Heermann K H, Seitz H, Thomssen R
Department of Medical Microbiology, University of Göttingen, Germany.
J Virol Methods. 1996 May;59(1-2):33-43. doi: 10.1016/0166-0934(96)02003-4.
The principle and practice of the polymerase chain reaction (PCR) has had a major impact on medical research. This is a powerful method but it does have its limitations, especially for clinical diagnostic work. We describe some improvements of hepatitis C virus (HCV) amplification such as simplification of specimen preparation, elimination of false negative reactions influenced by point mutations, and fluorimetric detection. The aim of the method is to make the procedure as easy and as inexpensive as possible for routine laboratories and for blood screening. After rapid chemical denaturation of the clinical specimen with guanidine thiocyanate and simultaneous hybridization of biotinylated primers to template HCV RNA, the product was fixed to streptavidin-coated magnetic beads and potential inhibitors were removed in easy washing steps. To eliminate the influence of point mutations within the primer binding sites, primer sets with different lengths at their 3'-end were developed for capture, reverse transcription, and amplification of genomic fragments by PCR. Positive results were identified by fluorescence staining. The low cost of the method allows the quantitation of templates by testing of dilution series as is common in microbiological laboratories.
聚合酶链反应（PCR）的原理与实践对医学研究产生了重大影响。这是一种强大的方法，但确实存在局限性，尤其是在临床诊断工作中。我们描述了丙型肝炎病毒（HCV）扩增的一些改进方法，如简化样本制备、消除点突变影响的假阴性反应以及荧光检测。该方法的目的是使常规实验室和血液筛查的操作尽可能简便且成本低廉。用硫氰酸胍对临床样本进行快速化学变性，同时将生物素化引物与模板HCV RNA杂交后，产物固定在链霉亲和素包被的磁珠上，并通过简单的洗涤步骤去除潜在抑制剂。为消除引物结合位点内点突变的影响，开发了3'-末端长度不同的引物组，用于通过PCR捕获、逆转录和扩增基因组片段。通过荧光染色鉴定阳性结果。该方法成本低廉，可像微生物实验室常见的那样通过检测稀释系列来定量模板。
