PMC:7068163 / 10999-14296 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":"Discussion\nOn 7 January 2020, a novel coronavirus was identified and shortly after, the first sequence of the new strain was published [9,10]. The main task of public health authorities is to react quickly to emerging pathogens of global threat to prevent spread. These responses include containment strategies, which means that close contacts of patients have to be identified and tested immediately. Reusken et al. identified the availability of positive control material and primer/probes as well as the lack of skilled personnel and time as the most prominent challenges in the implementation of the new SARS-CoV-2 assay [11].\nNational and local public health laboratories play a crucial role in testing emerging pathogens. The Public Health Microbiology Laboratory in Bavaria was confronted with SARS-CoV-2-related events very early: once the assays and control materials arrived and the PCR assays were performed for the first time, a large contact investigation around the first German COVID-19 patient (data not shown) was immediately started, with so far more than 700 samples. Evaluation and verification of the new assays and testing real samples had to take place simultaneously within a very short time.\nWe realised soon that the SARS-CoV E gene assay was more sensitive than the two RdRp gene assays combined with the one-step RT-PCR system available in our laboratory. However, our E gene assay showed high background levels hampering a clear evaluation of the assays. Each mastermix has its own proprietary composition, which may explain the differences in the performance of a certain PCR assay. Using commercial kits with optimised target regions and primer sequences (in the E gene and SARS-CoV-2-specific S gene) ruled out the unspecific signals completely. Hence, reasons for the observed unspecific signals may be dimerisation of primers and probes and/or unspecific primer binding and polymerase activity in the targeted region of the E gene, probably also depending on thermal profile and cycler-specific differences, or most likely a combination of these factors.\nContamination as a reason for unspecific signals was ruled out, as stringent prevention measures were taken, e.g. strict separation of working areas: oligonucleotides and PCR mastermix reagents were handled in one room under a PCR hood with specified laboratory coats. Sample preparation and RNA extraction took place in a second room. Sample RNA was added in a third room under a PCR hood. The synthetic E gene control was added last to the mastermix. All reagents were aliquoted and aliquots used once only. Contaminations from synthetic E gene present in primer batches upon delivery can be ruled out as well, although only one batch of E gene primers and probes was used with the QuantiTect and Superscript III setup, as only a certain proportion of samples showed the unspecific signals. Furthermore, the unspecific signals were significantly reduced in the Superscript III setup, which showed that its sensitivity was comparable to the QuantiTect setup. In addition, the initially used E gene primers and probe were separately used as templates with the RealStar kit and no amplification was observed, whereas the corresponding artificial E gene template delivered a clear S-shaped curve with this 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