血清中HBV cccDNA定量检测方法的建立及临床应用

Quantitative detection and application of HBV cccDNA

目的建立一种以TaqMan为探针检测乙型肝炎病毒共价闭合环状DNA(cccDNA)的实时定量PCR检测方法。方法根据rcDNA与cccDNA结构差异,在缺口两侧设计引物,应用TaqMan探针分析,对实时PCR产物进行克隆、测序以证实产物为目的片段;同时对89例符合病毒性乙型肝炎诊断标准的血清样本进行HBVcccDNA和HBV DNA含量检测,3例未感染乙肝病毒的血清作为阴性对照。结果测序结果显示扩增产物为目的片段,检出下限为500 copies/mL;89例样本检测HBV cccDNA的阳性率为55.0%,HBeAg阳性患者阳性率为67.8%,HBeAb阳性患者阳性率为30.0%,HBeAg阳性的样本的平均HBV cccDNA水平高于HBeAb阳性的样本(P<0.05),阴性对照组未检测出HBV cccDNA。应用ROC曲线图评价HBV cccDNA在抗病毒治疗中的价值,其价值为0.927。结论以TaqMan为探针检测血清HBV cccDNA的实时PCR检测方法,具有快速、敏感、特异性高等特点,应用该方法检测HBV cccDNA含量可作为判断临床抗病毒治疗效果的重要参考指标,指导临床用药。

Objective To set up a real-time PCR with Taq enzyme probe for the detection of hepatitis B virus covalently closed circular DNA（cccDNA）.Methods Two primers were designed according to structure difference between rcDNA and cccDNA for PCR and TaqMan probe was applied for analysis.Subsequently,PCR products were sequencing for confirmation.Eighty nine serum samples in line with the hepatitis B diagnostic criteria and three samples of healthy people were collected.The samples were quantified for HBV cccDNA and DNA.Results Amplified products were confirmed to be the target segment by sequencing.The lower detection limit was 500copies/ml.The HBV cccDNA positive rates were 55.0%,67.8% and 30.0% in all CHB patients,HBeAg positive patients and HBeAb positive patients,respeitively.The cccDNA positive rate in HBeAg positive patients was significantly higher than that in HBeAb positive patients（P〈0.05）.No HBV cccDNA was detected in healthy people.According to ROC curve evaluation,the value of th e HBV cccDNA for antiviral therapy was 0.927.Conclusion A successful real-time PCR detection method with the TaqMan probe to detect the serum of HBV cccDNA is set up with fast,high specificity and high reference value.Quantifying of HBV cccNDA by this method can provide important reference index of predicating the effect of clinical anti-viral treatment and guide clinical medication.