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      Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

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          Abstract

          Background

          The ongoing outbreak of the recently emerged novel coronavirus (2019-nCoV) poses a challenge for public health laboratories as virus isolates are unavailable while there is growing evidence that the outbreak is more widespread than initially thought, and international spread through travellers does already occur.

          Aim

          We aimed to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available.

          Methods

          Here we present a validated diagnostic workflow for 2019-nCoV, its design relying on close genetic relatedness of 2019-nCoV with SARS coronavirus, making use of synthetic nucleic acid technology.

          Results

          The workflow reliably detects 2019-nCoV, and further discriminates 2019-nCoV from SARS-CoV. Through coordination between academic and public laboratories, we confirmed assay exclusivity based on 297 original clinical specimens containing a full spectrum of human respiratory viruses. Control material is made available through European Virus Archive – Global (EVAg), a European Union infrastructure project.

          Conclusion

          The present study demonstrates the enormous response capacity achieved through coordination of academic and public laboratories in national and European research networks.

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          Most cited references 13

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          Detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction

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            Assays for laboratory confirmation of novel human coronavirus (hCoV-EMC) infections.

            We present a rigorously validated and highly sensitive confirmatory real-time RT-PCR assay (1A assay) that can be used in combination with the previously reported upE assay. Two additional RT-PCR assays for sequencing are described, targeting the RdRp gene (RdRpSeq assay) and N gene (NSeq assay), where an insertion/deletion polymorphism might exist among different hCoV-EMC strains. Finally, a simplified and biologically safe protocol for detection of antibody response by immunofluorescence microscopy was developed using convalescent patient serum.
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              Detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction.

              We present two real-time reverse-transcription polymerase chain reaction assays for a novel human coronavirus (CoV), targeting regions upstream of the E gene (upE) or within open reading frame (ORF)1b, respectively. Sensitivity for upE is 3.4 copies per reaction (95% confidence interval (CI): 2.5–6.9 copies) or 291 copies/mL of sample. No cross-reactivity was observed with coronaviruses OC43, NL63, 229E, SARS-CoV, nor with 92 clinical specimens containing common human respiratory viruses. We recommend using upE for screening and ORF1b for confirmation.
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                Author and article information

                Journal
                Euro Surveill
                Euro Surveill
                eurosurveillance
                Eurosurveillance
                European Centre for Disease Prevention and Control (ECDC)
                1025-496X
                1560-7917
                23 January 2020
                : 25
                : 3
                Affiliations
                [1 ]Charité – Universitätsmedizin Berlin Institute of Virology, Berlin, Germany and German Centre for Infection Research (DZIF), Berlin, Germany
                [2 ]Tib-Molbiol, Berlin, Germany
                [3 ]Department of Viroscience, Erasmus MC, Rotterdam, the Netherlands
                [4 ]National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands
                [5 ]University of Hong Kong, Hong Kong, China
                [6 ]Universite d Aix-Marseille, Marseille, France
                [7 ]Public Health England, London, United Kingdom
                [8 ]Department of Medical Microbiology, Vaccine and Infectious Diseases Institute, University of Antwerp, Antwerp, Belgium
                Author notes

                Correspondence: Christian Drosten ( christian.drosten@ 123456charite.de )

                Article
                2000045 2000045
                10.2807/1560-7917.ES.2020.25.3.2000045
                6988269
                31992387
                This article is copyright of the authors or their affiliated institutions, 2020.

                This is an open-access article distributed under the terms of the Creative Commons Attribution (CC BY 4.0) Licence. You may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence, and indicate if changes were made.

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