酶免疫法检测丙型肝炎病毒RNA的聚合酶链反应产物

EIA technique for detecting PCR products of HCV RNA

目的建立一灵敏、快速的检测丙型肝炎病毒（ＨＣＶ）ＲＮＡ逆转录－套式聚合酶链反应（ＰＣＲ）产物的酶免疫法。方法对第２次ＰＣＲ所用的引物进行双标记，使扩增产物同时被修饰有生物素和荧光素（ｆｌｕｏｒｅｓｃｅｉｎ）成分，再经过固相的链亲合素进行捕获、辣根过氧化物酶标记的抗－ｆｌｕｏｒｅｓ－ｃｅｉｎ反应后显色测定。结果所建立的方法与常规聚丙烯酰胺凝胶电泳法相比灵敏、快速、准确，结果判断客观，重复性也较好；所测结果可反映ＰＣＲ产物量的多少，但不能准确表示模板量的大小。结论建立的方法可取代电泳法而成为ＨＣＶＲＮＡ常规ＰＣＲ反应产物的检测技术。

Objective To establish a EIA method for detecting the RT nPCR products of HCV RNA.Methods The primers of the second PCR of HCV RNA were modified so that the products of amplification were labeled with biotin and fluorescein. The products were diluted and subsequently added into the streptavidin coated wells. The biotinylated amplification products were captured and others were removed. Then anti fluorescein HRP conjugates were added into the wells. After washing, OPD H 2O 2 were added and then measured on a microplate reader.Results Compared with the routine gel electrophoresis method, the established assay was sensitive, rapid, accurate and the precision was also acceptable. The ELISA data were correlated with the volume of the products, but no correlation with the amount of viral template was noted.Conclusion The established assay may be used as a routine method for detecting the products of amplification of HCV RNA instead of electrophoresis technique.