At the time SARS-CoV-2 was identified as the cause of coronavirus disease 2019 (COVID-19) no in vitro diagnostic (IVD) tests were available since it was a new virus. Very shortly after the release... Show moreAt the time SARS-CoV-2 was identified as the cause of coronavirus disease 2019 (COVID-19) no in vitro diagnostic (IVD) tests were available since it was a new virus. Very shortly after the release of the genomic sequence of SARS-CoV-2, laboratory-developed tests (LDTs) were developed, made available and implemented in several laboratories in the Netherlands and globally. In this study, the performance of an E-gene Sarbeco specific realtime reverse-transcriptase PCR (RT-PCR) was verified on the open modus of the geneLEAD VIII sample-to-answer platform. The results obtained from 134 clinical samples, of which 63 had been tested positive, showed almost complete concordance compared to the same PCR on the routine diagnostic systems and that was validated according to the national reference standard. The only discordant sample tested positive using the routine diagnostic workflow with a cycle threshold (CT) value of 37.7, while the sample tested negative using the geneLEAD VIII workflow. In addition, good performance was achieved in analyzing a blinded SARS-CoV-2 external quality assurance (EQA) panel. Implementation of the geneLEAD VIII platform as routine diagnostic tool resulted in testing 871 clinical samples with 115 positive results. In conclusion, the geneLEAD VIII SARSCoV-2 workflow presented in this study showed excellent diagnostic performance and with a rapid turnaround time of approximately two hours it proved a valuable option for STAT SARS-CoV-2 testing in the absence of (rapid, CE-IVD) point-of-care testing platforms. Show less
The RNA-dependent RNA polymerase (RdRp) of turnip yellow mosaic virus (TYMV) was isolated by a simple, new method. An active, template-dependent and specific enzyme was obtained. Although the... Show moreThe RNA-dependent RNA polymerase (RdRp) of turnip yellow mosaic virus (TYMV) was isolated by a simple, new method. An active, template-dependent and specific enzyme was obtained. Although the genomic RNA of TYMV could not be transcribed completely during an in vitro RdRp assay, a complete double-stranded product was obtained when a 3' terminal RNA fragment of 83 nucleotides was used as a template. The reaction product was identified as being of negative polarity by complete digestion With ribonuclease T1. Antibodies directed to part of the N-terminal (Ab140) or C-terminal (Ab66) in vitro autocleavage products of the large non-structural polyprotein of TYMV, could both partially inhibit RdRp activity. Further purification of the RdRp preparation by ion-exchange chromatography resulted in two activity peaks with different protein compositions. Both peak fractions retained high specificity for transcription of TYMV RNA. A protein of approximately 115 kDa was detected by both Ab140 and Ab66. (C) 1997 Elsevier Science B.V. Show less
