论文部分内容阅读
滚环扩增(RCA)是新近发展起来的一种能特异性扩增环形DNA的实验技术,自2008年以来被广泛用于HBV基因全长扩增及共价闭合环状DNA(cccDNA)耐药突变分析等研究。为了便于鸭乙型肝炎病毒(DHBV)cccDNA的分析,本研究建立了基于RCA的DHBV cccDNA的检测方法。通过针对DHBV高度保守序列设计的4对RCA硫化修饰引物,以血清DHBV DNA为阴性对照,从肝组织DHBV DNA标本中扩增得到DHBV cccDNA。然后用跨缺口引物扩增RCA产物测序替代限制性内切酶切分析进行DHBV cccDNA鉴定。应用该方法检测39份携带DHBV麻鸭肝组织与血清标本结果显示:全部肝组织标本均检出DHBV cccDNA,而全部血清标本则均无DHBV cccDNA检出,表明本研究建立的基于RCA的DHBV cccDNA检测法具有良好的特异性和灵敏性。该方法的建立为应用鸭乙型肝炎病毒动物模型研究cccDNA在病毒致病机制中的作用以及评价抗病毒疗效奠定了实验基础。
Rolling circle amplification (RCA) is a newly developed experimental technique for specific amplification of circular DNA. It has been widely used in HBV genome amplification and covalently closed circular DNA (cccDNA) resistance since 2008 Drug mutation analysis research. In order to facilitate the analysis of duck hepatitis B virus (DHBV) cccDNA, this study established a method for detecting DHBV cccDNA based on RCA. DHBV cccDNA was amplified from DHBV DNA samples of liver tissues by using 4 pairs of RCA vulcanization modified primers designed for highly conserved sequences of DHBV and serum DHBV DNA as a negative control. The amplified product of RCA was then sequenced using a cross-primed primer in place of the restriction endonuclease analysis for DHCV cccDNA identification. The results showed that DHBV cccDNA was detected in all of the liver samples and no DHBV cccDNA was detected in all the serum samples. The results showed that the RCA-based DHBV cccDNA Detection method has good specificity and sensitivity. The establishment of the method for the application of duck hepatitis B virus animal model of cccDNA in the pathogenesis of the virus and the evaluation of anti-viral efficacy has laid the experimental foundation.