地高辛标记引物酶显色法检测HBV基因多态性及应用  

Application of enzyme coloration with digoxin-labeled primers for detection of HBV gene polymorphism

作　　者：马达 王惠民[2] 张芹[2] 王万相 蒋玲 郭乃洲 张冬雷[2] 赵建龙[3] 孙悦[3] 

机构地区：[1]盐城市第一人民医院检验科,江苏盐城224006 [2]南通医学院附属医院 [3]上海微系统与信息技术研究所

出　　处：《临床检验杂志》2004年第6期408-411,共4页Chinese Journal of Clinical Laboratory Science

基　　金：江苏省"3 3 3工程"基金项目 (2 0 0 2 2 9)

摘　　要：目的　建立地高辛标记引物酶显色法 ,用于DNA芯片检测HBV基因多态性 ,并对实验条件进行优化。方法　用地高辛标记引物进行HBVDNA前C/C区和P区双重PCR ,制备含地高辛标记的DNA样品目的片段 ,并将目的片段杂交于含HBVDNA核苷酸序列野生型及突变型探针的DNA芯片上 ,用抗地高辛抗体 碱性磷酸酶进行显色 ,并转印于尼龙膜上 ,最后用光学扫描仪读取结果。结果　通过一次杂交反应同时获取HBVDNA前C/C区 (1896、1814 )、BCP区 (176 2、176 4 )和P区 (5 5 2 )位点的信息。杂交温度与时间为 4 2℃ 1h、酶作用和显色时间分别为 30min和 4 5min时结果较理想 ,采用高盐浓度洗涤获满意杂交信号 ,批内CV均在 15 %左右 ,敏感度为 2 .0× 10 3 拷贝 /ml。 5 0例乙型肝炎患者突变率 5 2 .0 % (2 6 / 5 0 ) ,以上各位点突变率分别为 34.0 %、2 2 .0 %、38.0 %、38.0 %、4 .0 %。结论　地高辛标记引物酶显色法用于芯片检测HBV基因常见位点多态性 ,简便易行 ,试剂成本显著降低 ,不需特殊设备 ,易于临床推广应用。Objective To establish an enzyme coloration method with digoxin-labeled primers and DNA microarray for detection of HBV gene polymorphism.Methods The pre-C/C-region and P-region were amplified by multiplex PCR with digoxin-labeled primers to prepare the target DNA fragments containing digoxin,and then the target fragments were hybridized with the probes of wild or mutative probe of HBV DNA sequences on the DNA chip.The alkaline phosphatase (ALP)-labeled antibody against digoxin was applied to produce color.The colored products which contained target DNA fragments on the DNA chip were transferred to a nylon membrane and read by optical scanner.Results The mutations of pre-C/C region (1 896,1 814),BCP region(1 762,1 764) and P region (552) of HBV gene were detected simultaneously by a single hybridization.The optimal experimental conditions were as follows:the hybridization time was one hour,the temperature of hybridization was 42℃,enzyme reaction and coloration time was 30 and 45 minutes respectively.DNA chip was washed by salt solution with high concentration.The CV value was about 15% and the sensitivity was 2.0×103 copies/ml.In 50 serum samples from hepatitis B patients,the general mutation rate was 52.0% and the point mutation rate of each region in HBV gene was 34.0%,22.0%,38.0%,38.0% and 4.0% respectively.Conclusion The method of enzyme coloration with digoxin-labeled primers for detection of HBV gene polymorphism is easily operated and popularized in clinical practice.The cost of reagents is lowered significantly and no special equipment is needed.

关 键 词：地高辛 酶显色法 基因多态性 芯片检测 乙型肝炎病毒 

分 类 号：R512.62[医药卫生—内科学] R446[医药卫生—临床医学]