宫颈癌中EGFR启动子甲基化荧光偏振技术检测方法研究

A Novel EGFR Promoter Methylation Status Assay Based on Fluorescence Polarization in Cervical Cancer Tissue Samples

目的:建立一种基于荧光偏振技术的宫颈癌组织标本中EGFR启动子甲基化检测的新方法。方法:设计通用引物,在封闭管中同时扩增EGFR启动子甲基化与非甲基化等位基因片段,再同时用序列特异的FAM或TAMRA荧光标记探针对扩增产物进行杂交检测,利用荧光偏振仪检测扩增杂交反应的荧光偏振值,确定EGFR启动子甲基化状态。应用荧光偏振法检测63例宫颈癌组织样本,并与直接测序法进行对比验证。结果:本方法检测EGFR启动子甲基化结果与直接测序法结果无统计学差异,且最低检测浓度为50拷贝/μl,最低检测含量可达10%,敏感度高。结论:本方法检测EGFR启动子甲基化灵敏度与准确度较高,为临床宫颈癌个体化治疗相关检测提供了新技术。

Objective： To develop the novel assay detecting the EGFR promoter methylation status in cervical cancer tissue sam-ples by using a hybridization-fluorescence polarization（FP） technique. Methods： A pair of primers was used to amplify a 156 bp fragment in the promoter region of EGFR. Two probes specific for either methylated or unmethylated EGFR promoter DNA labeled with different fluorophores hybridized, respectively, with their target amplicons. The EGFR promoter methylation status was determined by the FP val-ues. A total of 63 cervical cancer tissue samples were analyzed by using the new assay technique and sequencing in parellel.Results： The results of new assay showed no statistically significant difference with the results of sequencing. The minimum detection level established with the new assay was 50 copies/mL, and it was able to detect the minor population of the EGFR promoter methylation status even when its contents were as low as 10 %.Conclusions： The novel hybridization-FP assay for evaluating the EGFR promoter methylation status was suitable for the clinical setting and would provide an applicable pharmacogenomic tool for individualized management of cervical cancer patients.