Timely implementation of molecular diagnostics for SARS-CoV-2
In 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.
As 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.
We 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).
Each 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].
The 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.
Figure 1  Cumulative numbers of suspected COVID-19 samples tested during week 5 and 6 2020, Bavaria (n = 675)
COVID: coronavirus disease; PHEIC: public health emergency of international concern; LGL: Bavarian Health and Food Safety Authority; WHO: World Health Organization.
The 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 13.