应用肽核酸反向杂交技术检测HBV阿德福韦耐药突变株  

Application of peptide nucleic acid reverse hybridization technology in detecting adefovir-resistant mutations in hepatitis B virus

作　　者：单雪峰[1] 谢素兰[1] 黄爱龙[1] 胡源[1] 

机构地区：[1]重庆医科大学感染性疾病分子生物学教育部重点实验室,重庆400016

出　　处：《重庆医科大学学报》2014年第7期943-946,共4页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:81101310);教育部博士点新教师基金资助项目(编号:20115503120001);重庆市教委转化成果资助项目(编号:Kjzh11201)

摘　　要：目的:建立一种肽核酸(peptide nucleic acid,PNA)反向杂交技术,用于检测乙肝病毒阿德福韦酯(adefovir dipivoxil,ADV)常见耐药突变位点。方法:利用反向杂交技术,设计出特异性的PNA探针,与PCR扩增后带有生物素标记的样本进行杂交;通过对探针浓度,杂交温度和时间等条件的优化建立起最佳反应条件。应用该技术检测了临床ADV治疗慢性乙肝患者的72例血清样本,与测序法比较了其检测的准确性。结果:(1)通过共价结合能够将PNA探针固定在尼龙膜上,特异性检测ADV常见耐药突变位点。(2)与直接测序法相比,PNA反向杂交技术检测准确性为97.88%。结论:通过优化杂交反应,建立起快速、灵敏的检测乙肝病毒ADV常见耐药突变位点的PNA反向杂交技术。Objective ：To detect adefovir-resistant mutants of hepatitis B virus （HBV） using peptide nucleic acid （PINTA） probes. Methods： Specific PNA probes were designed for genotypes B, C D, which were then hybrized with biotinylated PCR products. Different hy- bridization factors such as probe concentration, hybridization temperature and time were investigated to establish the optimization conditions. Finally,72 hepatitis B virus（HBV） clinical samples with adefovir dipivoxil treatment were investigated using PNA hybriza- tion assay. Its usefulness was validated by comparing with sequencing data. Results ： （ 1 ）PNA probes could be fixed on the membrane by cross link and used for reverse hybridization. （2）Complete concordance of PNA hybrization assays was 97.88% with direct sequenc- ing. Conclusion：We develo~ an assav usin~ PNA orobes to detect adefovir-resistant mutants of HBV.

关 键 词：肽核酸 反向杂交 乙型肝炎病毒 阿德福韦酯 

分 类 号：R364.2[医药卫生—病理学]