摘要目的 对荧光PCR-优化寡核苷酸探针法与Sanger测序法检测肺癌、结直肠癌患者KRAS基因突变阳性率、符合率进行对比分析,探讨荧光PCR-优化寡核苷酸探针法检测KRAS基因突变的临床应用特点.方法 收集221例肺腺癌、131例结直肠癌标本.所有标本均经4％中性甲醛固定、石蜡包埋、组织切片后评估肿瘤细胞含量,必要时行刮取富集肿瘤细胞后提取基因组DNA,采用荧光PCR-优化寡核苷酸探针法及Sanger测序法检测标本中KRAS基因突变.统计两种方法检测突变阳性率、突变类型、与患者特征相关性及符合率等.结果 221例肺癌标本中通过荧光PCR-优化寡核苷酸探针法及Sanger测序法检出 KRAS基因突变阳性率分别为6.3％ (14/221)、4.5％(10/221);131例结直肠癌标本中检出 KRAS基因突变阳性率分别为41.2％ (54/131)、40.5％(53/131).不论何种方法检测KRAS基因突变阳性率均与患者性别、年龄无关(P＞0.05).两种方法检测KRAS基因突变的阳性率差异无统计学意义(P＞0.05).结直肠癌KRAS基因突变率显著高于肺癌(P＜0.01).KRAS基因第12位密码子突变比例显著高于第13位密码子(P＜0.05).两种方法检测结果总体符合率为97.4％.结论 荧光PCR-优化寡核苷酸探针法检测肺癌、结直肠癌患者KRAS基因突变检出率与Sanger测序法相当,可作为Sanger测序法之外的基因突变检测的备选方法.

abstractsObjective To investigate the feasibility of real-time PCR-optimized oligonucleotide probe method for detection of KRAS mutations in lung and colorectal carcinomas,as compared with Sanger sequencing method.Methods Genomic DNA was extracted from formalin fixed paraffin embedded samples of 221 lung carcinomas and 131 colorectal carcinomas after tumor cell content assessment and macrodissection.Real-time PCR-optimized oligonucleotide probe method and Sanger sequencing were performed to detect KRAS gene mutations.The frequency of KRAS mutation,mutation types,and their concordance were analyzed.Results KRAS mutation was detected in 6.3％ (14/221) and 4.5％ (10/221) of 221 lung cancer samples by using real-time PCR-optimized oligonucleotide probe method and Sanger sequencing,respectively,while in 41.2％ (54/131) and 40.5％ (53/131) of 131 colorectal cancer samples,respectively.There was no significant correlation between KRAS gene mutations and patients'gender and age (P ＞ 0.05).The positive rate of KRAS codon 12 mutation was significantly higher than that of KRAS codon 13 mutation (P ＜ 0.05).The overall concordance between real-time PCR-optimized oligonucleotide probe method and Sanger sequencing for KRAS mutation detection was 97.4％.Conclusion Real-time PCR-optimized oligonucleotide probe method provides an alternative method with high consistency and sensitivity as compared to Sanger sequencing in gene mutation detection.