荧光定量PCR检测HCVRNA两种核酸提取法.pdfVIP

荧光定量 PCR 检测 HCV RNA 两种核酸提取法 叶明奇 （厦门安普利生物工程有限公司，福建 厦门 343，技术工程部） 【摘要】目的探讨实时荧光PCR检测技术在丙型肝炎病原诊断中固相吸附法与磁珠分离技术 法运用在临床中提取HCV RNA核酸的提取率与去干扰物效果的比对。并通过对提取的RNA用于 RT—PCR荧光定量检测，从灵敏度及抗干扰能力方面进行评价：去除标本中常含有蛋白和脂 类等干扰核酸扩增的物质，特别是Rnast,因为RNase对 RNA 具有强烈降解作用的RNase，而 RNase较耐高温，不易失活。因此在提取 RNA 时，如何避免 RNase 对标本的污染及防止 RNase对提取的 RNA 的降解，所以对临床基因扩增检验中的标本进行核酸提取是核酸扩增前 不可或缺的一个步骤。采用简单而又高效的核酸提取方法对敏感的基因扩增检测技术的广泛 应用有重要意义。 结果：固相吸附法和磁珠分离技术法检测的敏感度和特异度可达99% 。 结论:采用固相吸附法和磁珠分离技术法提取血清HCV RNA具有较高的敏感度，在操作运用 上：固相吸附法更适用于标本量不多的手工核酸提取，磁珠分离技术法可运用于批量的标本 自动对核酸的提取，该两种方法在实时荧光定量PCR法检测中具有重要的临床价值。 【关键词】实时荧光定量 PCR；丙型肝炎；固相吸附法；磁珠分离技术；HCV RNA； Detection of HCV RNA by fluorescence quantitative PCR in two kinds of nucleic acid extraction method Ye Mingqi (Xiamen Amplly Biological Engineering Company Limited, Fujian Xiamen 343, the Engineering Department) [Abstract] technology of real-time fluorescence PCR detection of solid phase adsorption method and magnetic bead separation technique in the pathogenic diagnosis of hepatitis C in clinical use in extracting ratio on extraction rate of HCV RNA nucleic and to interference effect.And through the extraction of RNA for RT - PCR Fluorescence quantitative detection,evauation the sensitivity and the ability of anti-interference: the removal of samples of the contain protein and lipids interfering nucleic acid amplification substances, especially Rnast, because RNase has a strong role in the degradation of RNA RNase, while RNase is high temperature resistant, not easy to inactivation.Therefore in the extraction of RNA, how to avoid the pollution of specimen RNase and prevent the degradation of RNase on the extraction of RNA, so the nucleic acid extraction of clinical gene amplification test specimen is a nucleic acid amplification of an indispensable step.There is important significance in extensive application of sensitive PCR technique using nucleic acid extraction method is simple and efficient. Results: the solid phase adsorpt