CAJ | 학술논문

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LightCycler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265 bp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(x2=4.210,P＜0.004;x2=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.
목적 이용잡교탐침결합용해곡선분석기술,건립일충괄용우림상적쾌속、가고적실시PCR검측재지단백E(apoE)기인형적방법.방법 응용LightCycler기술,우화주요반응조건,설계재1개PCR중가동시분석2개밀마자적다태성방법.선취60세이상중심성비반증133례화체검정상노년자108명위연구대상.수선이LC-Red640화LC-Red750표기조침대돌변위점위112화158적검측탐침적5'단,상응적묘정탐침재3'단피형광표기,재종연구대상기인조DNA중쾌속순배PCR특이인물확증apoE기인265 bp편단,장기여특이탐침잡교후,인발형광공진능량전이,통과이원잡교적출현화착배감기류형적용해삼수래완성화평개야생형화돌변기인분형적특정성.결과 봉치대응착서렬특이적용해온도점,각기인형적모판균정현량호적봉형.중심성비반증조돌변형이E2/3、E3/4기인형빈솔최고,여정상조비교차이유통계학의의(x2=4.210,P＜0.004;x2=6.328,P＜0.012),중심성비반증조화대조조분포빈솔분별위E2/3형37례(27.8%)화18례(16.7%)、E3/4형33례(24.8%)화13례(12.0%)、E3/3형56례(42.1%)화74례(68.5%)、E2/4형3례(2.3%)화2례(1.9%)、E4/4형3례(2.2%)화0례(0)、E2/2형1례(0.8%)화1례(0.9%);기결과여측서결과일치;중심성비반증조apoE기인총농도(101.2±73.6)표체수평야교대조조(50.6±27.1)현저승고(t=3.359,P＜0.001).결론 해방법조작간편성시,불역교차오염;획득적각충기인류형특정성강,괄용우림상화류행병학조사적쾌속진단분형.apoE기인다태성여노년중심성비반증존재상관성,대휴대E3/4형、E2/3형혹E4/4형적개체,가제시결정개체노년중심성비반유전역감성방면기도중요작용.
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 codons'polymorphism after condition was optimized. The persons elder than 60 years including 133 patients with abdominal fat and 108 healthy elder were selected. The detection probes were labeled with L-Cred 640 and LC-Red 705 at 5'end covers codons 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(x2=4.210.P＜0.004,x2=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%),E4/4(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%),E4/4(O%)and E2/2(0.9%).It showed high agreement as compared with DNA sequencing analysis The 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).Conclusions Apolipoprotein E genotyping method by melting curve is faster and simpler than other technique. It can prevent the cross-contaminated 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.