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Arch Virol. 2017 Feb;162(2):477-486. doi: 10.1007/s00705-016-3131-1. Epub 2016 Nov 3.

A sensitive one-step TaqMan amplification approach for detection of rubella virus clade I and II genotypes in clinical samples.

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Institute of Virology, University of Leipzig, Leipzig, Germany.
Institute of Virology, University of Leipzig, Leipzig, Germany.
WHO European Regional Reference Laboratory for Measles and Rubella, Department of Infection and Immunity, Luxembourg Institute of Health, Esch-sur-Alzette, Luxembourg.
WHO European Regional Reference Laboratory for Measles and Rubella, Robert Koch Institute, Berlin, Germany.


Although teratogenic rubella virus (RV) causes a vaccine-preventable disease, it is still endemic in several countries worldwide. Thus, there is a constant risk of RV importation into non-endemic areas. RV monitoring, especially during measles and Zika virus outbreaks, requires reliable diagnostic tools. For this study, a TaqMan-based one-step reverse transcription-quantitative PCR (RT-qPCR) assay, with the p90 gene as a novel and so far unexplored target for detection of clade I and II genotypes, was developed and evaluated. Automated nucleic acid extraction was carried out. Performance characteristics of the TaqMan RT-qPCR assay were determined for a RV plasmid standard and RNA extracted from virus-infected cell culture supernatants representing clade I and II genotypes. Diagnostic specificity and sensitivity were validated against other RNA and DNA viruses, relevant for RV diagnostic approaches and for RV-positive clinical samples, respectively. The assay is specific and highly sensitive with a limit of detection as low as five to one copies per reaction or 200 infectious virus particles per ml. The coefficients of variation (CV) were specified as intra- (within one run) and inter- (between different runs) assay variation, and calculated based on the standard deviations for the obtained Ct values of the respective samples. Intra- and inter-assay CV values were low, with a maximum of 3.4% and 2.4%, respectively. The assay was shown to be suitable and specific for the analysis of clinical samples. With p90 as a novel target, the highly sensitive and specific TaqMan assay outlined in this study is suitable for RV diagnosis worldwide.

[Indexed for MEDLINE]

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