不同荧光定量PCR对于HBVcccDNA水平的检测差异对比

目的：研究对比不同荧光定量聚合酶链反应PCR对于乙型肝炎病毒HBVcccDNA水平的检测差异。方法选择20例从2007—2008年在广州市第八人民医院接受住院治疗的乙肝患者作为研究对象。提取其血清及肝组织进行实验分析，另取2份不含HBV感染的患者血清和肝组织作阴性对照组。将其中经或未经脱氧核糖核酸酶DNase处理的样本分别使用HBVc-ccDNA跨单缺口PCR和跨双缺口PCR检测。分析TaqMan探针跨单缺口PCR测定HBVcccDNA的结果，对比跨单、双缺口PCR测定结果以及不同荧光定量PCR的测定结果。分析血清和肝组织中HBVDNA与HBVcccDNA病毒载量的关系。结果血清及肝组织经过DNase消化后的有关拷贝数显著低于未消化者。血清和肝组织跨双缺口PCR测定结果较跨单缺口PCR测定结果均显著降低。血清中的HBVDNA及cccDNA实验结果均显著低于在肝组织中的测定结果，差异均有统计学意义（均P＜0．05）。阴性对照组经四种荧光定量PCR检测后均呈阴性。20例E抗原呈阳性的乙肝病人，其机体病毒载量总体趋势为血清HBVDNA的载量较肝组织更低，差异显著。结论 DNase 可有效减少 HBVcccDNA 在检测过程中的假阳性情况，经DNase处理后的跨双缺口PCR检测特异性最高。同时，血清中基本不含cccDNA。

Detective differences of different fluorescence quantitative PCR for HBVcccDNA levels

Objective To study detective differences of different fluorescence quantitative PCR for HBVcccDNA levels.Methods 20 cases of hepatitis b were selected from 2007 to 2008 in the Eighth People's Hospital.We analyzed the extracted serum and liver tissues from these patients,and used the other 2 samples of serum and liver tissues from patients without HBV infection as the negative control group.Samples with or without DNase treatment were tested with HBVcccDNA across double gap or single gap PCR detection.We ana-lyzed the testing results of HBVcccDNA by across single gap PCR TaqMan probe measurement,and compared the results between across the single and double gap PCR and different fluorescent quantitative PCR.We also investigated the relationship between HBVDNA and HBVcccDNA viral load in serum and liver tissues.Results The copy number of the serum and liver tissues after DNase digestion was significantly lower than the undigested ones.Results obtained using across double gap PCR were significantly reduced than that using single gap PCR The serum HBVDNA and cccDNA were significantly lower than liver tissues,which were statistically significantly differ-ent (all P <0.05).Testing results were all negative in negative control group using four kinds of fluorescent quantitative’s PCRs.In the 20 cases with hepatitis b positive E antigen,the overall trend of serum HBVDNA viral load capacity was relatively lower than liver tissues,which showed significant difference.Conclusions DNase can effectively reduce HBVcccDNA false positives in the testing process,and cross double gap PCR shows highest detection specificity after DNase treatment.In addition,no cccDNA was observed in serum.