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首页> 外文期刊>Journal of Medical Virology >Application of new assays for rapid confirmation and genotyping of isolates of rubella virus.
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Application of new assays for rapid confirmation and genotyping of isolates of rubella virus.

机译：新方法在风疹病毒分离株快速确认和基因分型中的应用。

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Rubella virus (RV) isolation is recommended by the WHO Measles and Rubella Labnet for studying the etiology and epidemiology of rubella. However, the absence of cytopathologic effects (CPE) in many of the cell lines used commonly makes it difficult to confirm RV growth. In this study, two assays amplifying RV cDNA were developed and validated in order to confirm and genotype RV isolates after cell culture. A SYBR Green I-based real-time PCR (Rtime-SGE317) was established for initial rapid detection of RV in Vero cells and a nested PCR (PCR-E860) was used for amplifying further the 739 nt window of the E1 gene for the identification of RV genotype as recommended by the WHO. Sensitivities of the two assays were evaluated using eight RV isolates, two from infants with the congenital rubella syndrome (CRS) and six from patients with acute rubella. All the isolates had cycle threshold (C(t)) values <37 after the third passage, which is recommended as the cut-off for the confirmation of a viable RV isolate. Phylogenetic analysis based on the 739 nt window generated by the PCR-E860 showed that the eight RV isolates belonged to genotypes 1E, 1G, and 2B. The Rtime-SGE317 assay can be carried out in local public health laboratories, which would extend the molecular surveillance of rubella and contribute to the WHO goal of eradicating rubella worldwide.
机译：WHO麻疹和风疹Labnet建议使用风疹病毒（RV）分离来研究风疹的病因和流行病学。但是，通常使用的许多细胞系都缺乏细胞病理学作用（CPE），因此很难确认RV的生长。在这项研究中，开发并验证了两种扩增RV cDNA的检测方法，以便在细胞培养后确认RV分离株并对其进行基因分型。建立了基于SYBR Green I的实时PCR（Rtime-SGE317），用于对Vero细胞中的RV进行初始快速检测，并使用巢式PCR（PCR-E860）进一步扩增了E1基因的739 nt窗口。根据世界卫生组织的建议鉴定RV基因型。使用八种RV分离株评估了两种测定的敏感性，其中两种来自先天性风疹综合症（CRS）婴儿，另外六种来自急性风疹患者。在第三次传代后，所有分离株的循环阈值（C（t））值均<37，建议将其作为确认可行RV分离株的临界值。基于PCR-E860产生的739 nt窗口的系统发育分析表明，八个RV分离株分别属于基因型1E，1G和2B。 Rtime-SGE317测定法可以在当地公共卫生实验室中进行，这将扩展风疹的分子监测，并有助于世界卫生组织消灭风疹的目标。

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