铕标记基因探针半定量检测丙型肝炎病毒RNA

目的 利用自行设计的引物、探针和新型铕螯合物BHHCT,建立一种半定量丙型肝炎病毒(HCV)RNA的检测方法。方法 收集44份丙型肝炎病人血清,20份正常人血清。利用中山大学达安基因有限公司RNA提取试剂提取HCV RNA,用自行设计的引物进行逆转录聚合酶链反应(RT-PCR)。扩增产物cDNA与微孔板上的捕捉探针杂交后,借助于其上游引物5′端带有的生物素,与标记有铕的链霉亲合素特异性结合,将铕连接到微孔板上,在特定波长激发光激发下,发出荧光,进行检测。结果 铕标记HCV RNA检测法的线性范围为102-106cDNA拷贝,敏感性、特异性均为100％。结论 铕标记RT-PCR检测HCV RNA法的线性范围宽,敏感性、特异性好,检测时间短,无放射性污染,有推广应用的价值。

Semi-quantitative measurement of HCV RNA by Europium-labeled gene probe

Objective To establish a semi-quantitative method for measurement of HCV RNA by use of primers and probe, which was sensitive and designed by ourselves, a new europium fluorescent chelate BHHCT. Methods 44 serum samples of HCV infected patients and 20 samples of the healthy people were collected. HCV RNA in serum sample was extracted by HCV fluorescence PCR diagnostic kit produced by Zhongshan University DAAN Gene Co Ltd, and amplified by RT-PCR in which one PCR primer was pre-labeled with biotin. The amplified products were hybridized with capture probes pre-fixed on the microplate. The biotin in the amplified products was conjugated with europium labeled streptavidin. So europium was linked to the target DNA. Then the fluorescent of europium was measured. Results The linear range of this assay was 10 - 10 copies/ml. Both sensitivity and specificity were 100%. Conclusion Europium labeled RT-PCR assay is a sensitive, specific, fast and non-radioactive contaminant method for the measurement of HCV RNA.