摘要
目的解决ＲＴ－ＰＣＲ检测ＨＣＶ ＲＮＡ方法的重复性较差问题，使其成为常规实验室检测方法。方法采用亲合素生物素系统将ＨＣＶ特异的核酸片段包被在微孔板上，利用特异核酸捕获样本中的ＨＣＶ ＲＮＡ然后进行逆转录和ＰＣＲ扩增，从而建立了特异核酸捕获ＲＴ－ＰＣＲ检测ＨＣＶ ＲＮＡ方法，并与酶消化法和抽提法进行比较，评价其敏感性、特异性、重复性及抗污染能力。结果特异核酸捕获ＲＴ－ＰＣＲ获得的电泳条带较为清晰，其特异性、敏感性与酶消化法、抽提法无显著差异，但重复性高于抽提法，抗污染能力显著高于酶消化法和抽提法。结论特异核酸捕获ＲＴ－ＰＣＲ检测ＨＣＶ ＲＮＡ方法能有效去除非特异核酸和提高电泳条带清晰度，并具有极强的抗产物污染能力。因此，特异核酸捕获ＲＴ－ＰＣＲ具有广泛推广应用的明景。
Objective To solve the poor reproducibility of RT--PCR method in detection of HCV RNA in order to make it a routine laboratory assay. Methods Specific nuclear acid captured RT--PCR was established through capturing HCV RNA to microplate using specific nuclear acid employed avidin--biotin system, and amplifying captured HCV RNA thereafter. The sensitivity, specificity, reproducibility and the ability against contamination were evaluated, and compared with enzyme digestion method and extraction method. Results Specific nuclear acid captured RT--PCR could gain sharper electrophoresis bands. There was no significant difference in sesitivity and specificity as compared with enzyme digestion method and extraction method. But the reproducibility was better than that of extraction methods, and the ability against contamination was better than that of both enzyme digestion method and extraction method. Conclusion Specific nuclear acid captured PCR could promote the sharpness of clectrophoresis bands and efficiently discharge most of non--specific nuclear acids. And it possessed wonderful ability againot contamination. Thus, specific nuclear acid captured RT--PCR will be a very useful assay in the future.
出处
《中国实验诊断学》
2000年第1期7-9,共3页
Chinese Journal of Laboratory Diagnosis
基金
广东省卫生厅青年基金!97031
广东省自然科学基金!970079
关键词
丙型肝炎病毒
聚合酶链反应
HCV-RNA
Hepatitis C virus
Reverse transcription--polymerase chain reaction
Methodology
