乙型肝炎病毒前C区及基本核心启动子变异检测方法的比较研究  

Comparative study on detection methods of mutations in hepatitis B virus precore and basic core promoter

作　　者：丁静娟[1] 刘悦晖[1] 王梅[1] 

机构地区：[1]贵阳医学院附院感染科实验室,550004

出　　处：《中华检验医学杂志》2006年第5期402-406,共5页Chinese Journal of Laboratory Medicine

基　　金：贵州省优秀科技教育人才省长基金资助[黔省专合字(2005)71号]

摘　　要：目的建立错配聚合酶链反应-限制性片段长度多态性(mPCR-RFLP)检测乙型肝炎病毒(HBV)前C区A1896、基本核心启动子(BCP)T1762/A1764双变异的方法,与直接测序法比较,评估其应用价值。方法利用错配PCR的原理扩增HBV前C区长194bp、C启动子区长184bp的基因片段,扩增产物分别经限制性内切酶Bsu36I、BclI酶切,琼脂糖凝胶电泳,根据酶切图谱多态性,建立检测HBV前C A1896、BCP T1762/A1764双变异的方法,对127份HBsAg、HBV DNA阳性的HBV感染者血清进行分析,酶切同时测序。2份标本用克隆测序以验证酶切鉴定变异株、野株混合感染的准确性。结果127份血清中125(98·42%)份能用酶切、121(95·21%)份能用测序分析HBV前CA1896、BCP T1762/A1764状况。酶切与测序均成功的119份血清中,16份为前C区A1896变异株,34份为BCP T1762/A1764双变异株,21份为前C区A1896、BCP T1762/A1764联合变异株,48份为前C区G1896、BCP A1762/G1764野株,酶切与测序的结果完全一致。1份酶切鉴定为前C区A1896、G1896混合感染标本的5个克隆子中,1个为前C区A1896变异株,另外4个为前C区G1896野株;另1份酶切鉴定为单纯前C区A1896变异标本的5个克隆子均为前C区A1896变异株,酶切分析结果与克隆测序结果完全相符。结论与测序法相比,本方法较简便、特异性强,能区分野毒株、变异株混合感染,适合较大样本分析,可用于流行病学调查及临床筛查。Objects To establish a mismatched polymerase chain reaction restricted fragment length polymorphism（mPCR-RFLP） method for the detection of hepatitis B virus （HBV） mutations in preeore A1896 ,basic core promoter（BCP） T1762/A1764 and compare the method with direct sequencing to evaluate its application value. Methods According to principle of mPCR, 196 bp and 184 bp gene fragments in HBV precore and core promoter region were amplified, respectively. The products of PCR were digested by Bsu36I or BclI and subjected to agarose gel eleetrophoresis. The method for detection precore A1896, BCP T1762/ A1764 mutation was established by restricted fragment length plymorphism. 127 sera were analyzed by both mPCR-RFLP and direct sequencing method. Two sera which were identified mixture infection of preeore wild and mutant strains by mPCR-RFLP,were analyzed by cloning sequencing. Result From 127 sera ,125 sera could be analyzed HBV preeore 1896 ,BCP T1762/A1764 situation by mPCR-RFLP method, 121 sera could be analyzed by sequencing. In 119 sera which could be analyzed by two methods, 16 were mutant strains of precore A1896,34 were BCP T1762/A1764 mutant strains, 21 were precore A1896 associated with BCP T1762/A1764 mutant strains and 48 were wild strains. The results from both methods were completely compatible. There was no significant difference in detective rate of HBV precore A1896,BCP T1762/A1764 mutation between two methods （ 98.42 % vs. 95. 21% ,χ^2 = 0. 2668, P 〉 0. 05 ）. The sequence of five clones from one serum which was identified preeore mutation by mPCR-RFLP were all A1896 mutant strains. Another serum identified mixture infection by mPCR-RFLP , one clone was A1896 mutant strain and four were G1896 wild strains. The results of mPCR-RFLP were verified by cloning. Conclusions Comparison with sequencing, the mPCR-RFLP method is simple,accurate and can be used in large-scale surveys and clinical research.

关 键 词：肝炎病毒 乙型 变异 限制性片段长度多态性 测序 

分 类 号：R450[医药卫生—治疗学]