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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 references21

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          Identification of a Novel Coronavirus in Patients with Severe Acute Respiratory Syndrome

          The severe acute respiratory syndrome (SARS) has recently been identified as a new clinical entity. SARS is thought to be caused by an unknown infectious agent. Clinical specimens from patients with SARS were searched for unknown viruses with the use of cell cultures and molecular techniques. A novel coronavirus was identified in patients with SARS. The virus was isolated in cell culture, and a sequence 300 nucleotides in length was obtained by a polymerase-chain-reaction (PCR)-based random-amplification procedure. Genetic characterization indicated that the virus is only distantly related to known coronaviruses (identical in 50 to 60 percent of the nucleotide sequence). On the basis of the obtained sequence, conventional and real-time PCR assays for specific and sensitive detection of the novel virus were established. Virus was detected in a variety of clinical specimens from patients with SARS but not in controls. High concentrations of viral RNA of up to 100 million molecules per milliliter were found in sputum. Viral RNA was also detected at extremely low concentrations in plasma during the acute phase and in feces during the late convalescent phase. Infected patients showed seroconversion on the Vero cells in which the virus was isolated. The novel coronavirus might have a role in causing SARS. Copyright 2003 Massachusetts Medical Society
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            Origin and evolution of pathogenic coronaviruses

            Severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are two highly transmissible and pathogenic viruses that emerged in humans at the beginning of the 21st century. Both viruses likely originated in bats, and genetically diverse coronaviruses that are related to SARS-CoV and MERS-CoV were discovered in bats worldwide. In this Review, we summarize the current knowledge on the origin and evolution of these two pathogenic coronaviruses and discuss their receptor usage; we also highlight the diversity and potential of spillover of bat-borne coronaviruses, as evidenced by the recent spillover of swine acute diarrhoea syndrome coronavirus (SADS-CoV) to pigs.
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              Hosts and Sources of Endemic Human Coronaviruses

              The four endemic human coronaviruses HCoV-229E, -NL63, -OC43, and -HKU1 contribute a considerable share of upper and lower respiratory tract infections in adults and children. While their clinical representation resembles that of many other agents of the common cold, their evolutionary histories, and host associations could provide important insights into the natural history of past human pandemics. For two of these viruses, we have strong evidence suggesting an origin in major livestock species while primordial associations for all four viruses may have existed with bats and rodents. HCoV-NL63 and -229E may originate from bat reservoirs as assumed for many other coronaviruses, but HCoV-OC43 and -HKU1 seem more likely to have speciated from rodent-associated viruses. HCoV-OC43 is thought to have emerged from ancestors in domestic animals such as cattle or swine. The bovine coronavirus has been suggested to be a possible ancestor, from which HCoV-OC43 may have emerged in the context of a pandemic recorded historically at the end of the 19th century. New data suggest that HCoV-229E may actually be transferred from dromedary camels similar to Middle East respiratory syndrome (MERS) coronavirus. This scenario provides important ecological parallels to the present prepandemic pattern of host associations of the MERS coronavirus.
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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
                : 2000045
                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
                f6cd37e4-7130-44ff-b2f7-9cf10376e23a
                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.

                History
                : 21 January 2020
                : 22 January 2020
                Categories
                Research
                Custom metadata
                5

                novel coronavirus,rt-pcr,testing,laboratory,diagnostics,wuhan,2019-ncov,outbreak

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