竞争性等位基因特异的TaqMan聚合酶链反应法和DNA直接测序法检测KRAS G12D突变的对比分析

Comparative analyses of competitive allele-specific TaqMan polymerase chain reation and DNA direct sequencing for detection of KRAS G12D mutation

目的对比分析竞争性等位基因特异的TaqMan聚合酶链反应(castPCR)法和DNA直接测序法检测肠镜活组织检查标本KRAS G12D突变的差异。方法应用活组织检查钳收集肠镜下结直肠腺瘤和结直肠癌组织,抽提组织DNA,分别采用castPCR法和DNA直接测序法获得KRAS G12D的突变情况。结果腺瘤组和腺癌组castPCR法检测的KRAS G12D突变率均显著高于同组DNA直接测序法(P值均<0.05),两组间castPCR法和DNA直接测序法检测的KRAS G12D突变率的差异均无统计学意义(P值均>0.05)。腺瘤组两种检测方法的突变型符合率为55.6%,野生型符合率为100.0%,阴性符合率为100.0%,总体符合率为87.5%,一致性检验Kappa值为0.643 5;腺癌组两种检测方法的突变型符合率为50.0%,野生型符合率为100.0%,阴性符合率为100.0%,总体符合率为85.0%,一致性检验Kappa值为0.583 3。提示两种方法检测阴性的一致性程度均较高,但检测阳性的一致性程度一般。Mutation Detector分析结果显示,castPCR法能够检测出突变量<1%的突变,灵敏度高达0.1%。检测突变量分析结果显示,当KRAS G12D突变量≥4%时,castPCR法和DNA直接测序法检测KRAS G12D均为阳性;当突变量<4%时,仅castPCR法能够检测出KRAS G12D为阳性。提示castPCR法能够检测出DNA直接测序法检测不出的KRAS G12D突变,较DNA直接测序法效率高。结论 castPCR法能够高效地检测肠镜活组织检查标本中低突变量的KRAS G12D,其临床实用价值较DNA直接测序法高。

Objective To compare competitive allele-specific TaqMan polymerace chain reaction （castPOR） with DNA direct sequencing for detection of KRAS G12D in endoscopic biopsy samples. Methods DNA were extracted from lesion tissues in colorectal adenoma and adenocarcinoma by endoscopic biopsy forceps. KRAS G12D mutation was detected by castPOR and DNA direct sequencing, respectively. Results In both colorectal adenoma and adenocarcinoma tissues, the mutation rate of KRAS G12D tested by castPCR was higher than DNA direct sequencing （P〈0.05）. However, there was no significant difference in the mutation rate of KRAS G｜2D between colorectal adenoma tissues and adenocarcinoma tissues （both P〉0.05）. Negative coincidence rate of these two methods was 100%. Positive coincidence rate of the two methods was 55.6% in colorectal adenoma and 50. 0% in adenocarcinoma samples. The overall coincidence rate was 87. 5% （Kappa value 0. 643 5） and 85% （Kappa value 0. 583 3） in colorectal adenoma and adenocarcinoma, respectively. CastPOR could to detect mutant allele at 0.1% according to Mutation Detector system. The result of mutation was positive by both castPCR and DNA direct sequencing when KRAS G12D mutation rate≥4%, while only castPCR could detect positive results when KRAS G12D mutation rate 〈 4% sequencing, castPCR has higher sensitMty for detecting low mutation and is a more proper method for clinical application. （Shanghai Med Conclusion Compared with DNA direct of KRAS GI2D Jn endoscopic biopsy samples J, 2015, 38： 820-823）