摘要
目的探讨结合酶切及片段分析技术建立稳定的高灵敏度EGFR19外显子缺失突变检测技术检测血浆EGFR19外显子缺失突变的价值。方法设计针对野生型片段的限制性内切酶予以降低野生型DNA背景。通过野生型和突变型序列的分析,以野生型序列为酶切底物,选择工具内切酶Tru1Ⅰ,设计内外两个PCR反应引物,且内侧引物一端予以标记绿色荧光。采用PCR-酶切-PCR-片段分析步骤,优化各反应条件,得到稳定的技术。以野生型DNA稀释突变型DNA检测该方法的灵敏度。采用上述方法检测42例肺癌患者外周血浆中EGFR19外显子突变情况。结果采用野生型DNA稀释突变型DNA模拟检测本方法的灵敏度,能够检测出1∶1000(Mt∶Wt)突变型DNA。42例肺癌患者中5例血浆EGFR19外显子存在缺失,其中4例为15bp的缺失,1例为24bp的缺失。结论本研究建立了一种联合酶切与片段分析的高灵敏度血浆EGFR 19外显子缺失突变检测技术,能够有效从外周血中检测出EGFR19外显子缺失突变。
Objective To establish a stable, high-sensitive detection technology of EGFR19 exon deletion mutation through combined restriction fragment length polymorphism（RFLP） and fragment analysis techniques. Methods Wide type DNA was digested to reduce the background. Fragment analysis was used to assess the length of DNA. The wild-type DNA was used to dilute mutant DNA to test the sensitivity of the method. Using this method, we detected the status of EGFR 19 exon in 42 non-small cell lung cancer （ NSCLC） peripheral blood plasma. Results The mutant DNA diluted in wide-type DNA was used to test the sensitivity of the method and the highest sensitivity was 1∶1000（ Mt∶Wt） . For the 42 plasma samples of NSCLC, 5 samples contained EGFR19 exon deletion mutation, with four cases 15bp deletion, 1 cases 24bp deletion. Conclusion We established a restriction enzyme digestion and frag-ment analysis based high sensitive method to detect plasma EGFR exon 19 deletion. The method can effectively identify EGFR19 exon deletion mutations in the peripheral blood.
出处
《临床肿瘤学杂志》
CAS
2014年第5期407-410,共4页
Chinese Clinical Oncology
基金
国家自然科学基金资助项目(81101816)
南京市医学科技发展资助项目(YKK12063)
