Establishment and application of a real-time PCR method to detect hepatitis B virus cccDNA quantitatively

目的 建立一种HBV cccDNA定量检测方法,并定量检测慢性乙型肝炎患者肝组织内的cccDNA.方法 分析A-G亚型HBV DNA序列,根据其结构特点,于DNA缺口两侧高度保守区域设计引物和探针,并摸索该方法最佳反应条件,以期实现不同亚型HBV cccDNA的特异性扩增.对该方法进行特异性、敏感性及重复性检测.将扩增产物进行测序以检测方法的特异性.用102～1010拷贝/ml的标准质粒检测方法的敏感度.106拷贝/ml标准质粒30复管,以检测其重复性.取32例慢性乙型肝炎患者抗病毒治疗前后的肝穿组织,提取DNA,用该方法进行cccDNA定量,观察抗病毒治疗前后cccDNA的变化及与总HBV DNA的关系.结果 测序结果显示扩增产物为目的片段;该定量方法的敏感度为103～1010拷贝/ml;标准质粒30复管后其Ct值为29.69±0.31,变异系数为1.04%.肝内cccDNA占肝内总HBV DNA的47%～98%,平均81.5%.结论 本方法操作简便,特异性高,定量线形范围宽及重复性好,可用于科研中cccDNA的定量检测.To establish a new method to detect HBV cccDNA quantitatively and to apply it to detect cccDNA in liver needle biopsy specimens of chronic hepatitis B patients.The sequences of HBV DNA genotypes A through G were analyzed. According to the different sequence structure of cccDNA and rcDNA, primes and probe were designed in highly conservative region outside the nick of cccDNA in order to amplify cccDNA but not rcDNA. The best conditions of this method were found after testing experiments. Also we checked its specificity and sensitivity and reproducibility. The products of PCR were sequenced in order to ascertain if it was the right region expected. To amplify with standard plasmid ranged from 10(2) to 10(10) copies/ml to measure the sensitivity and amplify in parallel with standard plasmid of 10(6) copies/ml for 30 replicates so as to measure its reproducibility. DNA was extracted from 32 needle liver biopsy specimens of chronic hepatitis B patients. The cccDNA was quantitatively detected with this method. The data of cccDNA obtained before and after therapy and their relationship with total HBV DNA were analyzed. RESULTS Results of sequencing showed that the PCR product was from the right region. The sensitivity was 10(3)-10(10) copies/ml. The Ct value was 29.69+/-0.31 and the coefficient of variability was 1.04 percent calculated from the data of 30 PCR reactions with standard plasmid. The percentage of decrease in serum HBV DNA, total HBV DNA in liver and cccDNA in liver were 0.49+/-0.17, 0.22+/-0.18 and 0.16+/-0.28 respectively. There is 47 percent-98 percent cccDNA in total HBV DNA in liver and the mean is 81.5 percent.The method is good because of the simple and convenient operation, the high specificity, the wide linear detection range and the fine reproducibility. Therefore it can be used for both scientific research and clinical purpose. Lamividine can significantly inhibit serum HBV DNA by, but its inhibitory effect on cccDNA in liver was rather weak.北京市科委科研项目; 北京大学211工程循证医学学科课题PubMed中文核心期刊要目总览(PKU)中国科技核心期刊(ISTIC)中国科学引文数据库(CSCD)02182-1842