反向杂交检测肝癌相关乙型肝炎病毒前C区突变方法的建立和应用  

Development and application of a reverse hybridization method for detection of hepatitis B virus precore mutation associated with hepatocellular carcinoma

作　　者：赵芝梅[1] 朱宇[1] 吴燕[1] 樊春笋[1] 陈陶阳[2,3] 甘愉[1] 屠红[1] 

机构地区：[1]上海交通大学医学院附属仁济医院上海市肿瘤研究所癌基因及相关基因国家重点实验室,上海200032 [2]启东肝癌防治研究所 [3]启东肿瘤医院病因室,江苏启东226200

出　　处：《中国癌症杂志》2014年第4期266-272,共7页China Oncology

基　　金：国家十二五重大科技专项(No:2012ZX10002-008-002)

摘　　要：背景与目的：日益增多的研究表明，乙型肝炎病毒（hepatitis B virus, HBV）DNA前C区G1896A和G1899A突变是肝癌发生的危险因素。本研究旨在建立简单、快速、灵敏和准确的检测HBV前C区突变的反向杂交方法，并应用于检测江苏省启东地区HBV DNA前C区突变与肝癌发生的关系。方法：设计并合成HBV DNA前C区1896和1899位点的特异性探针，通过优化条件建立特异、敏感的杂交体系，并与直接测序检测结果进行比较。将该方法应用于检测启东100例肝癌和100例慢性HBV携带者（对照组），分析HBV DNA前C区突变与肝癌的关系。结果：反向杂交对血清样本的最低检测下限为HBV DNA 103 copy/mL，检测混合感染时比直接测序更占优势，混合株中10％以上的突变株均可被检测。启东地区HBV DNA前C区G1899A突变与肝癌高发具有相关性（P=0.000, OR=4.846, 95%CI：2.240～10.485），而G1896A突变未见其相关性。结论：反向杂交检测HBV DNA前C区突变方便、快速、准确，可有效监控肝癌的发生，适合临床大规模推广应用。Background and purpose：It was reported that G1896A and G1899A mutation in the hepatitis B virus （HBV） precore region were all signiifcantly associated with hepatocellular carcinoma （HCC）. Simple, sensitive and reliable methods to detect precore mutations are needed in order to prevent the occurrence of HCC in clinical detection. The aim of this study was to develop a simple and sensitive reverse hybridization method for the detection of HBV precore mutation in HBV carriers. This method was applied for exploring the relationship between the precore mutations and HCC in patients of Qidong, Jiangsu Province.Methods：The speciifc probes of nt.1896 and nt.1899 were designed and synthesized. In order to improve the sensitivity and specificity, reaction conditions of reverse hybridization were optimized. We used reverse hybridization to detect 50 HCC serum samples and compared the results with direct sequencing. Then we used this method to assess the association between HBV precore mutations and HCC in serum samples from 50 HCC patients and 50 non-HCC controls. Results：The detection limit of reverse hybridization for HBV DNA level was 103 copy/mL and the sensitivity was 10%within a mixed virus population. The coincidence rate of reverse hybridization analysis was 98%compared with the direct sequencing results. It was showed that G1899A mutation was signiifcantly associated with HCC compared to non-HCC controls （P=0.000, OR=4.846, 95%CI：2.240-10.485）, while G1896A mutation was not associated with HCC. Conclusion：Reverse hybridization is a simple and accurate approach for the detection of low amounts of HBV precore mutants among a mixed viral population. It has potential usage in the large-scale screening of precore mutations in chronic hepatitis B carriers.

关 键 词：乙型肝炎病毒 前C区 反向杂交 突变 肝癌 

分 类 号：R735.7[医药卫生—肿瘤]