检测乙型肝炎病毒“a”决定簇热点突变基因芯片的制备和初步应用  

Establishment and preliminary application of a gene chip for detection of hepatitis B virus ＂a＂ determinant hotpoint mutation

作　　者：张瑞[1] 李荣成 李艳萍 王升启[3] 梁争论[1] 李河民[1] 庄辉[4] 

机构地区：[1]中国药品生物制品检定所,北京100050 [2]广西壮族自治区疾病预控制中心 [3]军事医学科学院 [4]北京大学医学部

出　　处：《中华肝脏病杂志》2007年第2期103-106,共4页Chinese Journal of Hepatology

基　　金：国家“十五”科技攻关资助项目（2004BA718802）

摘　　要：目的为快速、高通量检测HBV“a”决定簇热点突变，制备基因芯片，并进行了初步应用。方法应用HBV基因保守序列设计PCR扩增引物，针对“a”决定簇突变设计简并探针，制备了检测126A、126S、144A、145R、145E、144A＋145R和144A＋145E突变的基因芯片。通过对质粒参考品或样本进行测定，以评价芯片的特异性、灵敏度、重复性和检测劣势株的能力，并对45例乙型肝炎患者的血清样本同时应用基因芯片和PCR产物直接测序法进行检测比较。结果所制备的基因芯片能特异性检测出质粒参考品，灵敏度为5×10^3拷贝／μl。同一样本检测10次，芯片内和芯片间变异系数均小于15％。当劣势株占HBV毒株10％以上时，基因芯片即可检出。芯片法测得45例样本中126A、126S-1和126S-2的阳性率（分别为46．67％、35．56％和24．44％）显著高于PCR产物直接测序法（分别为9．00％、4．44％和2．22％;P值分别为0．000、0．000和0．002），克隆测序验证了基因芯片法的特异性。结论基因芯片法可快速、有效地检测HBV特定位点突变株，为HBV突变的大规模筛查提供了方法。Objective To develop a gene chip for rapid detection of the ＂a＂ determinant hotpoint mutation of hepatitis B virus （HBV）. Methods Primers were designed in the HBV conservative region, and probes for detecting 126A, 126S, 144A, 145R, 145E, 144A＋145R, and 144A＋145E mutants were developed for that gene chip. PCR amplification and gene chip technology were optimized. The performance of the gene chip was evaluated by detecting the reference plasmids. Forty five samples of serum obtained frompatients with chronic hepatitis B were used to compare the sensitivity of the gene chip and the direct sequencing of PCR products. Results The oligonucleotide microarray was specific for mutant and native plasmids. The sensitivity of the gene chip was 5 × 103 copies/μl with a high reproducibility. The gene chip could detect minor variants when they were more than 10% among the HBV strains. The positive rates of 126A, 126S- 1, 126S-2 detected in the 45 specimens by the gene chip （46.67%, 35.56% and 24.44%, respectively） were higher than those detected by direct sequencing of PCR products （9.00%, 4.44% and 2.22%; P = 0.000, P = 0.000 and P = 0.002, respectively）. The sequencing of cloned PCR products demonstrated that the gene chip was specific for the ＂a＂ determinant hotpoint mutation detection. Condusion HBV ＂a＂ determinant hotpoint mutations can be detected by oligonucleotide microarray with high sensitivity and specificity, providing a method for large scale screening of the mutants.

关 键 词：肝炎病毒 乙型 基因芯片 突变 “a”决定簇 

分 类 号：R686[医药卫生—骨科学]