检测人巨细胞病毒抗原的原位免疫PCR技术

Detection of human cytomegalovirus antigen with immuno in situ PCR assay

目的 :建立用于诊断HCMV活动性感染的原位免疫PCR方法。方法 :HCMVAD16 9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 :本方法具有敏感性高、特异性好及精确组织定位等特点 。

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.