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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。
Objective To establish a multiplex PCR system for the detection of 7 mutations in the 12th and 13th codons of KRAS exon 2 in plasma-free DNA (cfDNA). Methods Seven kinds of KRAS mutant plasmids were constructed to establish multiplex digital PCR detection system. The sensitivity, specificity and dynamic range were used as the main parameters to evaluate the performance of the system. This system was used to detect the mutation of 12 and 137 codons of KRAS exon 2 in plasma cfDNA of 15 patients with surgically resectable pancreatic ductal adenocarcinoma (PDAC) and to compare the results with those of ARMs. Results Self-built multiplex digital PCR was linear over the dynamic range of 0.01% to 10%. The sensitivity of the two detection systems can reach 0.025% and 0.043% respectively. In 15 PDAC patients, the positive rate of KRAS mutation reached 100%. Digital PCR detection of plasma cfDNA results and the results of tissue matching rate of 40%, ARMs test tissue and plasma cfDNA matching rate of 20%. The lowest abundance of plasma cfDNA was found to be 0.09% using multiplex digital PCR in 15 samples. Conclusion Multiplex PCR is highly sensitive and specific and can be used to accurately determine the level of KRAS-related mutations in peripheral blood cfDNA.