong Special Administrative Region</wicri:noRegion></affiliation></author><author><name sortKey="Zou, Zijiao" sort="Zou, Zijiao" uniqKey="Zou Z" first="Zijiao" last="Zou">Zijiao Zou</name><affiliation wicri:level="1"><nlm:affiliation>Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region</wicri:regionArea><wicri:noRegion>Hong Kong Special Administrative Region</wicri:noRegion></affiliation></author><author><name sortKey="Tsoi, Hoi Wah" sort="Tsoi, Hoi Wah" uniqKey="Tsoi H" first="Hoi-Wah" last="Tsoi">Hoi-Wah Tsoi</name><affiliation wicri:level="1"><nlm:affiliation>Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region</wicri:regionArea><wicri:noRegion>Hong Kong Special Administrative Region</wicri:noRegion></affiliation></author><author><name sortKey="Choi, Garnet Kwan Yue" sort="Choi, Garnet Kwan Yue" uniqKey="Choi G" first="Garnet Kwan-Yue" last="Choi">Garnet Kwan-Yue Choi</name><affiliation wicri:level="1"><nlm:affiliation>Department of Microbiology, Queen Mary Hospital, Pokfulam, Hong Kong Special Administrative Region, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Department of Microbiology, Queen Mary Hospital, Pokfulam, Hong Kong Special Administrative Region</wicri:regionArea><wicri:noRegion>Hong Kong Special Administrative Region</wicri:noRegion></affiliation></author><author><name sortKey="Tam, Anthony Raymond" sort="Tam, Anthony Raymond" uniqKey="Tam A" first="Anthony Raymond" last="Tam">Anthony Raymond Tam</name><affiliation wicri:level="1"><nlm:affiliation>Department of Medicine, Queen Mary Hospital, Hong Kong Special Administrative Region, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Department of Medicine, Queen Mary Hospital, Hong Kong Special Administrative Region</wicri:regionArea><wicri:noRegion>Hong Kong Special Administrative Region</wicri:noRegion></affiliation></author><author><name sortKey="Cheng, Vincent Chi Chung" sort="Cheng, Vincent Chi Chung" uniqKey="Cheng V" first="Vincent Chi-Chung" last="Cheng">Vincent Chi-Chung Cheng</name><affiliation wicri:level="1"><nlm:affiliation>Department of Microbiology, Queen Mary Hospital, Pokfulam, Hong Kong Special Administrative Region, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Department of Microbiology, Queen Mary Hospital, Pokfulam, Hong Kong Special Administrative Region</wicri:regionArea><wicri:noRegion>Hong Kong Special Administrative Region</wicri:noRegion></affiliation></author><author><name sortKey="Chan, Kwok Hung" sort="Chan, Kwok Hung" uniqKey="Chan K" first="Kwok-Hung" last="Chan">Kwok-Hung Chan</name><affiliation wicri:level="1"><nlm:affiliation>State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region</wicri:regionArea><wicri:noRegion>Hong Kong Special Administrative Region</wicri:noRegion></affiliation></author><author><name sortKey="Tsang, Owen Tak Yin" sort="Tsang, Owen Tak Yin" uniqKey="Tsang O" first="Owen Tak-Yin" last="Tsang">Owen Tak-Yin Tsang</name><affiliation wicri:level="1"><nlm:affiliation>Department of Medicine and Geriatrics, Princess Margaret Hospital, Hong Kong Special Administrative Region, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Department of Medicine and Geriatrics, Princess Margaret Hospital, Hong Kong Special Administrative Region</wicri:regionArea><wicri:noRegion>Hong Kong Special Administrative Region</wicri:noRegion></affiliation></author><author><name sortKey="Yuen, Kwok Yung" sort="Yuen, Kwok Yung" uniqKey="Yuen K" first="Kwok-Yung" last="Yuen">Kwok-Yung Yuen</name><affiliation wicri:level="1"><nlm:affiliation>Department of Clinical Microbiology and Infection, The University of Hong Kong-Shenzhen Hospital, Shenzhen, Guangdong Province, China. kyyuen@hku.hk.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Department of Clinical Microbiology and Infection, The University of Hong Kong-Shenzhen Hospital, Shenzhen, Guangdong Province</wicri:regionArea><wicri:noRegion>Guangdong Province</wicri:noRegion></affiliation></author></analytic><series><title level="j">Journal of clinical microbiology</title><idno type="eISSN">1098-660X</idno><imprint><date when="2020" type="published">2020</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">On 31<sup>st</sup> 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 as 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 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 <i>in vitro</i> (1.8 TCID<sub>50</sub>/ml with genomic RNA and 11.2 RNA copies/reaction with <i>in vitro</i> 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 RdRd-P2-negative specimens [119/273 (43.6%) vs 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 3.21×10<sup>4</sup> RNA copies/ml (range, 2.21×10<sup>2</sup> to 4.71×10<sup>5</sup> 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.</div></front></TEI><affiliations><list><country><li>République populaire de Chine</li></country></list><tree><country name="République populaire de Chine"><noRegion><name sortKey="Chan, Jasper Fuk Woo" sort="Chan, Jasper Fuk Woo" uniqKey="Chan J" first="Jasper Fuk-Woo" last="Chan">Jasper Fuk-Woo Chan</name></noRegion><name sortKey="Chan, Kwok Hung" sort="Chan, Kwok Hung" uniqKey="Chan K" first="Kwok-Hung" last="Chan">Kwok-Hung Chan</name><name sortKey="Cheng, Vincent Chi Chung" sort="Cheng, Vincent Chi Chung" uniqKey="Cheng V" first="Vincent Chi-Chung" last="Cheng">Vincent Chi-Chung Cheng</name><name sortKey="Choi, Garnet Kwan Yue" sort="Choi, Garnet Kwan Yue" uniqKey="Choi G" first="Garnet Kwan-Yue" last="Choi">Garnet Kwan-Yue Choi</name><name sortKey="Fung, Agnes Yim Fong" sort="Fung, Agnes Yim Fong" uniqKey="Fung A" first="Agnes Yim-Fong" last="Fung">Agnes Yim-Fong Fung</name><name sortKey="Leung, Kit Hang" sort="Leung, Kit Hang" uniqKey="Leung K" first="Kit-Hang" last="Leung">Kit-Hang Leung</name><name sortKey="Ng, Anthony Chin Ki" sort="Ng, Anthony Chin Ki" uniqKey="Ng A" first="Anthony Chin-Ki" last="Ng">Anthony Chin-Ki Ng</name><name sortKey="Tam, Anthony Raymond" sort="Tam, Anthony Raymond" uniqKey="Tam A" first="Anthony Raymond" last="Tam">Anthony Raymond Tam</name><name sortKey="Tang, Tommy Hing Cheung" sort="Tang, Tommy Hing Cheung" uniqKey="Tang T" first="Tommy Hing-Cheung" last="Tang">Tommy Hing-Cheung Tang</name><name sortKey="To, Kelvin Kai Wang" sort="To, Kelvin Kai Wang" uniqKey="To K" first="Kelvin Kai-Wang" last="To">Kelvin Kai-Wang To</name><name sortKey="Tsang, Owen Tak Yin" sort="Tsang, Owen Tak Yin" uniqKey="Tsang O" first="Owen Tak-Yin" last="Tsang">Owen Tak-Yin Tsang</name><name sortKey="Tsoi, Hoi Wah" sort="Tsoi, Hoi Wah" uniqKey="Tsoi H" first="Hoi-Wah" last="Tsoi">Hoi-Wah Tsoi</name><name sortKey="Wong, Sally Cheuk Ying" sort="Wong, Sally Cheuk Ying" uniqKey="Wong S" first="Sally Cheuk-Ying" last="Wong">Sally Cheuk-Ying Wong</name><name sortKey="Yip, Cyril Chik Yan" sort="Yip, Cyril Chik Yan" uniqKey="Yip C" first="Cyril Chik-Yan" last="Yip">Cyril Chik-Yan Yip</name><name sortKey="Yuen, Kwok Yung" sort="Yuen, Kwok Yung" uniqKey="Yuen K" first="Kwok-Yung" last="Yuen">Kwok-Yung Yuen</name><name sortKey="Zou, Zijiao" sort="Zou, Zijiao" uniqKey="Zou Z" first="Zijiao" last="Zou">Zijiao Zou</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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