检测人巨细胞病毒抗原的原位免疫PCR技术

目的：建立用于诊断ＨＣＭＶ活动性感染的原位免疫ＰＣＲ方法。方法：ＨＣＭＶ ＡＤ１６９株感染的人胚肺成纤维细胞或先天畸形胎儿肾组织石蜡切片，依次加入抗ＨＣＭＶ单抗，生物素化二抗，链霉亲合素以及作为指示元件的生物素化ＤＮＡ，随后进行原位ＰＣＲ扩增，经酶显色反应判断结果。结果：应用本法检测ＨＣＭＶ感染的细胞可于感染后８ｈ清晰检出阳性信号；检测先天畸形胎儿肾组织ＨＣＭＶ感染，阳性率显著高于免疫组化法。结论

Detection of human cytomegalovirus antigen with immuno-in situ PCR assay

Objective:This study was to establish a immuno in situ PCR method for the diagnosis of HCMV active infection. Methods:The cells infected with HCMV or renal tissues sections of paraffin embedded were used. A mouse monoclonal anti HCMV primary antibody and biotinylated secondary antibody are stepwise immobilized. After that, free streptavidin and a marker biotinylated pBV220 DNA are immunobilized consecutively. A segment of the immunobilized DNA are amplified by in situ PCR with the appropriate primers. Antigens induced by HCMV were visualised with a purple blue precipitate obtained. Results:The strong positive signals can be detected as early as 8 h postinfection by immuno in situ PCR, the positive signal was much more intensive with infection time. 6 of 23 case renal tissues of congenital malformed foetus were shown to be positive. Conclusion:This immuno in situ PCR assay was sensitive, specific and exact location and can be used in diagnostic laboratories to detect active infection caused by HCMV.