Young K K, Archer J J, Yokosuka O, Omata M, Resnick R M
Department of Infectious Diseases, Roche Molecular Systems, Inc., Alameda, California 94501, USA.
J Clin Microbiol. 1995 Mar;33(3):654-7. doi: 10.1128/jcm.33.3.654-657.1995.
Many of the current reverse transcription (RT)-PCR assays for the detection of hepatitis C virus (HCV) RNA are multistep processes which use multiple enzymes and buffers. The assays are also often suboptimal, requiring nested amplification to achieve the desired levels of sensitivity. As a result, these assays are cumbersome and prone to false-positive results. The susceptibility to contamination is further aggravated by the lack of carryover controls. We have previously reported the development of a combined RT-PCR assay for HCV RNA detection which is sensitive and simple to perform. We have since successfully integrated dUTP-uracil-N-glycosylase carryover prevention into the combined assay. Restriction of as much as 0.5 microliter of deoxyuridine-containing amplification products has been achieved. The performance of the improved combined assay was compared directly with conventional nested RT-PCR and antibody detection. The combined assay was found to have sensitivity similar to that of nested RT-PCR in detecting HCV RNA from HCV antibody-positive specimens. In an analysis of hepatitis B virus antibody-positive specimens, nested amplification had false-positive rates ranging from 8 to 31%, while no false-positive results were seen with the combined assay. In comparison with serological methods, the combined assay had specificity and sensitivity of 100 and 95%, respectively.
目前许多用于检测丙型肝炎病毒（HCV）RNA的逆转录（RT）-PCR检测方法都是多步骤过程，需要使用多种酶和缓冲液。这些检测方法往往也不尽人意，需要进行巢式扩增才能达到所需的灵敏度水平。因此，这些检测方法繁琐且容易出现假阳性结果。由于缺乏残留对照，污染的易感性进一步加剧。我们之前报道了一种用于HCV RNA检测的联合RT-PCR检测方法的开发，该方法灵敏且操作简单。此后，我们已成功将dUTP-尿嘧啶-N-糖基化酶残留预防技术整合到联合检测中。已实现对多达0.5微升含脱氧尿苷的扩增产物的限制。将改进后的联合检测方法的性能与传统巢式RT-PCR和抗体检测方法直接进行了比较。结果发现，在检测HCV抗体阳性标本中的HCV RNA时，联合检测方法的灵敏度与巢式RT-PCR相似。在对乙型肝炎病毒抗体阳性标本的分析中，巢式扩增的假阳性率在8%至31%之间，而联合检测方法未出现假阳性结果。与血清学方法相比，联合检测方法的特异性和灵敏度分别为100%和95%。
