实时荧光定量聚合酶链反应检测B7-H4基因表达的方法学构建  

Establishment of real-time PCR method on detecting costimulatory molecule B7-H4 expression

作　　者：郑晓[1] 吴昌平[1] 吴骏[1] 鲁常青[1] 季枚[1] 徐斌[1] 陈陆俊[1] 蒋敬庭[1] 

机构地区：[1]江苏省苏州大学附属第三医院肿瘤生物诊疗中心,常州213003

出　　处：《中华实验外科杂志》2011年第4期621-623,共3页Chinese Journal of Experimental Surgery

基　　金：基金项目：国家自然科学基金资助项目（30950022、30972703）;江苏省卫生厅医学科技发展基金项目（P200932、P200933）;江苏省卫生厅“科教兴卫工程”开放课题（KF200972）;常州市社会发展计划资助项目（CS20092019、CS20102020）;江苏省“333工程”培养资金资助项目（BRA2010037）;常州市卫生局重大科技资助项目（ZD201009）

摘　　要：目的建立实时荧光定量聚合酶链反应（PCR）检测协同刺激分子B7-H4基因表达的方法。方法采用自行设计的检测协同刺激分子B7-H4及内参照基因GAPDH的引物及TaqMan探针，经优化反应体系及反应条件后，检测B7-H4及GAPDH的基因表达。将B7-H4和内参照基因GAPDH的PCR扩增产物经测序鉴定正确并纯化后分别与pMDl9-T载体连接，克隆构建含B7-H4及GAPDH基因片段的重组质粒，作为实时荧光定量PCR检测B7-H4及GAPDH基因表达的标准品。将重组质粒标准品进行定量及梯度稀释，建立标准曲线。结果PCR扩增产物分别经测序证实为B7-H4及GAPDH基因的特异性片段。该法检测B7-H4基因表达的灵敏度为527copies／ml，线性范围为5．27X×10^2～5．27×10^7copies／ml，标准曲线方程为：Y=-3．1395X＋41．805，直线回归相关系数r=0．995，批间变异系数范围为2．39％～3．59％，扩增效率108．2％。该法检测GAPDH基因表达的灵敏度为386copies／ml，线性范围为3．86×10^2～3．86×10^7 copies／ml，标准曲线方程为：Y=-3．2436X＋41．083，直线回归相关系数r=0．999，批间变异系数范围为2．26％～3．86％，扩增效率103．4％。结论成功构建实时荧光定量PCR检测协同刺激分子B7-H4基因表达的方法，该方法特异性好、灵敏度高、重复性好。Objective To establish a real-time polymerase chain reaction （PCR） method to detect costimulatory molecule B7-H4 gene expression. Methods The mRNA levels of costimulatory molecule B7- H4 and the reference gene GAPDH were detected by using real-time PCR using TaqMan technology. The primers and TaqMan probes of B7-H4 and GAPDH were designed. The B7-H4 and internal reference gene GAPDH fragments in pure form from classical PCR amplification were cloned into pMD19-T vector, and re- combinant plasmids were used as the standard substances of B7-H4 and GAPDH quantitative detection. Standard curves were established using a serial dilution of quantified plasmids to measure B7-H4 and GAP- DH. The reaction systems were optimized, and the sensitivity, specificity and reproducibility were evalua- ted. Results The amplification products were confirmed as the specific fragments of B7-H4 and GAPDH by DNA sequencing instrument. For B7-H4, the sensitivity was 527 copies/ml. The linear range was from 5.27 ×10^2 to 5.27 ×10^7 copies/ml, the standard curve equation was Y = -3. 1395X ＋41. 805, the corre- lation coeffecient was 0. 995, the interassay coefficient of variations was 2. 39% -3.59%, and the amplifi- cation efficiency was 108.2% ; For GAPDH, the sensitivity was 386 copies/ml. The linear range was from 3.86 x 102 to 3.86 x 107 copies/ml, the standard curve equation was Y = -3. 2436X ＋41. 083, the corre- lation coeffecient was 0. 999, the interassay coefficient of variations was 2. 26% -3.86% , and the amplifi- cation efficiency was 103.4%. Conclusion The real-time PCR system for quantifying costimulatory mole- cule BT-H4 mRNA levels has been established successfully with high specificity, sensitivity and good re- peatability.

关 键 词：B7-H4 协同刺激分子 实时荧光定量PCR 

分 类 号：R440[医药卫生—诊断学]