应用LNA-PCR法检测乙型肝炎病毒阿德福韦酯耐药位点基因突变 <sup>*</sup>

doi:10.13523/j.cb.20180907

中国生物工程杂志

2018, Vol. 38

Issue (9): 48-54 DOI: 10.13523/j.cb.20180907

技术与方法

应用LNA-PCR法检测乙型肝炎病毒阿德福韦酯耐药位点基因突变 ^*

张晓勇¹,罗前程^2,^**(

)

¹ 上海默礼生物医药科技有限公司上海 201203
² 上海市浦东新区公利医院上海 200135

Establishment and Clinical Application of LNA-PCR Assay Detecting Hepatitis B Virus Adefovir Dipivoxil Resistance

Xiao-yong ZHANG¹,Qian-cheng LUO^2,^**(

)

¹ Shanghai MOLE Diagnostics Co. Ltd, Shanghai 201203,Chain
² Gongli Hospital Affiliated to the Second Military Medical University, Shanghai 200135,Chain

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摘要：

目的: 建立简便、快速、灵敏的锁核酸(locked nucleic acid,LNA)探针实时荧光聚合酶链反应(PCR)检测方法,检测乙型肝炎病毒(hepatitis B virus,HBV) 阿德福韦酯(Adefovir dipivoxil,ADV)耐药相关位点(rtA181V、rtN236T)突变。方法: 通过基因测序筛选阳性样本,进而构建ADV rt181和rt236位点野生株和突变株重组质粒,设计包含扩增阿德福韦酯rtA181V和rtN236T耐药位点在内的特异性引物和LNA荧光探针,以构建的重组质粒为标准品建立实时荧光PCR反应体系,并通过与基因测序平行检测血清样本以判断检测方法的可行性与准确性。结果:所建立的LNA-PCR法能够检测10 ²copies/ml的HBV中ADV基因突变,同时具备较高的特异性。通过对89例ADV治疗一年后HBV阳性临床样本进行检测,有8例(8.98%)rtA181V突变、5例(5.61%)rtN236T突变、2例(2.24%)rtA181V和rtN236T混合突变,检测结果与测序结果一致。结论:所建立的LNA-PCR法是一种简便、快速、灵敏的基因突变检测方法,能有效的区分单碱基突变,对慢性乙型肝炎患者德福韦治疗过程中耐药突变的监控和抗病毒药物的调整具有指导意义。

关键词： 乙型肝炎病毒; 阿德福韦酯; 锁核酸; 突变; 耐药性

Abstract:

Objective: To develop a simple, quick and sensitive method based on the LNA (locked nucleic acid) PCR for detection of rtA181V and rtN236T mutations associated with adefovir dipivoxil (ADV) resistance of hepatitis B virus (HBV). Method: Built wild strains and the mutant recombinant plasmids of ADV rt181 and rt236 by gene sequencing screening positive samples, then designed the specific primers and LNA fluorescent probes (rtA181V, rtN236T) to construct recombinant plasmid for standard real-time fluorescent PCR reaction system, and determined the feasibility and accuracy of the detection method through the parallel to gene sequencing to detect serum samples. Result: LNA-PCR assay could detect 10 ² copies/ml mutant template, with high specificity. Eighty-nine HBV DNA positive samples were from patients with ADV therapy over one year. LNA-PCR detected two (2.24%) rtA181V and rtN236T dual mutations, eight (8.98%) rtA181V mutations, five (5.61%) N236T mutations. Complete concordance between LNA-PCR and sequencing were observed with all samples. Conclusion: LNA-PCR test is a simple, fast, and sensitive method for monitoring ADV resistant mutations of HBV in patients with chronic hepatitis B.

Key words: Hepatitis B virus Adefovir dipivoxil Locked nucleic acid Mutation Drug resistance

收稿日期: 2018-03-28 出版日期: 2018-10-12

基金资助: *上海市卫生和计划生育委员会科研项目(201640405);上海市浦东新区卫生系统优秀青年医学人才培养计划(PWRq2017-09)

通讯作者: 罗前程 E-mail: luoqiancheng19@163.com

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引用本文:

张晓勇,罗前程. 应用LNA-PCR法检测乙型肝炎病毒阿德福韦酯耐药位点基因突变 ^*[J]. 中国生物工程杂志, 2018, 38(9): 48-54.

Xiao-yong ZHANG,Qian-cheng LUO. Establishment and Clinical Application of LNA-PCR Assay Detecting Hepatitis B Virus Adefovir Dipivoxil Resistance. China Biotechnology, 2018, 38(9): 48-54.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180907 或 https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I9/48

表1 ADV耐药rtA181V和rtN236T位点引物及探针序列

图1 rt181A/rt181V/rt236N/rt236T重组质粒酶切鉴定结果

图2 突变质粒测序图

图3 LNA-PCR检测能力测试

表2 LNA-PCR方法重复性试验结果

图4 临床样本LNA-PCR检测结果

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