肺癌中EGFR外显子突变丰度的荧光偏振检测

A novel EGFR 19,21 exon mutation level detection based on fluorescence polarization in lung cancer tissue samples

目的:建立一种基于荧光偏振技术快速、特异的检测体系,并将该体系用于检测肺癌标本中EGFR基因19、21外显子的突变丰度,以便更加全面、准确地预测靶向药物疗效以及指导临床靶向药物选择。方法:应用Primer premier 5.0软件在EGFR基因19、21号外显子突变区域上下游处设计19、21外显子各自的通用PCR引物,并设计覆盖各自突变区域的两对荧光标记探针(突变型和野生型)。以包含野生型(19、21)和突变型(19、21)的质粒分别为4种阳性标准品,空载体为阴性标准品,分别作为模板进行PCR扩增反应,将扩增结果应用荧光偏振仪进行检测,以建立突变丰度测量体系。最后,对51例肺腺癌标本进行检测,并计算出各自的突变丰度。结果:荧光偏振检测EGFR基因19、21外显子突变结果与直接测序法结果无统计学差异,且最低检测浓度为40拷贝/μl,最低检测含量可达10%。本法可提供更全面的瘤内基因变异丰度分析,有利于指导临床用药。结论:荧光偏振检测EGFR基因19、21外显子突变结果不仅灵敏度与特异性高,还能提供全面的瘤内异质性分析指标,可为临床肺癌个体化治疗效果提供预测依据。

Objective：To raise a new concePtion for the descriPtion of the intratumor heterogeneity in the regard of gene mutations-mutation level,and establish a novel assay detecting the EGFR 19,21 excon mutation level in lung cancer tissue samPles based on a hybridization -fluorescence Polarization（ FP）technique. Methods：Two Pairs of Primers was designed by Primer Premier 5. 0 to amPlify a fragment in the 19,21 excon region of EGFR. Two Pairs of Probes sPecific for either mutation or non-mutation of the EGFR 19,21 exon regions were also designed and labeled with different fluoroPhores hybridized. Then,using the recombinants containing mutation or non-mutation of the EG-FR 19,21 exon region as the four Positive temPlates of the PCR reaction according to the gradient dilution. The PCR Products were determined by the FP values,so did the negative temPlates used by the emPty vector. A total of 51 lung cancer tissue samPles were analyzed by using the new assay technique to check with sequencing analysis and gained the Precise mutation level numbers. Results：The results of this new assay showed almost no statistically significant difference with the results of sequencing analysis. The minimum detection level established with the new assay could reach to 40 coPies/μl,and it was able to detect the mutation DNA of the EGFR even when its contents were as low as 10%. Conclusion：The results of the detection of EGFR 19,21 excon mutation in lung cancer was raPid,simPle,sen-sitive,sPecific,and could Provide mutation level to guide the cilinical theraPy.