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目的:通过实验验证并建立一种用荧光定量PCR技术准确检测血清中HBV cccDNA的方法,同时能够消除HBV rcDNA非特异性扩增的干扰。方法:用两步法提取20例乙肝患者血清中的total HBVDNA,分别用荧光PCR试剂盒定量检测HBV DNA和cccDNA。然后通过质粒保护型的DNA酶对提取的DNA模板进行消化处理,使rcDNA得到一定程度的降解后,再分别进行HBV DNA和cccDNA的定量检测。结果:未经过酶消化处理直接定量cccDNA时存在一定的非特异性扩增。这种非特异性扩增在HBV DNA含量>106copies/ml时比较明显,消化前后cccDNA定量值的对数平均相差1.94。经过酶消化后的定量cccDNA值基本消除了rcDNA非特异性扩增的干扰。结论:通过质粒保护型的DNA酶降解rcDNA后再进行cccDNA检测是一个方便可行的解决cccDNA准确定量问题的方法,非特异性干扰几乎可以完全消除。血清cccDNA与HBeAg及HBV-DNA有关,提示它可能也是HBV复制的血清标志。乙肝患者血清中cccDNA的含量与total HBV DNA含量成正相关。
OBJECTIVE: To establish a method for the accurate detection of HBV cccDNA in serum by fluorescence quantitative PCR and to eliminate the interference of non-specific amplification of HBV rcDNA. Methods: Total HBVDNA was extracted from 20 patients with hepatitis B by two-step method. HBV DNA and cccDNA were detected quantitatively by fluorescence PCR kit. Then, the extracted DNA template was digested with plasmid-protected DNase to degrade rcDNA to a certain degree, and then quantitative detection of HBV DNA and cccDNA respectively. Results: There was some nonspecific amplification when cccDNA was directly quantified without enzymatic digestion. This non-specific amplification of HBV DNA content> 106copies / ml obvious, before and after digestion cccDNA quantitative logarithm average difference of 1.94. Quantitative cccDNA values after enzymatic digestion basically eliminated the interference of rcDNA nonspecific amplification. Conclusion: It is a convenient and feasible method to solve the problem of accurate quantification of cccDNA by the degradation of rcDNA by plasmid-protected DNase followed by cccDNA detection. Non-specific interference can be almost eliminated completely. Serum cccDNA is associated with HBeAg and HBV-DNA, suggesting that it may also be a serum marker of HBV replication. The content of cccDNA in serum of hepatitis B patients is positively correlated with total HBV DNA content.