焦磷酸测序技术检测k-ras基因突变方法的建立  

Establishment of pyrosequencing method for detection of k-ras mutation

作　　者：周霞辉[1] 吴成就[2] 肖乐东[2] 孙立贤[1] 曹忠[1] 

机构地区：[1]长沙理工大学化学与生物工程学院 [2]中南大学湘雅医学检验所

出　　处：《中国临床药理学与治疗学》2015年第4期430-434,共5页Chinese Journal of Clinical Pharmacology and Therapeutics

基　　金：国家自然科学基金(21275022;21075011);湖南省科技计划(2013FJ3075);国家科技支撑计划(2012BAC17B01)资助课题

摘　　要：目的:建立k-ras基因突变的焦磷酸测序方法,并分析该基因在结直肠癌中的突变率。方法:制备10份卫生部临床检验中心2013年全国kras基因突变检测室间质评模板DNA,对焦磷酸测序技术检测k-ras基因突变方法进行验证;制备230例结直肠癌标本g DNA,应用Pyro Mark Q24焦磷酸测序仪进行k-ras基因的焦磷酸测序。结果:建立了k-ras基因突变的焦磷酸测序新方法,检测正确率100%;230例结直肠癌标本中共检出基因突变型33例,总突变率14.34%(33/230),其中12号密码子上34G>T的突变率为1.74%(4/230),35G>A的突变率为5.22%(12/230),35G>T的突变率为3.04%(7/230),37G>A的突变率为0.43%(1/230),13号密码子上38G>A的突变率为3.91%(9/230)。结论:k-ras基因突变的焦磷酸测序新方法具有快速、准确和高通量的优点,适合于在科研和临床基因扩增检验中推广。AIM： To establish a pyrosequencing method for detecting k-ras mutation and to investi- gate the frequency and characteristic of k-ras of pa- tients with colorectal cancer. METHODS： A total of 10 DNA specimens from the ministry of health clinical test center of China for external quality as- sessment were prepared for k-ras mutation detecting method verification. A total of 230 DNA specimens from patients with colorectal cancer were used for k- ras mutation detection. RESULTS ：The overall pos- itive rate of k-ras gene mutations obtained by pyrose- quencing was 14.34% （33/230）, and the positive rate of mutations was 10% （23/230） at codon 12 [5.22% （12/230）for35G〉A, 3.04% （7/230） for35G〉T, 1.74% （4/230） for 34G〉T] and 4.34% （10/230） at codon 13 [0.43% （1/230） for 37G 〉 A, 3.91% （9/230） for 38G 〉 A]. CONCLUSION： This pyrosequencing method to detect k-ras mutation is proved to be a rapid, accu- rate and high throughput method alternative to con- ventional method, and it can be a preferred option in research and clinical application.

关 键 词：结直肠癌 K-RAS基因 基因突变 焦磷酸测序 

分 类 号：R969[医药卫生—药理学]