HBV阿德福韦酯耐药位点基因突变检测方法的建立  

Establishment of method for detection of adefovir dipivoxil resistance locus gene mutation in patients with HBV infection

作　　者：利振坤 何吕芬 李欢 李沙 王身华 LI Zhen-kun;HE Lyu-fen;LI Huan;LI Sha;WANG Shen-hua(Sanya People′s Hospital,Sanya,Hainan 572000,China)

机构地区：[1]三亚市人民医院检验科,海南三亚572000 [2]三亚市人民医院感染科,海南三亚572000

出　　处：《中华医院感染学杂志》2020年第20期3041-3046,共6页Chinese Journal of Nosocomiology

基　　金：海南省自然科学基金青年基金项目(8180N326)。

摘　　要：目的探究并评价Taqman锁核酸(Locked nucleic acid,LNA)探针实时荧光聚合酶链式反应(Polymerase chain reaction,PCR)在检测乙型肝炎病毒(Hepatitis B virus,HBV)感染患者阿德福韦酯(Adefovir dipivoxil,ADV)耐药位点基因突变中的应用价值。方法通过基因测序筛选阳性样本,进而构建ADV重组质粒标准品、标准曲线等,以构建的重组质粒为标准品建立实时荧光定量PCR反应体系,评价其线性范围、扩增效率、灵敏度及重复性等指标。结果rtA181V、rtN236T位点野生型和突变型重组质粒被成功构建;Taqman-LNA荧光PCR检测的线性范围为1×10^9/μl^1×10^2/μl;rtA181V、rtN236T位点野生型及突变型质粒标准品的扩增效率均高于75;rtA181V位点可稳定准确检测到最低为0.04%的突变比例,rtN236T位点可稳定准确检测出最低为0.05%的突变比例,则Taqman-LNA荧光PCR检测rtA181V和rtN236T位点临床标本的灵敏度分别为0.04%和0.05%;rtA181V、rtN236T位点野生型及突变型质粒标准品在高、中浓度中的变异系数均<5%。结论Taqman-LNA荧光PCR检测法线性范围广、扩增效率高、灵敏度高、重复性好,是一种快速、简便、灵敏的基因突变检测方法,在检测HBV感染患者ADV耐药位点基因突变中具有较高的应用价值和临床意义。OBJECTIVE To explore and evaluate the application of Taqman locked nucleic acid(LNA)probe realtime fluorescent polymerase chain reaction(PCR)in the detection of drug resistance gene mutation of Adefovir dipivoxil(ADV)in patients with hepatitis B virus(HBV)infection.METHODS The positive samples were screened by gene sequencing,and then the standard and standard curve of ADV recombinant plasmid were constructed.Using the constructed recombinant plasmid as the standard,a real-time fluorescence quantitative PCR reaction system were established to evaluate its linear range,amplification efficiency,sensitivity and repeatability and other indicators.RESULTS The wild and mutant recombinant plasmids at rtA181Vand rtN236Tsites were successfully constructed.The linear range of Taqman-LNA fluorescence PCR detection was 1×10^9/μl^1×10^2/μl.The amplification efficiency of wild type and mutant plasmid standards at rtA181V,rtN236Tsites was higher than 75.The rtA181Vsite could be stably and accurately detected with the mutation rate as low as 0.04%,and rtN236T site could be stably and accurately detected with the mutation rate as low as 0.05%.Therefore,the sensitivity of Taqman-LNA fluorescence PCR for the detection of rtA181V site and rtN236T site was 0.04%and 0.05%,respectively.The coefficient of variation of wild-type and mutant plasmid standards at RtA181V,rtN236Tsites at high and medium concentrations were both less than 5%.CONCLUSION Taqman-LNA fluorescence PCR had a wide linear range,high amplification efficiency,high sensitivity and good repeatability,which was a rapid,simple and sensitive method for gene mutation detection,and had high application value and clinical significance in detecting ADV resistance site gene mutations in patients infected with HBV.

关 键 词：Taqman-LNA荧光PCR 乙型肝炎病毒感染 阿德福韦酯 基因突变 

分 类 号：R512.62[医药卫生—内科学]