慢性乙型肝炎患者HBV cccDNA定量方法的建立及应用

目的 建立慢性乙型肝炎患者HBV cccDNA荧光定量检测方法 ,并检测慢性乙型肝炎患者血清中HBV cccDNA含量.方法 根据HBV DNA结构特点,于HBV cicada缺口两侧高度保守区域设计巢式PCR引物和探针,并优化PCR反应条件;选取175例慢性乙型肝炎患者,提取抗病毒治疗前后血清DNA,以不降解质粒的ATP依赖的DNA酶(PSAD)进行酶切,进行rcDNA定量检测;分析影响血清HBV cccDNA检出率的相关因素,探讨血清HBV cccDNA载量与慢性乙型肝炎患者临床特征的关系.结果 慢性乙型肝炎患者血清中可检测到HBV cccDNA.血清HBV cccDNA检出率与血清HBV DNA载量相关.HBeAg阳性慢性乙肝患者血清HBV cccDNA载量高于HBeAg阴性慢性乙肝患者.结论 本方法 具有较好的敏感性,可用于HBV cccDNA的检测.

Quantitative detection of hepatitis B virus covalently closed circular DNA in sera of chronic hepatitis B patients with a newly established assay

Objective To quantitatively detect hepatitis B virus covalently closed circular DNA (HBV cccDNA) in sera of chronic hepatitis B patients with a newly established assay. Methods Primers and probe were designed in highly conservative region of HBV DNA. DNA was extracted from 175 sera samples of chronic hepatitis B patients, and was treated with plasmid-Safe-ATP-dependent Dnase (PSAD) to eliminate the relaxed circular DNA (rcDNA). The products were amplified by real-time PCR with primers spanning. Results The detection rate of serum HBV cccDNA was found to correlate directly with serum HBV DNA loading. HBeAg positive chronic hepatitis B patients had higher serum HBV cccDNA levels than HBeAg negative chronic hepatitis B patients. Conclusion The method is good because of the high specificity. It can be used for detection of HBV cccDNA.