Evaluation of a TaqMan PCR assay to detect rabies virus RNA: Influence of sequence variation and application to quantification of viral loads
Evaluation of a TaqMan PCR assay to detect rabies virus RNA: Influence of sequence variation and application to quantification of viral loads
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DOI：
10.1128/jcm.42.1.299-306.2004
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发表时间：
2004-01-01
影响因子：
9.4
通讯作者：
Rupprecht, CE
中科院分区：
文献类型：
--
作者：
Hughes, GJ;Smith, JS;Rupprecht, CE
已发表的使用TaqMan PCR的检测试剂盒具有一致的灵敏度、快速性和可转移性。在这里，我们描述了一种基于TaqMan PCR的方法，用于检测组织样本中的狂犬病病毒（RV）RNA。我们表明，该方法具有可接受的线性范围，是敏感性和特异性，重要的是，与感染性病毒的浓度相关。此外，RV特异性扩增的水平可根据内源性对照（β-肌动蛋白mRNA）的水平进行调节，从而可以计算相当的量。我们测试了该测定处理靶序列变异的能力。基因特异性寡核苷酸和靶序列之间的序列错配数量显著影响扩增（P <0.001），并且探针中心的点突变可通过阻止探针结合和随后的荧光而导致假阴性结果。这项研究表明，RV的遗传异质性可能证明是一个严重的障碍，在发展的诊断试验的基础上TaqMan PCR，然而，RV水平的定量可能被证明是一个有价值的应用，这种方法。
Published assays that use TaqMan PCR are consistently sensitive, rapid, and readily transferable. Here we describe a TaqMan PCR-based method for the detection of rabies virus (RV) RNA in tissue samples. We show that the method has an acceptable linear range, is both sensitive and specific, and, importantly, correlates with the concentration of infectious virus. In addition, the levels of RV-specific amplification are adjustable according to the levels of an endogenous control (beta-actin mRNA), allowing the calculation of comparable quantities. We tested the capacity of this assay to cope with target sequence variations. The number of sequence mismatches between gene-specific oligonucleotides and the target sequence significantly affects amplification (P < 0.001), and point mutations at the center of the probe can result in false-negative results through the prevention of probe binding and subsequent fluorescence. This study demonstrates that the genetic heterogeneity of RVs may prove a serious obstacle in the development of a diagnostic assay based on TaqMan PCR; however, the quantification of RV levels may prove to be a valuable application of this assay.