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castPCR法和DNA直接测序法检测KRASG12D突变的对比分析
Comparative analyses of castPCR and DNA direct sequencing for detection of KRAS G12D*
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DOI:
作者:
郑晴晴1 贾伟平2张蓉2 胡承2 罗彦丽3 常英1#
ZHENG Qingqing,JIA Weiping,ZHANG Rong,HU Cheng,Luoyanli,CHANG Ying
作者单位:
1.上海交通大学附属第六人民医院消化内镜室,上海,200233;2.上海交通大学附属第六人民医院上海市糖尿病研究所,上海,200233;3. 上海交通大学附属第六人民医院病理科,上海,200233
1Department of Digestive Endoscopy; 2Shanghai Diadetes Institute; 3Department of Pathology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, 200233, China
关键词:
CastPCR;肠镜活检标本;KRAS G12D
CastPCR; Endoscopic biopsy sampels; KRAS G12D
摘要:
目的 对比分析castPCR法和DNA直接测序法检测肠镜活检标本KRAS G12D突变的差异。方法 采用活检钳收集肠镜下结直肠腺瘤和结直肠癌组织,抽提组织DNA,分别使用castPCR法和DNA直接测序法获得KRAS G12D突变情况。结果 结直肠腺瘤组castPCR法检测KRAS G12D突变率28.1%,比DNA直接测序法高12.5%;结直肠癌组castPCR法检测KRAS G12D突变率30%,比DNA直接测序法高15%。两种方法阴性符合率100%。Mutation Detector分析结果显示castPCR法能够检测出突变量<1%的突变。从检测突变率分析,两种方法无统计学意义(P>0.05),结直肠腺瘤组总体符合率87.5%(Kappa值0.6429),结直肠癌组总体符合率85%(Kappa值0.5833)。结论 castPCR法灵敏度高达0.1%,能够高效的检测肠镜活检标本中低突变量的KRAS G12D,而且操作简单、耗时短、可重复性强,比DNA直接测序法临床实用价值更高。
Objective: Comparative analysis of Competitive Allele-Specific TaqMan PCR (castPCR) and DNA direct sequencing for detection of KRAS G12D in endoscopic biopsy samples. Methods: DNA were extracted from lesions tissue, which obtained in colorectal adenoma and adenocarcinoma by endoscopic biopsy forceps. KRAS G12D mutation were detected by castPCR and DNA direct sequencing respectively. Results: In both colorectal adenoma and adenocarcinoma, the mutation rate of KRAS G12D by castPCR was higher than DNA direct sequencing (28.1% vs 12.5% for colorectal adenoma; 30% vs 15% for colorectal adenocarcinoma). Negative coincidence rate of these two methods is 100%. CastPCR was enabled to detect mutant allele as little as 0.1% according to Mutation Detector system. However, statistics analysis indicated that the mutation rate of KRAS G12D is no significantly differences by castPCR and DNA direct sequencing in our samples (P>0.05). The overall coincidence rate was 87.5% (Kappa value 0.6429) and 85% (Kappa value 0.5833) in colorectal adenoma and adenocarcinoma, respectively. Conclusions: Our data suggested that castPCR was clinic practical, with highly sensitive to detect mutation in biopsy samples, as well as simple operation and good repeatability.
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