A sensitive one-step TaqMan amplification approach for detection of rubella virus clade I and II genotypes in clinical samples.
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.
Institute of Virology, University of Leipzig, Leipzig, Germany. claudia.claus@medizin.uni-leipzig.de.
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Claus
Institute of Virology, University of Leipzig, Leipzig, Germany.
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Bergs
Institute of Virology, University of Leipzig, Leipzig, Germany.
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Emmrich
WHO European Regional Reference Laboratory for Measles and Rubella, Department of Infection and Immunity, Luxembourg Institute of Health, Esch-sur-Alzette, Luxembourg.
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Hübschen
WHO European Regional Reference Laboratory for Measles and Rubella, Robert Koch Institute, Berlin, Germany.
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Mankertz
Institute of Virology, University of Leipzig, Leipzig, Germany.
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Liebert
D001483:Base Sequence; D015870:Gene Expression; D005838:Genotype; D006801:Humans; D015588:Observer Variation; D010802:Phylogeny; D012367:RNA, Viral / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012409:Rubella / Q000175:diagnosis* / Q000821:virology; D012412:Rubella virus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D014764:Viral Proteins / Q000235:genetics*
D012367:RNA, Viral; D014764:Viral Proteins