PMC:7068163 / 1128-4777 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7068163","sourcedb":"PMC","sourceid":"7068163","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7068163","text":"Timely implementation of molecular diagnostics for SARS-CoV-2\nIn connection with the ongoing outbreak of a novel coronavirus in the province Hubei and surrounding areas in China, it was expected that Europe would also be confronted with the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as infections in travellers in several Asian countries outside of China were confirmed shortly after the announcement of the outbreak in Wuhan [1-4]. Therefore, it was necessary to rapidly implement adequately quick and sensitive diagnostic assays for outbreak management of SARS-CoV-2 in public health laboratories.\nAs soon as the World Health Organization (WHO) published the first protocols for real-time RT-PCR assays, the Bavarian Food and Health Authority started to implement them. We ordered control material and oligonucleotides (see details below) in week 4 and ran our first SARS-CoV-2 assays on 27 January (week 5). On the same day, the first German case of coronavirus disease 2019 (COVID-19) was diagnosed in Bavaria [1]. In the following days, health authorities implemented comprehensive measures to prevent further transmission of SARS-CoV-2, including testing of contact persons.\nWe initially used the protocol based on the E gene and RdRp gene developed by the German Consiliary Laboratory for Coronaviruses hosted at the Charité in Berlin [5]. Real-time RT-PCR was initially performed with the QuantiTect Virus +Rox Vial kit (QIAGEN, Hilden, Germany) on the Bio-Rad CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Feldkirchen, Germany). The kit was chosen for its frequent and successful use in our laboratory with other assays and its immediate availability. Primers and probes were used as described [5] and provided by TIB Molbiol (Berlin, Germany). Control material was ordered from the European Virus Archive (EVAg) and consisted of synthetic Wuhan coronavirus 2019 E gene control (reference number 026N-03866) and SARS-CoV Frankfurt 1 RNA (reference number 004N-02005) [6]. In addition, the control of LightMix Modular Wuhan CoV RdRP-gene (TibMolbiol, Berlin, Germany) was used for the SARS-CoV-2 specific assay. Respiratory samples (nasopharyngeal swabs or sputum) were obtained from patients and contact persons. Sputum samples were diluted in 2 mL phosphate buffered saline (PBS). RNA was extracted using the QIAamp Bio Robot kit (QIAGEN) on a Hamilton Microlab Star (Hamilton, Bonaduz, Switzerland).\nEach sample was tested by three PCRs: the screening assay for the E gene and two confirmatory assays targeting the RdRp gene, all performed as recommended in [5], with either both probes (RdRP_SARSr-P1 and RdRP_SARSr-P2), detecting SARS-CoV and SARS-CoV-2, or only with the SARS-CoV-2-specific probe RdRP_SARSr-P2 [5]. We used human RNAse P as control for RNA extraction [7].\nThe cumulative numbers of all tested samples are shown in Figure 1. Two weeks after starting the SARS-CoV-2 diagnostics, we had analysed 669 respiratory samples at the Bavarian Health and Food Safety Authority (LGL). Among them, seven showed positive results for SARS CoV-2 RNA. By 11 February, 14 COVID-19 cases had been detected in Bavaria.\nFigure 1 Cumulative numbers of suspected COVID-19 samples tested during week 5 and 6 2020, Bavaria (n = 675)\nCOVID: coronavirus disease; PHEIC: public health emergency of international concern; LGL: Bavarian Health and Food Safety Authority; WHO: World Health Organization.\nThe figure shows all samples tested at LGL, negative and positive, as well as six positive samples tested in other laboratories in Bavaria. All data are cumulative, with a final total of positive tests of 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