基于滚环扩增的鸭乙型肝炎病毒共价闭合环状DNA检测方法的建立

Establishment of A Method to Detect Duck Hepatitis B Virus Covalently Closed Circular DNA Based on Rolling Circle Amplification

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 that can specifically amplify circular DNA.Since 2008,RCA has been extensively used in hepatitis B virus（HBV）research,such as the amplification of the full-length sequence of the HBV genome,and the analysis of the drug-resistant mutations of HBV covalently closed circular DNA（cccDNA）,amongst others.To create an easy assay for the analysis of duck hepatitis B virus（DHBV）cccDNA,this study established an RCAbased method.DHBV cccDNA was amplified from the DHBV DNA samples of duck liver with four pairs of sulfur-modified primers,which were designed according to the highly conserved sequence of DHBV using sera DHBV DNA as the negative control.DHBV cccDNA was detected in the obtained RCA products by the sequencing of RCA amplicons that were amplified with primer pairs on both sides of the gap of DHBV relaxed circular DNA,rather than by digesting RCA products with a restriction enzyme.The liver and sera DHBV DNA samples of 39ducks infected with DHBV were examined with the RCA-based DHBV cccDNA detection method,and the results showed that while DHBV cccDNA was detected from all 39liver DHBV DNA samples,no DHBV cccDNA was found in any of the sera DHBV DNA samples.These results suggest that the method established in the study is highly specific and sensitive for the detection of DHBV cccDNA.The establishment of this RCA-based DHBV method for cccDNA detection lays the groundwork for using a DHBV model to study the role of cccDNA in the pathogenesis of hepatitis B and to evaluate the effect of anti-virus therapies.