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
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...
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| Veröffentlicht in: | Journal of clinical microbiology 2020-04, Vol.58 (5) |
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| creator | Chan, Jasper Fuk-Woo Yip, Cyril Chik-Yan To, Kelvin Kai-Wang Tang, Tommy Hing-Cheung Wong, Sally Cheuk-Ying Leung, Kit-Hang Fung, Agnes Yim-Fong Ng, Anthony Chin-Ki Zou, Zijiao Tsoi, Hoi-Wah Choi, Garnet Kwan-Yue Tam, Anthony Raymond Cheng, Vincent Chi-Chung Chan, Kwok-Hung Tsang, Owen Tak-Yin Yuen, Kwok-Yung |
| description | 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
(1.8 50% tissue culture infective doses [TCID
]/ml with genomic RNA and 11.2 RNA copies/reaction with
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%];
< 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
RNA copies/ml (range, 2.21 × 10
to 4.71 × 10
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_str_mv | 10.1128/JCM.00310-20 |
| format | Article |
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(1.8 50% tissue culture infective doses [TCID
]/ml with genomic RNA and 11.2 RNA copies/reaction with
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%];
< 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
RNA copies/ml (range, 2.21 × 10
to 4.71 × 10
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.</description><identifier>ISSN: 0095-1137</identifier><identifier>EISSN: 1098-660X</identifier><identifier>DOI: 10.1128/JCM.00310-20</identifier><identifier>PMID: 32132196</identifier><language>eng</language><publisher>United States: American Society for Microbiology</publisher><subject>Adult ; Aged ; Animals ; Betacoronavirus - isolation & purification ; China ; Chlorocebus aethiops ; Clinical Laboratory Techniques ; Coronavirus Infections - diagnosis ; Coronavirus Infections - virology ; COVID-19 ; COVID-19 Testing ; COVID-19 Vaccines ; Female ; Humans ; In Vitro Techniques ; Male ; Middle Aged ; Molecular Diagnostic Techniques - standards ; Pandemics ; Pneumonia, Viral - diagnosis ; Pneumonia, Viral - virology ; Reverse Transcriptase Polymerase Chain Reaction - standards ; SARS-CoV-2 ; Sensitivity and Specificity ; Vero Cells ; Viral Proteins - genetics ; Virology</subject><ispartof>Journal of clinical microbiology, 2020-04, Vol.58 (5)</ispartof><rights>Copyright © 2020 Chan et al.</rights><rights>Copyright © 2020 Chan et al. 2020 Chan et al.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c427t-51f9f3ef73ac3edba42d516b6bcda4634c20f4f861057e6d7ebb040e41ef98f53</citedby><cites>FETCH-LOGICAL-c427t-51f9f3ef73ac3edba42d516b6bcda4634c20f4f861057e6d7ebb040e41ef98f53</cites><orcidid>0000-0002-1921-5824</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7180250/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7180250/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,724,777,781,882,3175,27905,27906,53772,53774</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/32132196$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>McAdam, Alexander J.</contributor><creatorcontrib>Chan, Jasper Fuk-Woo</creatorcontrib><creatorcontrib>Yip, Cyril Chik-Yan</creatorcontrib><creatorcontrib>To, Kelvin Kai-Wang</creatorcontrib><creatorcontrib>Tang, Tommy Hing-Cheung</creatorcontrib><creatorcontrib>Wong, Sally Cheuk-Ying</creatorcontrib><creatorcontrib>Leung, Kit-Hang</creatorcontrib><creatorcontrib>Fung, Agnes Yim-Fong</creatorcontrib><creatorcontrib>Ng, Anthony Chin-Ki</creatorcontrib><creatorcontrib>Zou, Zijiao</creatorcontrib><creatorcontrib>Tsoi, Hoi-Wah</creatorcontrib><creatorcontrib>Choi, Garnet Kwan-Yue</creatorcontrib><creatorcontrib>Tam, Anthony Raymond</creatorcontrib><creatorcontrib>Cheng, Vincent Chi-Chung</creatorcontrib><creatorcontrib>Chan, Kwok-Hung</creatorcontrib><creatorcontrib>Tsang, Owen Tak-Yin</creatorcontrib><creatorcontrib>Yuen, Kwok-Yung</creatorcontrib><title>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</title><title>Journal of clinical microbiology</title><addtitle>J Clin Microbiol</addtitle><description>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
(1.8 50% tissue culture infective doses [TCID
]/ml with genomic RNA and 11.2 RNA copies/reaction with
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%];
< 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
RNA copies/ml (range, 2.21 × 10
to 4.71 × 10
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.</description><subject>Adult</subject><subject>Aged</subject><subject>Animals</subject><subject>Betacoronavirus - isolation & purification</subject><subject>China</subject><subject>Chlorocebus aethiops</subject><subject>Clinical Laboratory Techniques</subject><subject>Coronavirus Infections - diagnosis</subject><subject>Coronavirus Infections - virology</subject><subject>COVID-19</subject><subject>COVID-19 Testing</subject><subject>COVID-19 Vaccines</subject><subject>Female</subject><subject>Humans</subject><subject>In Vitro Techniques</subject><subject>Male</subject><subject>Middle Aged</subject><subject>Molecular Diagnostic Techniques - standards</subject><subject>Pandemics</subject><subject>Pneumonia, Viral - diagnosis</subject><subject>Pneumonia, Viral - virology</subject><subject>Reverse Transcriptase Polymerase Chain Reaction - standards</subject><subject>SARS-CoV-2</subject><subject>Sensitivity and Specificity</subject><subject>Vero Cells</subject><subject>Viral Proteins - genetics</subject><subject>Virology</subject><issn>0095-1137</issn><issn>1098-660X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2020</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpVkV1v0zAUhiME2srYHdfIl1zMmx3H-bhBmjKgRftAXam4sxznuDVy7MxOi_rr-Gtk66iYZMmWzuPnPdKbJO8pOac0LS--1TfnhDBKcEpeJRNKqhLnOfn5OpkQUnFMKSuOk7cx_iKEZhnnR8kxS-l4qnyS_Jl1ffBbaNGNt6A2VgZ0ZeTK-Wgi8hrVd8vZFaYVanZoWAO6HWF7hqZmtbY7dA8umsFsAUnXovselNFGHT7heTvvL6Zg0RykxQvTwfjaQoiAFkG6qILpB-Md_l7P0WWMcoeW0ppWDuNGM4eWZgj-yf3bDGtUW-OMknaf1I3h75I3WtoIp8_3SfLjy-dFPcXXd19n9eU1VllaDJhTXWkGumBSMWgbmaUtp3mTN6qVWc4ylRKd6TKnhBeQtwU0DckIZBR0VWrOTpJPe2-_aTpoFbghSCv6YDoZdsJLI15OnFmLld-KgpYk5WQUfHwWBP-wgTiIzkQF1koHfhNFykaSc1bSET3boyr4GAPoQwwl4rFzMXYunjoX6aP5w_-rHeB_JbO_uhqpLQ</recordid><startdate>20200423</startdate><enddate>20200423</enddate><creator>Chan, Jasper Fuk-Woo</creator><creator>Yip, Cyril Chik-Yan</creator><creator>To, Kelvin Kai-Wang</creator><creator>Tang, Tommy Hing-Cheung</creator><creator>Wong, Sally Cheuk-Ying</creator><creator>Leung, Kit-Hang</creator><creator>Fung, Agnes Yim-Fong</creator><creator>Ng, Anthony Chin-Ki</creator><creator>Zou, Zijiao</creator><creator>Tsoi, Hoi-Wah</creator><creator>Choi, Garnet Kwan-Yue</creator><creator>Tam, Anthony Raymond</creator><creator>Cheng, Vincent Chi-Chung</creator><creator>Chan, Kwok-Hung</creator><creator>Tsang, Owen Tak-Yin</creator><creator>Yuen, Kwok-Yung</creator><general>American Society for Microbiology</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0002-1921-5824</orcidid></search><sort><creationdate>20200423</creationdate><title>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</title><author>Chan, Jasper Fuk-Woo ; Yip, Cyril Chik-Yan ; To, Kelvin Kai-Wang ; Tang, Tommy Hing-Cheung ; Wong, Sally Cheuk-Ying ; Leung, Kit-Hang ; Fung, Agnes Yim-Fong ; Ng, Anthony Chin-Ki ; Zou, Zijiao ; Tsoi, Hoi-Wah ; Choi, Garnet Kwan-Yue ; Tam, Anthony Raymond ; Cheng, Vincent Chi-Chung ; Chan, Kwok-Hung ; Tsang, Owen Tak-Yin ; Yuen, Kwok-Yung</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c427t-51f9f3ef73ac3edba42d516b6bcda4634c20f4f861057e6d7ebb040e41ef98f53</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2020</creationdate><topic>Adult</topic><topic>Aged</topic><topic>Animals</topic><topic>Betacoronavirus - isolation & purification</topic><topic>China</topic><topic>Chlorocebus aethiops</topic><topic>Clinical Laboratory Techniques</topic><topic>Coronavirus Infections - diagnosis</topic><topic>Coronavirus Infections - virology</topic><topic>COVID-19</topic><topic>COVID-19 Testing</topic><topic>COVID-19 Vaccines</topic><topic>Female</topic><topic>Humans</topic><topic>In Vitro Techniques</topic><topic>Male</topic><topic>Middle Aged</topic><topic>Molecular Diagnostic Techniques - standards</topic><topic>Pandemics</topic><topic>Pneumonia, Viral - diagnosis</topic><topic>Pneumonia, Viral - virology</topic><topic>Reverse Transcriptase Polymerase Chain Reaction - standards</topic><topic>SARS-CoV-2</topic><topic>Sensitivity and Specificity</topic><topic>Vero Cells</topic><topic>Viral Proteins - genetics</topic><topic>Virology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Chan, Jasper Fuk-Woo</creatorcontrib><creatorcontrib>Yip, Cyril Chik-Yan</creatorcontrib><creatorcontrib>To, Kelvin Kai-Wang</creatorcontrib><creatorcontrib>Tang, Tommy Hing-Cheung</creatorcontrib><creatorcontrib>Wong, Sally Cheuk-Ying</creatorcontrib><creatorcontrib>Leung, Kit-Hang</creatorcontrib><creatorcontrib>Fung, Agnes Yim-Fong</creatorcontrib><creatorcontrib>Ng, Anthony Chin-Ki</creatorcontrib><creatorcontrib>Zou, Zijiao</creatorcontrib><creatorcontrib>Tsoi, Hoi-Wah</creatorcontrib><creatorcontrib>Choi, Garnet Kwan-Yue</creatorcontrib><creatorcontrib>Tam, Anthony Raymond</creatorcontrib><creatorcontrib>Cheng, Vincent Chi-Chung</creatorcontrib><creatorcontrib>Chan, Kwok-Hung</creatorcontrib><creatorcontrib>Tsang, Owen Tak-Yin</creatorcontrib><creatorcontrib>Yuen, Kwok-Yung</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Journal of clinical microbiology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Chan, Jasper Fuk-Woo</au><au>Yip, Cyril Chik-Yan</au><au>To, Kelvin Kai-Wang</au><au>Tang, Tommy Hing-Cheung</au><au>Wong, Sally Cheuk-Ying</au><au>Leung, Kit-Hang</au><au>Fung, Agnes Yim-Fong</au><au>Ng, Anthony Chin-Ki</au><au>Zou, Zijiao</au><au>Tsoi, Hoi-Wah</au><au>Choi, Garnet Kwan-Yue</au><au>Tam, Anthony Raymond</au><au>Cheng, Vincent Chi-Chung</au><au>Chan, Kwok-Hung</au><au>Tsang, Owen Tak-Yin</au><au>Yuen, Kwok-Yung</au><au>McAdam, Alexander J.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>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</atitle><jtitle>Journal of clinical microbiology</jtitle><addtitle>J Clin Microbiol</addtitle><date>2020-04-23</date><risdate>2020</risdate><volume>58</volume><issue>5</issue><issn>0095-1137</issn><eissn>1098-660X</eissn><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
(1.8 50% tissue culture infective doses [TCID
]/ml with genomic RNA and 11.2 RNA copies/reaction with
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%];
< 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
RNA copies/ml (range, 2.21 × 10
to 4.71 × 10
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.</abstract><cop>United States</cop><pub>American Society for Microbiology</pub><pmid>32132196</pmid><doi>10.1128/JCM.00310-20</doi><orcidid>https://orcid.org/0000-0002-1921-5824</orcidid><oa>free_for_read</oa></addata></record> |
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| source | American Society for Microbiology; MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central |
| subjects | Adult Aged Animals Betacoronavirus - isolation & purification China Chlorocebus aethiops Clinical Laboratory Techniques Coronavirus Infections - diagnosis Coronavirus Infections - virology COVID-19 COVID-19 Testing COVID-19 Vaccines Female Humans In Vitro Techniques Male Middle Aged Molecular Diagnostic Techniques - standards Pandemics Pneumonia, Viral - diagnosis Pneumonia, Viral - virology Reverse Transcriptase Polymerase Chain Reaction - standards SARS-CoV-2 Sensitivity and Specificity Vero Cells Viral Proteins - genetics Virology |
| title | 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 |
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