利用多重数字PCR检测KRAS相关突变

发布时间：2018-03-25 20:41

本文选题：多重数字PCR　切入点：血浆游离DNA　出处：《复旦学报(医学版)》2016年06期

【摘要】：目的建立多重数字PCR体系检测血浆细胞游离DNA(cell-free DNA,cfDNA)中KRAS第2外显子12、13密码子的7种突变。方法构建7种KRAS突变型质粒用以建立多重数字PCR检测体系,以灵敏度、特异度和动态范围为主要参数评估体系的性能。利用该体系检测15例手术可切除的胰腺导管腺癌(pancreatic ductal adenocarcinoma,PDAC)患者血浆cfDNA中KRAS第2外显子12、13密码子突变情况,并与ARMs的检测结果进行比对。结果自建的多重数字PCR在0.01%~10%的动态范围内线性良好。两个检测体系的灵敏度分别可达到0.025%和0.043%。15名PDAC患者组织中KRAS突变的阳性率达到100%。数字PCR检测血浆cfDNA所得的结果与组织结果的匹配率达到40%,ARMs检测组织和血浆cfDNA结果的匹配率为20%。15例样本中使用多重数字PCR检测到血浆cfDNA的最低丰度达到0.09%。结论多重数字PCR具备高灵敏度和特异性,可用于准确定量外周血cfDNA中KRAS相关突变的水平。
[Abstract]:Objective to establish a multiplex digital PCR system for the detection of 7 mutations in codon 12 of KRAS exon 2 in plasma cell free DNA(cell-free DNA. Methods Seven KRAS mutants were constructed to establish a multiplex digital PCR detection system. Specificity and dynamic range were the main parameters to evaluate the performance of the system. The mutation of codon 1213 in exon 2 of KRAS in plasma of 15 patients with resectable pancreatic ductal adenocarcinoma (PDAC) was detected. The results were compared with the results of ARMs. Results the linear range of the self-made multiplex digital PCR was 0.0110%. The sensitivity of the two detection systems could reach 0.025% and 0.043%, respectively. The positive rate of KRAS mutation in the tissues of 15 PDAC patients was up to 0.043%. The matching rate between the results of digital PCR detection of plasma cfDNA and tissue results was 40. The matching rate of tissue and plasma cfDNA results was 20. 15 samples. The lowest abundance of plasma cfDNA detected by multi digital PCR was 0. 09%. Conclusion\\\; Heavy digital PCR has high sensitivity and specificity. It can be used to accurately quantify the level of KRAS related mutations in peripheral blood cfDNA.
【作者单位】：复旦大学附属中山医院检验科;复旦大学附属中山医院普外科;
【基金】：国家自然科学基金面上项目(81572064) 上海市卫生计生系统重要薄弱学科建设项目(2015ZB0201)~~
【分类号】：R440

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