检测人巨细胞病毒的免疫-PCR方法的建立

Development of a immunoPCR assay for the detection of HCMV

目的：依据免疫－ＰＣＲ基本原理，建立检测人巨细胞病毒（ＨＣＭＶ）活动性感染的免疫－ＰＣＲ方法。方法：我们建立的免疫－ＰＣＲ方法与酶联免疫ＥＬＩＳＡ不同之处是，用ＰＣＲ扩增生物素化线性ｐＢＶ２２０ＤＮＡ代替ＥＬＩＳＡ的酶催化底物，经琼脂糖凝胶电泳溴化乙锭染色观察分析扩增产物特异条带，判断结果。结果：用本法检测ＨＣＭＶ的敏感性高于ＥＬＩＳＡ法１０３～１０５倍，可检测到５个感染ＨＣＭＶ细胞；而检测ＨＳＶ－１、ＶＺＶ、ＢＫＶ均呈阴性反应，显示良好特异性。结论：建立的免疫－ＰＣＲ方法具有高度敏感性，可用于检测微量ＨＣＭＶ抗原。

Objective:The objective of this study was to establish a newly immunoPCR method for the detection of HCMV.Methods:The immunoPCR approach developed in the study is similar to conventional enzymelinked immunosorbent assay(ELISA). We immobilized various amounts of HCMV antigen on the surface of microtiter plate wells. A mouse monoclonal antiHCMV primary antibody and biotinylated secondary antibody are stepwise immobilized. After that, free streptavidin and a marker biotinylated linear pBV220 DNA are immunobilized consecutively. A segment of the immunobilized DNA are amplified by PCR with the appropriate primers. Analysis of the PCR products by agarose gel electrophoresis after staining with ethidium bromide. Viral antigen present in the sample is analyse by PCR amplification of the marker DNA. Results:Comparison with ELISA demonstrates that enhancement (approximately 1×103￣105) in detection sensitivity was obtained with the use of immunoPCR. 5 infected cells of HCMV could be detected by the immunoPCR. While HSV1、VZV、BKV antigen could not be detected with the immunoPCR.Conclusion:This immunoPCR assay has a extremely high sensitivity, could be applied to measure low level viral antigen in biological samples for diagnostic infection of HCMV.