【作者】 贾双荣；

【导师】 陈鸣；

【作者基本信息】 第三军医大学 ， 临床检验诊断学， 2014， 博士

【摘要】 乙型肝炎病毒(hepatitis B virus，HBV)耐药的产生成为长期治疗慢性乙型肝炎成功与否的主要障碍。目前检测HBV耐药的方法多种多样，但都因存在一些缺陷，其临床应用受到限制，例如灵敏度低、耗时长、成本高等缺点。在本论文中，我们建立了一种可以同时检测HBV拉米夫定(lamivudine, LAM)及阿德福韦(adefovir, ADV)耐药突变株(rtM204V, rtM204I, rtA181T, rtA181V, rtN236T)的多重连接探针-实时荧光PCR(multiplex ligation-dependent probe real-time PCR, MLP-RT-PCR)方法。该方法结合了多重连接探针扩增(multiplex ligation-dependent probe amplification，MLPA)技术的灵敏、特异、高通量和实时PCR方便快捷、实时检测的特点。通过检测临床116例慢性乙肝患者DNA样本对MLP-RT-PCR方法进行了方法性能评价，其中检测出LAM耐药突变41例(35.3%)，ADV耐药突变17例(14.7%)及两者共同耐药突变5例(4.3%)。检测结果与金标准直接测序方法比较，MLP-RT-PCR检测rtM204V、rtM204I、rtA181T、rtA181V和rtN236T的符合率分别为95.7%(111/116)、98.3%(114/116)、99.1%(115/116)、98.3%(114/116)和99.1%(115/116)。MLP-RT-PCR方法检测低比例突变株的灵敏度比直接测序方法更高，能够检测0.1%以上的rtM204V、rtM204I、rtA181T和rtN236T突变株，1%以上的rtA181V突变株。MLP-RT-PCR发现了4例低比例临床突变株，并进一步被TA克隆测序方法确证。MLP-RT-PCR能够快速、灵敏检测HBV多重耐药突变位点，为临床提供了一种成本低廉、检测快速的乙型肝炎耐药检测方法。更多还原

【Abstract】 Drug-resistant mutations of hepatitis B virus (HBV) are the major obstacles tosuccessful therapy for chronic hepatitis B. Although there are many methods to detect theantiviral drug-resistant mutations of HBV, their applications are restricted because of theshortcomings such as low sensitivity, time required and high cost. Here a multiplexligation-dependent probe real-time PCR (MLP-RT-PCR) method was developed to detectlamivudine (LAM) and adefovir (ADV) resistant HBV mutants (rtM204V/I, rtA181V/T andrtN236T) simultaneously. The new method combined the high-throughout of multiplexligation-dependent probe amplification (MLPA) with the rapid and sensitive detection ofreal-time PCR. In this report, the MLP-RT-PCR was evaluated by detecting drug-resistantmutants in116patients with chronic hepatitis B. By MLP-RT-PCR analysis, LAM-resistantmutations were detected in41patients (35.3%), ADV-resistant mutations were detected in17patients (14.7%), LAM and ADV-resistant mutations were detected in5patients (4.3%).Based on the results of MLP-RT-PCR, the mutations rtM204V, rtM204I, rtA181T, rtA181Vand rtN236T were95.7%(111/116),98.3%(114/116),99.1%(115/116),98.3%(114/116),and99.1%(115/116), respectively, in concordance with those of direct sequencing. TheMLP-RT-PCR assay was more sensitive than direct sequencing when detecting mutationswith low percentage.The channels for rtM204V, rtM204I, rtA181T and rtN236T detectedmutants whose percentage were over0.1%, while the channels for rtA181V detectedmutants whose percentage was over1%. Four samples containing the low percentage(<10%) mutants were identified by MLP-RT-PCR and further confirmed by clonalsequencing. MLP-RT-PCR is a rapid and sensitive method that enables the detection ofmulti-drug-resistant HBV mutations in clinical practices.更多还原