杂交探针技术结合熔解曲线快速检测载脂蛋白E基因型  

High-speed apolipoprotein E genotyping by hybridization probes and melting curves

作　　者：樊锦秀[1] 朱慧民[3] 张黎明[2] 陈琪[1] 金国健[3] 王爱华[4] 朱红[5] 

机构地区：[1]浙江省台州市中心医院老年医学重点实验室,318000 [2]浙江省台州市中心医院检验中心,318000 [3]浙江省台州市中心医院老年科,318000 [4]浙江省台州市中心医院B超室,318000 [5]浙江省台州市中心医院中医科,318000

出　　处：《中华检验医学杂志》2008年第2期152-158,共7页Chinese Journal of Laboratory Medicine

基　　金：浙江省医药卫生科学研究基金项目计划资助课题（200713230）;台州市科技局科技计划基金资助项目（043244）

摘　　要：目的利用杂交探针结合熔解曲线分析技术，建立一种适用于临床的快速、可靠的实时PCR检测载脂蛋白E（apoE）基因型的方法。方法应用Light Cyeler技术，优化主要反应条件，设计在1个PCR中可同时分析2个密码子的多态性方法。选取60岁以上中心性肥胖症133例和体检正常老年者108名为研究对象。首先以LC-Red640和LC-Red750标记两条针对突变位点为112和158的检测探针的5′端，相应的锚定探针在3′端被荧光标记，再从研究对象基因组DNA中快速循环PCR特异引物扩增apoE基因265bp片段，将其与特异探针杂交后，引发荧光共振能量转移，通过异源杂交的出现和错配碱基类型的熔解参数来完成和评价野生型和突变基因分型的特征性。结果峰值对应着序列特异的熔解温度点，各基因型的模板均呈现良好的峰形。中心性肥胖症组突变型以E2／3、E3／4基因型频率最高，与正常组比较差异有统计学意义（χ^2=4．210，P〈0．004；χ^2=6．328，P〈0．012），中心性肥胖症组和对照组分布频率分别为E2／3型37例（27．8％）和18例（16．7％）、E3／4型33例（24．8％）和13例（12．0％）、E3／3型56例（42．1％）和74例（68．5％）、E2／4型3例（2．3％）和2例（1．9％）、E4／4型3例（2．2％）和0例（0）、E2／2型1例（0．8％）和1例（0．9％）；其结果与测序结果一致；中心性肥胖症组apoE基因总浓度（101．2±73．6）表达水平也较对照组（50．6±27．1）显著升高（t=3．359，P〈0．001）。结论该方法操作简便省时，不易交叉污染；获得的各种基因类型特征性强，适用于临床和流行病学调查的快速诊断分型。apoE基因多态性与老年中心性肥胖症存在相关性，对携带E3／4型、E2／3型或E4／4型的个体，可提示决定个体老年中心性肥胖遗传易感性方面起到重要作用。Objective To establish a high speed and effective real-time PCR assay to analyze apolipoprotein E （apoE） genotyping in Chinese population using hybridization probes and melting curves. Methods Lightcycler was used to analyzed two cedous＇ polymorphism after condition was optimized. The persons eider than 60 years including 133 patients with abdominal fat and 108 healthy eider were selected. The detection probes were labeled with L-Cred 640 and LC-Red 705 at 5＇ end covers cedons 112 and 158 with the corresponding anchor probes labeled with fluorescein at 3＇ ends. A 265-bp fragment of the apoE gene was amplified from human genomic DNA to produce FRET. Depending on the various types of base-pair mismatch in the heteroduplex, wild type and mutant type were differentiated. Results The peaks represented the sequence-specific melting points （Tm） and each genetype showed perfect peak. E2/3 and E3/4 in abdominal fat group were much more common allele than health persons （χ^2=4. 210,P 〈0. 004,χ^2=6. 328,P〈0.012）. The frequencies of abdominal fat group was E2/3 （27.8%）, E3/4 （24. 8% ）, E3/3（42. 1%）, E2/4（2.3%）, EA/4（2.2%） and E2/2（0.8%）. The frequencies of healthy controls were E2/3（ 16. 7% ）, E3/4（ 12% ）, E3/3（68. 5% ）, E2/4（ 1.9% ）, EA/4 （0%） and E2/2（0. 9% ）. It high agreement as compared with DNA sequencing analysis rlhe expression of apoE in abdominal fat group （ 101.5 ±73. 6 ）was up-regulated than the healthy group （50. 6± 27. 1 ,P 〈0. 01 ）. Coneluslons Apolipoprotein E genotyping method by melting curve is faster and simpler than other technique. It can prevent the crosscontaminated and is suitable to be applied in clinical diagnosis. There was significant difference between the two groups. There was positive relationship between the elder＇s abdominal fat and apoE gene polymorphism. The genotyping of E3/4, E2/3 or E4/4 had the important role in the elder＇s abdominal fat on genetic susceptibility.

关 键 词：载脂蛋白E类 基因型 荧光共振能量转移 聚合酶链反应 DNA探针 肥胖症 

分 类 号：R686[医药卫生—骨科学]