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k-ras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gDNA,应用PyroMark 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.