利用多重数字PCR检测KRAS相关突变

 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。

KRAS related mutations detected by multiplex digital PCR

Objective To establish amultiplex digital PCR panel detecting 7 most common mutations in codon 12, 13 of KRAS exon 2 from plasma cell-free DNA (cfDNA). Methods Plasmids prepared were applied to the establishment of multiplex digital PCR panels. We regarded sensitivity, specificity and dynamic range as main parameters to evaluate the performance. Plasma cfDNA and tissue sample from 15 resectable patients of pancreatic ductal adenocarcinoma (PDAC) were tested with dPCR and ARMs respectively and consistency between these two methods was also evaluated. Results Our self-built multiplex dPCR showed a good linearity performance in the dynamic range of 0.01%-10%. The sensitivity of two panels reached 0.025% and 0.043%, respectively. Among 15 PDAC patients, the consistency between plasma cfDNA given by digital PCR and tissue samples was 40%, while the result was 20% when we used ARMs to detect plasma cfDNA. The lowest abundance of KRAS mutations in plasma cfDNA among 15 PDAC patients was 0.09% according to digital PCR. Conclusions Multiplex digital PCR system shows high sensitivity and specificity in detecting and quantifying mutations of KRAS simultaneously in cfDNA.