Infection and Immunity Division, Roslin Institute, University of Edinburgh, Edinburgh, United Kingdom.
J Clin Microbiol. 2012 Sep;50(9):2910-7. doi: 10.1128/JCM.01172-12. Epub 2012 Jun 27.
Nucleic acid amplification methods such as the PCR have had a major impact on the diagnosis of viral infections, often achieving greater sensitivities and shorter turnaround times than conventional assays and an ability to detect viruses refractory to conventional isolation methods. Their effectiveness is, however, significantly influenced by assay target sequence variability due to natural diversity and rapid sequence changes in viruses that prevent effective binding of primers and probes. This was investigated for a diverse range of enteroviruses (EVs; species A to D), human rhinoviruses (HRVs; species A to C), and human parechovirus (HPeV) in a multicenter assay evaluation using a series of full-length prequantified RNA transcripts. RNA concentrations were quantified by absorption (NanoDrop) and fluorescence methods (RiboGreen) prior to dilution in buffer supplemented with RNase inhibitors and carrier RNA. RNA transcripts were extremely stable, showing minimal degradation after prolonged storage at temperatures between ambient and -20°C and after multiple freeze-thaw cycles. Transcript dilutions distributed to six referral laboratories were screened by real-time reverse transcriptase PCR assays using different primers and probes. All of the laboratories reported high assay sensitivities for EV and HPeV transcripts approaching single copies and similar amplification kinetics for all four EV species. HRV detection sensitivities were more variable, often with substantially impaired detection of HRV species C. This could be accounted for in part by the placement of primers and probes to genetically variable target regions. Transcripts developed in this study provide reagents for the ongoing development of effective diagnostics that accommodate increasing knowledge of genetic heterogeneity of diagnostic targets.
核酸扩增方法，如 PCR，对病毒感染的诊断产生了重大影响，通常比传统检测方法具有更高的灵敏度和更短的周转时间，并且能够检测到对传统分离方法具有抗性的病毒。然而，由于天然多样性和病毒快速序列变化导致的检测靶序列变异性，其有效性受到显著影响，这会阻止引物和探针的有效结合。本研究使用一系列全长预定量 RNA 转录本，通过多中心检测评估，对多种肠道病毒（EV；A 至 D 种）、人鼻病毒（HRV；A 至 C 种）和人副肠孤病毒（HPeV）进行了研究。通过吸收（NanoDrop）和荧光方法（RiboGreen）对 RNA 浓度进行定量，然后在含有 RNase 抑制剂和载体 RNA 的缓冲液中进行稀释。RNA 转录本非常稳定，在环境温度至-20°C 之间长时间储存后，以及经过多次冻融循环后，降解最小。将转录本稀释液分发给六家参考实验室，通过使用不同引物和探针的实时逆转录 PCR 检测进行筛选。所有实验室报告的 EV 和 HPeV 转录本的检测灵敏度都非常高，接近单拷贝，并且所有四种 EV 种的扩增动力学相似。HRV 的检测灵敏度差异较大，通常对 HRV C 种的检测能力明显受损。这在一定程度上可以归因于引物和探针置于遗传变异的靶区。本研究中开发的转录本为正在开发的有效诊断试剂提供了试剂，以适应诊断靶标遗传异质性不断增加的知识。
