摘要
目的以丙型肝炎病毒（ＨＣＶ）ＲＮＡ（ＲＮＡ）核酸扩增产物的量化酶免疫（ＥＩＡ）通用型检测方法及试剂，以准确评价疗效，指导治疗。方法选择合适的扩增循环数使聚合酶链反应（ＰＣＲ）在“平台期效应”出现前即停止；同时将第２次ＰＣＲ所用的引物进行生物素标记，使扩增产物带有生物素标记成分，再通过固相包被的链霉亲合素进行捕获、氢氧化钠解链变性、荧光素标记的ＨＣＶ特异性探针杂交以及辣根过氧化物酶（ＨＲＰ）标记的抗荧光素酶标抗体反应后，ＴＭＢ显色测定，通过显色反应颜色的深浅，反映样品中ＨＣＶＲＮＡ模板的情况。结果ＨＣＶＲＮＡ扩增产物的ＥＩＡ检测结果表明：所建立的方法操作简便、重复性好、特异性强、敏感度高、结果判断客观准确，而且可以反映样品中病原体核酸模板量的多少；干扰素治疗期间，ＨＣＶ感染的慢性丙型肝炎患者不同时间系列血的动态检测结果提示，在临床监测治疗、评价疗效上，本方法具有较大的指导意义。结论所建立的方法可以成为一种通用型检测技术，用于量化分析病毒、细菌等致病因子及人类某些基因（如ＨＬＡＢ２７）的核酸扩增产物的研究，具有广阔的应用前景。
Objective To develop a universal quantitative immunoenzyme assay (EIA) for detecting amplified products of nucleic acid according to the study of hepatitis C virus (HCV), so as to guide the clinical treatment.Methods The appropriate cycle number of amplification was selected to stop polymerase chain reaction (PCR) before the “plateau”. At the same time, primers of the second PCR were modified with biotin so that the amplified products were labeled. The products were diluted and subsequently added into the streptavidin coated wells, and the biotinylated products were captured, followed by denaturation of NaOH, and non biotinlylated strands were removed. Hybridization was performed by adding the specific probe labeled with fluorescein. Finally anti fluorescein horse radish peroxidase (HRP) conjugates were added. After washing, 3,3′,5,5′, tetramethylbenzidine was added into the wells and then measured on a microplate reader. Results EIA detection of amplified products of HCV showed that this assay was rapid, sensitive, specific, and accurate. The correlation between the initial number of viral template and the EIA results of amplified products was good. We also prospectively investigated the response to interferon in five patients with HCV infection. The results showed that the assay could be used as a guidance to the clinical therapy in directing the use of antiviral drugs. Conclusion The established assay could be widely used as a universal technique for the quantitative detection of amplified products of nucleic acid ( such as virus, bacterium) and other human genes (such as HLA B 27 ).
基金
美国中华医学会基金
关键词
丙型肝炎病毒
RNA
量化酶免疫法
基因扩增
Immunoenzyme techniques Gene amplification Polymerase chain reaction Hepatitis C viruses
