三种检测乙型肝炎病毒YMDD自然变异方法的比较

实用肝脏病杂志 ›› 2010, Vol. 13 ›› Issue (1): 16-18.

三种检测乙型肝炎病毒YMDD自然变异方法的比较

闵晓春, 赵书民, 杨德刚

201800上海市嘉定区中心医院感染病科（闵晓春）; 上海第二军医大学附属长征医院感染病科（赵书民,杨德刚）

收稿日期:2009-08-13 出版日期:2010-02-10 发布日期:2016-04-18
作者简介:闵晓春男,32岁,医学硕士。主要从事病毒性肝炎诊断及治疗的研究。E-mail：zqmxcaexshq@126.com

The comparison of three assays on the spontaneous YMDD mutation in Hepatitis B Virus

MIN Xiao- chun, ZHAO Shumin, YANG Degang

Department of Infectious Disease,Jiading District Central Hospital,Shanghai 201800,China

Received:2009-08-13 Online:2010-02-10 Published:2016-04-18

摘要/Abstract

摘要： 目的筛选一种特异性及灵敏性较高的检测乙型肝炎病毒YMDD自然变异的方法。方法通过分子克隆的方法,构建YMDD、YVDD及YIDD三种质粒;通过检测不同比例混合的质粒,比较PCR产物直接测序法、单探针特异性荧光PCR技术及引物特异性荧光PCR技术三种检测方法。结果经酶切鉴定及直接测序鉴定,显示三种质粒构建成功;当待测模板占总模板的比例大于20%时,直接测序法才能准确检测;当突变株占总毒株的比例占50/51时,单探针特异性荧光PCR法才能判定该样品为YMDD变异型;当突变株占总毒株的比例高于1/11时,引物特异性荧光PCR技术非特异结合的影响可忽略,具有很好的特异性,同时具有较高的灵敏性,检测突变株占总毒株的最低比例可达到1/11。结论引物特异性荧光PCR技术特异性及灵敏性较高,适合用于乙型肝炎病毒 YMDD自然突变株的检测。

关键词: 乙型肝炎病毒, YMDD自然变异, PCR

Abstract: Objective To sieve a assay of higher specificity and sensitivity on the spontaneous YMDD mutation in hepatitis B virus. Method Plasmids of YMDD,YVDD,YIDD were constructed with molecular methods. The sensitivity and specificity of the three assays （direct sequencing of PCR products,single probe-specific fluorescent PCR and primer-specific fluorescent PCR）were compared by detecting coexisting strains. Result Plasmids of YMDD,YVDD,YIDD which were constructed successfully were identified by restriction enzyme methods and direct sequence analysis. In mixed plasmids,a certain plasmid can be detected by direct sequencing if it covers more than 20% of the total plasmids. YMDD mutant strains only can be tested by single probe-specific fluorescent PCR when the proportion of the total strains is 50/51. The method of primer-specific fluorescent PCR has higher specificity and sensitivity when mutant strains account for more than 1/11 of the total strains. Conclusions This method of primer-specific real-time PCR is fit for the detection of the spontaneous HBV YMDD mutation.

Key words: Hepatitis B virus, YMDD spontaneous mutation, PCR

闵晓春, 赵书民, 杨德刚. 三种检测乙型肝炎病毒YMDD自然变异方法的比较[J]. 实用肝脏病杂志, 2010, 13(1): 16-18.

MIN Xiao- chun, ZHAO Shumin, YANG Degang. The comparison of three assays on the spontaneous YMDD mutation in Hepatitis B Virus[J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2010, 13(1): 16-18.

参考文献

[1] OKAMOTO H,IMAI M,KAMETANI M,et al. Genomic heterogeneity of hepatitis B virus in a 54-year-old woman who contracted the infection through materno-fetal transmission[J]. J Exp Med,1987,57（4）：231-236.
[2] KOBAYASHI S,LDE T,SATA M. Detection of YMDD motif mutations in some lamivudine-untreated asymptomatic hepatitis B virus carriers[J]. J Hepatol,2001,34：584-586.
[3] 刘传苗,张欣欣,陆志檬. 拉米夫定治疗前后乙型肝炎病毒YMDD野毒株和变异株的变化[J]. 肝脏,2004,9（2）：73-76.
[4] AKARSU M,SENGONUL A. YMDD motif variants in inactive hepatitis B carriers detected by Inno-Lipa HBV DR assay[J]. J Gastroenterol Hepatol,2006,21（12）：1783-1788.
[5] LOK AS,ZOULIM F,LOCARNINI S,et al. Antiviral drug-resistant HBV：standardization of nomenclature and assays and recommendations for management[J]. Hepatology,2007,46：254-265.
[6] 季秀玲,沈雪芳,陈宇明,等. 应用实时荧光PCR技术检测HBV YMDD变异[J]. 医学分子生物学杂志,2004,1：279-281.
[7] CANE PA,COOK P,RATCLIFFE D,et al. Use of real-time PCR and fluorimetry to detect lamivudine resistance-associated mutations in hepatits B virus[J]. Antimicrob Agents Chemother,1999,43：1600-1608.
[8] MLMSTROM S,HANNOUN C,M.LINDH. Mutation analysis of lamivudine resistant hepatitis B virus strains by TaqMan PCR[J]. J Virol Methods,2007,143：147-152.
[9] SHI M,WANG RS,YANG ZJ,et a1. Rapid quantitation of lamivudine-resistant mutants in lamivudine treated and untreated patients with chronic hepatitis B virus infection[J]. Clinica Chimica Acta,2006,373：172-175.
[10] 霍红,赵书民,蔡雄,等. YMDD自然变异毒株流行病学调查[J]. 肝脏,2006,11（4）：232-235.
[11] DEGERTEKIN B,HUSSAIN M,TAN J,et al. Sensitivity and accuracy of an updated line probe assay（HBV DR v.3） in detecting mutations associated with hepatitis B antiviral resistance[J]. J Hepatol,2009,50：42-48.
[12] HUSSAIN M,FUNG S,LIBBRECHT E,et al. Sensitive line probe assay that simultaneously detects mutations conveying resistance to lamivudine and adefovir[J]. J Clin Microbiol,2006,44：1094-1097.

[1]	吴海义, 陈建新. 四种诊断模型诊断乙型肝炎病毒携带者肝纤维化的效能分析^*[J]. 实用肝脏病杂志, 2019, 22(6): 832-835.
[2]	周积云,马勇,丁体龙,涂远航,陈策. 慢性乙型肝炎患者外周血CD8⁺CD28⁺T淋巴细胞百分比的变化及其临床意义探讨[J]. 实用肝脏病杂志, 2019, 22(2): 180-183.
[3]	谭静, 叶晓林, 王慧. 妊娠中晚期应用替比夫定阻断HBsAg阳性孕妇HBV母婴传播效果及对婴儿接种乙肝疫苗应答的影响^*[J]. 实用肝脏病杂志, 2019, 22(1): 49-52.
[4]	张利宁, 刘志刚, 高彩霞, 白桂林, 张晓玲. 授权教育饮食干预在乙型肝炎相关性肾炎患者中的应用研究^*[J]. 实用肝脏病杂志, 2018, 21(5): 799-800.
[5]	王萍, 周小佳, 刘新华. 视频化健康教育在乙型肝炎病毒相关性肾炎患者中的应用效果研究*[J]. 实用肝脏病杂志, 2018, 21(3): 451-452.
[6]	翁叶蕊, 符爱贞. HBV感染对孕妇产后母乳喂养的影响及护理策略[J]. 实用肝脏病杂志, 2018, 21(3): 457-458.
[7]	占国清, 谭华炳, 李芳, 李儒贵, 张薇薇, 郭鹏. 恩替卡韦抗病毒对TACE治疗的HBV DNA阴性乙型肝炎相关性肝癌的影响[J]. 实用肝脏病杂志, 2018, 21(1): 96-99.
[8]	包依夏姆·阿巴拜克力, 努力比亚·阿不杜克尤木, 张跃新. 乙肝疫苗联合HBIG接种阻断HBsAg/HBeAg双阳性孕妇HBV母婴传播的效果研究[J]. 实用肝脏病杂志, 2017, 20(4): 481-482.
[9]	张伟, 董政, 孔慧芳, 张百龙, 毛威, 高旭东, 荣义辉. 肝癌组织HNF4α mRNA定量检测方法的建立与初步应用[J]. 实用肝脏病杂志, 2017, 20(3): 302-306.
[10]	郭立杰，王艳，王敏，杜婧，王超，叶立红，张海丛，王晓静. 国产恩替卡韦治疗慢性乙型肝炎患者早期抗病毒疗效观察*[J]. 实用肝脏病杂志, 2017, 20(2): 226-227.
[11]	包依夏姆·阿巴拜克力, 努力比亚·阿不杜克尤木, 张跃新. 乙肝疫苗联合HBIG接种阻断HBsAg/HBeAg双阳性孕妇HBV母婴传播的效果研究[J]. 实用肝脏病杂志, 2017, 20(2): 248-249.
[12]	朱甜甜综述，李军，朱传龙审校. 模式识别受体在HBV感染过程中的作用研究进展[J]. 实用肝脏病杂志, 2016, 19(6): 749-752.
[13]	杜景华, 张宇, 郝祎卓. 慢性乙型肝炎患者血清乙型肝炎病毒表面大蛋白水平检测意义探讨[J]. 实用肝脏病杂志, 2016, 19(5): 599-600.
[14]	沈显元, 陈金香, 喻春红, 卢芝君, 王丹凤. 血清HBsAg定量变化预测干扰素α-2b治疗慢性乙型肝炎停药后疗效研究^*[J]. 实用肝脏病杂志, 2016, 19(4): 476-477.
[15]	王毅，杨振华. miRNAs在HBV感染及肝癌发病中的作用[J]. 实用肝脏病杂志, 2016, 19(3): 381-384.