荧光PCR检测乙型肝炎病毒cccDNA方法的建立

A real-time PCR assay for detection of hepatitis B virus covalently closed circular DNA in serum from hepatitis B virus-infected patients

目的:建立特异、灵敏的荧光PCR检测乙型肝炎病毒cccDNA的方法。方法:首先用煮沸法提取血清中的HBVDNA,然后用绿豆核酸酶(Mung Bean Nuclease)消化松弛环状HBVDNA(rcDNA),保留共价闭合环状DNA(cccDNA)。设计一条嵌合引物,该引物分为两部分序列,近5′部分为人为设定的序列,近3′部分是与正链缺口下游互补序列。先用该嵌合引物以正链为模板,自HBV cccDNA1590nt开始向上游延伸,然后以嵌合引物中人为设定的序列为下游引物、与负链互补的上游引物以及一条在两引物之间与正链互补的Taqman探针,扩增检测嵌合引物延伸的产物。由于带有缺口的HBV rcDNA无法被嵌合引物延伸,因此最终只能扩增检测到cccDNA。结果:用含有相应HBV DNA片端的PUC19质粒模拟HBV cccDNA,该检测方法的检测灵敏度为1×103拷贝/ml;以模拟松弛环状DNA(rcDNA)与模拟HBV cccDNA按不同比例混合,在rcDNA与cccDNA的比为1×109拷贝/ml:1×103拷贝/ml时,该方法仍可检测到模拟cccDNA;单独检测模拟rcDNA浓度为1×109拷贝/ml时,该方法检测未出现假阳性。结论:该检测乙型肝炎病毒cccDNA的方法操作简单、灵敏、特异,适用于检测血清中HBV cccDNA。

Objective：To establish a high specificity and sensitivity real - time PCR assay for the detection of hepatitis B virus covalently closed circular DNA in serum. Methods：To remove HBV rcDNA from sample, DNA template was digested by Mung Bean Nuclease. A combination primer was designated in which segment A near 3′ end is complementary to HBV plus strand just before the DR2 region gap, and segment B near 5′ end is fabricative sequence, without homogenetic relationship to HBV genome. Promoted by taq DNA polymerase, a single nucleotide strand is elongated from combination primer generated by HBV cccDNA. In contrast, other HBV DNA forms do not produce a single nucleotide strand due to the cessation of elongation reaction at the DR2 gap. The newly formed single nucleotide strand is subsequently amplified by a new polymerase chain reaction system （PCR）, in which a primer, identical to combination primer segments B, is used to ensure specific amplification, avoiding other HBV DNA format inference. In addition, a taqman probe was used in the PCR system to report the detection signal. Results：The sensitivity of the method was 1 × 10^3 copy/ml HBV cccDNA in serum, and 1 × 10^3 copy/ml HBV cccDNA can be detected from 1 × 10^9 copy/ ml simulation HBV rcDNA. The false postive results not happened when 1 × 10^9 copy/ml simulation HBV rcDNA was detected. Conclusion ：This method proved to be effective for rapid and sensitive detection of HBV cccDNA with high specificity and efficacy.