摘要
背景与目的表皮生长因子受体(epidermal growth factor receptor,EGFR)基因突变是决定表皮生长因子受体酪氨酸激酶抑制剂(EGFR tyrosine kinase inhibitor,EGFR-TKI)疗效最重要的预测因子,对EGFR基因突变进行检测,对指导患者个体化治疗具有重要意义。EGFR基因突变检测方法有很多,每种方法各有优缺点,本研究拟采用扩增阻滞突变系统(ampli cation refractory mutation system,ARMS)技术与Taqman探针相结合的方法,建立一种能快速、敏感及特异检测非小细胞肺癌EGFR基因突变的方法。方法首先,应用Primer Premier5.0软件在EGFR基因E746_A750和L858R处设计ARMS引物及Taqman水解探针。然后,以包含E746_A750缺失和L858R点突变的质粒为研究对象,进一步分析所建立方法的灵敏度、敏感性以及特异性。最后,用所建立的ARMS-Taqman法检测100例非小细胞肺癌(non-small cell lung cancer,NSCLC)临床标本。结果在无背景DNA干扰的情况下,ARMS-Taqman法检测灵敏度可达10copies。对于检测敏感性,在500copies/L野生型基因背景下,其敏感性达1%;在5,000copies/l野生型基因背景下,其敏感性高达0.1%-0.5%。对于检测特异性,以正常人白细胞DNA为研究对象,21L858R突变存在一定程度的非特异性扩增,但其最小Ct高达14.89,而19Del未见非特异性扩增。对100份临床标本进行检测,19Del21例,21L858R18例,总突变率为39.0%。结论我们所构建的ARMS-Taqman法是一种快速、简便以及具有较高灵敏度和特异性的EGFR基因突变检测方法,值得在临床上进一步推广和验证。
[ Abstract ] Background and objective Epidermal growth factor receptor （EGFR） gene mutation is the most im- portant predictor of the efficiency ofEGFR-tyrosine kinase inhibitors in the treatment of non-small cell lung cancer （NSCLC）. The detection ofEGFR gene mutations can guide individual therapies for NSCLC. Numerous methods are used to detect EGFR gene mutation and each method has different features. This study aims to establish a real-time polymerase chain reaction （PCR） method for the detection ofEGFR gene mutations using amplification refractory mutation system （ARMS） specific primers and Taqman fluorescence probes. Methods ARMS specific primers for the two EGFR gene mutations （E746 A750 and L858R） and Taqman fluorescence probes for the detection of the target sequence were carefully designed by the Primer Premier 5.0 software. Then, using the recombinants containing E746_A750 and L858R mutations as the study objects, we further analyzed the sensitivity and lower limit of this method, and then determined the cutoff ACt value to evaluate specific or non-specific amplification. A total of 100 clinical samples were collected and used to detect the EGFR gene mutations using this method. Results The lower limit of this method for the detection ofEGFR gene mutation was 10 copies if no interference of wild-type EGFR gene or background DNA existed. Regarding the method sensitivity, the detection resolution was as high as1% and 0.1%-0.5% in the background of 500 and 5,000 copies/~L wild-type EGFR gene, respectively. Regarding the method specificity, non-specific amplifications were found when it was used to detect 21 L858R mutations in leukocyte DNA samples from healthy volunteers. However, the minimal ACt value was 14.48. Non-specific amplifications were not found when detect- ing 19 Del mutations. Among the 100 clinical samples, 39 mutations were detected （19 Del and 21 L858R were 21 and 18, respectively） using this method. The total mutation rate was 39.0%. Conclusion The proposed ARMS-TaqMan real-time PCR method for the detection of 19 Del and 21 L858R mutations in EGFR gene was rapid, simple, sensitive, specific, and applicable in the clinical setting.
出处
《中国肺癌杂志》
CAS
北大核心
2013年第1期25-32,共8页
Chinese Journal of Lung Cancer
基金
教育部博士点基金(20091106120014)资助~~
关键词
扩增阻滞突变系统
TAQMAN探针
表皮生长因子受体
突变检测
肺肿瘤
Amplification refractory mutation system
Taqman probe
Epidermal growth factor receptor
Mutation detection
Lung neoplasms
