A sensitive one-step TaqMan amplification approach for detection of rubella virus clade I and II genotypes in clinical samples
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- 作者：C. Claus ; S. Bergs ; N. C. Emmrich ; J. M. Hübschen ; A. Mankertz…
- 刊名：Archives of Virology
- 出版年：2017
- 出版时间：February 2017
- 年：2017
- 卷：162
- 期：2
- 页码：477-486
- 全文大小：1611KB
- 刊物类别：Biomedical and Life Sciences
- 刊物主题：Virology; Medical Microbiology; Infectious Diseases;
- 出版者：Springer Vienna
- ISSN：1432-8798
- 卷排序：162
文摘
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.