应用锁核酸探针技术检测肿瘤样本中K—ras基因突变

目的应用锁核酸探针技术检测肿瘤样本中K—ras基因突变，探讨其诊断价值。方法结合锁核酸TaqMan探针与实时荧光PCR技术建立K一ras基因突变快速检测法，与传统聚合酶链反应一单链构象多态性分析法（PCR—SSCP）及测序结果进行比较，评价新方法的诊断价值。结果锁核酸探针PCR法可对肿瘤样本中K—ras基因突变进行有效检测，操作简便；可在104倍的野生型K—rag基因背景下检测出突变，灵敏度为0．1％，可检测突变基因最低限为10拷贝/mL，相对应传统方法，本方法可同时进行较大规模的样品检测，更快捷和灵敏。结论锁核酸探针实时荧光PCR法较PCR—SSCP法有明显优势，适用于大规模临床样本的K—ras基因突变检测，可作为肿瘤筛查和预后判断有效手段。

Assay for K-ras Mutation in Tumor Samples by Locked Nucleic Acid Probe Technique

Objective To establish a locked nucleic acid（ LNA ）probe mediated PCR assay for discriminating K-ras mutation, and to evalu- ate its diagnostic value. Methods We developed real-time PCR combined with LNA probe to detect the genotype in codon 12 of K-ras. Meanwhile, the mutation of K-ras was also analyzed by PCR-SSCP and was validated by sequencing. Results Our method could discriminate the genotype of K-ras effectively and was convenient to perform. The sensitivity of our method was 0.1% in a 105-fold excess of wild-type K-ras. The detection limit was 10copies/mL. Our method could be used for large sample test,with the time of a single test being 1 hour. Compared with the traditional method such as PCR-SSCP,this method could simultaneously carry out large-scale sample testing, more efficient and sensitive. Conclusion LNA-real time PCR has obvious advantage over PCR-SSCP and it is suitable for detecting K-ras mutation in large samples. This method can serve as an effective means for tumor screening and prognosis.