多重PCR法检测外周血单个核细胞中的HBVDNA与HBVcccDNA

目的建立多重聚合酶链反应法(Multiplex Polymerase chain reaction，M—PCR)，揭示慢性乙肝患者外周血单个核细胞(PBMC)中HBVDNA的存在状况。方法采用一种把一套扩增乙肝病毒基因组DNA(HBV genome DNA)的引物和一套特异性扩增乙肝病毒共价闭合环状DNA(HBVcccDNA)的引物掺入到一个体系中的PCR法，检测PBMC中的HBVDNA核酸分子。结果PCR法可使处于同一PBMC中的总HBVDNA和HBVcccDNA同时分别良好的扩增出来；30例慢性乙肝患者的PBMC中，总HBVDNA与HBVcccDNA同耐检出者23例，检出率76．6％；检出HBVcccDNA者占检出总HBVDNA者的82．1％。结论PBMC中的HBVDNA部分参与复制。成功的建立了能同时检测HBV基因组DNA与HBVcccDNA的M—PCR方法。

Detection of HBVDNA and HBVcccDNA in Peripheral Blood Mononuclear Cells by a Multiplex Polymerase Chain Reaction

Objective To establish a multiplex polymerase chain reaction (M-PCR) assay to detect HBV DNA in the peripheral blood mononuclear cells(PBMCs) of chronic HB patients. Methods One pair of primer amplifying HBV genome DNA and another pair of primer amplifying HBV covalently closed circular DNA (cccDNA ) were added to one PCR reaction to detect HBV DNA in PBMCs. Results Various forms of HBVDNA including total DNA and cccDNA could be amplified simultaneously. Among the 30 chronic HB patients, both the HBVDNA and HBVcccDNA in the PBMCs of 23 patients were detected, the positive rate of which was 76.6%. The positive rate of HBV cccDNA accounted for 82.1% of total HBV DNA positive rate. Conclusion HBVDNA in the PBMCs could partially replicate. The M-PCR was successfully set up to amplify HBV genome DNA and HBV cccDNA simultaneously.