滚环扩增在乙型肝炎病毒cccDNA检测中的应用

Detection of hepatitis B virus covalently closed circular DNA by rolling cycle amplification method

目的建立慢性乙型肝炎病毒(HBV)共价闭合环状DNA(cccDNA)的滚环扩增(RCA)方法。方法以27例慢性乙型肝炎患者的肝组织cccDNA和血清松弛环状DNA(rcDNA)为研究对象。根据中国患者HBV基因序列特点设计4对引物,分别可与HBVcccDNA的正、负链结合。在Phi29DNA聚合酶的作用下,获得线性多拷贝cccDNA扩增产物,以该产物为模板获得cccDNA全序列;通过对模板的系列稀释鉴定RCA方法的灵敏度;以RCA产物为模板扩增肝组织cccDNA的反转录酶区基因,并与同时扩增的同时间采集的同一患者血清中的rcDNA序列进行比较。结果成功建立了HBVcccDNA全序列的RCA方法,最低可检测到10个拷贝的cccDNA分子,并获得了所有扩增cccDNA的全基因序列。27例患者中,18例患者的肝组织cccDNA和血清rcDNA反转录酶区的序列完全一致,9例患者中共检出84个不同的核苷酸位点,平均每个患者9.3个,84个不同核苷酸中只有5个引起了氨基酸的改变。结论滚环扩增方法对肝组织HBVcccDNA的检测具有较好的灵敏度。该方法的建立对深入了解cccDNA在HBV致病机制中的作用以及评价抗病毒疗效有重要意义。

Objective To establish a method of rolling cycle amplification （RCA） assay for analyzing hepatitis B virus （HBV） covalently closed circular DNA （cccDNA） from patients with chronic hepatitis B. Methods Liver biopsy-derived cccDNA and serum-derived relax circle DNA （rcDNA） obtained from 27 patients with HBV were studied. Four pairs of primers targeting either plus or minus chain of cccDNA were designed based on the HBV sequence profile popular in Chinese patients. The cccDNA was amplified to get a product with linear, multiple copies of cccDNA in ramification by Phi29 DNA polymerase. Entire cccDNA sequence was obtained by a following PCR for full-length HBV genome. The sensitivity of RCA assay was evaluated with testing serially diluted cccDNA templates. The reverse transcriptase（RT）region of HBV genomes was amplified from both liver biopsy-derived cccDNA and serum-derived rcDNA from the same patients at the same sampling time The direct sequencing was performed to compare the sequence consensus between them. Results RCA assay for HBV cccDNA amplification was set up which was capable of detecting as low as 10 copies of cccDNA. Full-length cccDNA was obtained from all tested samples. Sequence of the RT gene between cccDNA and rcDNA was completely identical in 18 out of 27 patients. For other 9 patients, 84 discrepant nuclcotides were identified in total, and 9. 3 in average for each patient. Only 5 discrepant nucleotides induced amino acid alteration. Conclusion The RCA assay with high specificity and sensitivity has been established for analyzing HBV cccDNA, and it is very helpful for better understanding of HBV pathogenesis and evaluating antiviral therapeutic efficacy.