ARMS技术联合Taqman探针检测100例非小细胞肺癌EGFR基因突变
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,...
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| Published in | 中国肺癌杂志 Vol. 16; no. 1; pp. 25 - 32 |
|---|---|
| Main Author | |
| Format | Journal Article |
| Language | Chinese |
| Published |
100730北京,中国医学科学院,北京协和医学院,北京协和医院呼吸内科%中国科学院北京基因组研究所,北京,100029
2013
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| Subjects | |
| Online Access | Get full text |
| ISSN | 1009-3419 1999-6187 |
| DOI | 10.3779/j.issn.1009-3419.2013.01.05 |
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| Abstract | 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。 |
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| AbstractList | R734.2; 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(amplification 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 Premier 5.0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10 copies.对于检测敏感性,在500 copies/μL野生型基因背景下,其敏感性达1％；在5,000 copies/μl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,21 L858R突变存在一定程度的非特异性扩增,但其最小ΔCt高达14.89,而19 Del未见非特异性扩增.对100份临床标本进行检测,19 Del 21例,21 L858R 18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. 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。 |
| Author | 赵静 赵肖 钟巍 张力 李龙芸 王孟昭 赵金银 陈唯军 |
| AuthorAffiliation | 中国医学科学院,北京协和医学院,北京协和医院呼吸内科,北京100730 中国科学院北京基因组研究所,北京100029 |
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| Author_FL | Li ZHANG Wei ZHONG Jing ZHAO Weijun CHEN Xiao ZHAO Jinyin ZHAO Longyun LI Mengzhao WANG |
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| DocumentTitleAlternate | Detection of EGFR Gene Mutations in 100 Non-small Cell Lung Cancer Clinical Samples by a Real-time Polymerase Chain Reaction Method Using Amplification Refractory Mutation System Specific Primers and Taqman Fluorescence Probes |
| DocumentTitle_FL | Detection of EGFR Gene Mutations in 100 Non-small Cell Lung Cancer Clinical Samples by a Real-time Polymerase Chain Reaction Method Using Amplification Refractory Mutation System Specific Primers and Taqman Fluorescence Probes |
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| Keywords | Taqman探针 表皮生长因子受体 肺肿瘤 扩增阻滞突变系统 突变检测 |
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| Notes | [ 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 determine |
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| PublicationTitle | 中国肺癌杂志 |
| PublicationTitleAlternate | Chinese Journal of Lung Cancer |
| PublicationTitle_FL | Chinese Journal of Lung Cancer |
| PublicationYear | 2013 |
| Publisher | 100730北京,中国医学科学院,北京协和医学院,北京协和医院呼吸内科%中国科学院北京基因组研究所,北京,100029 |
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| Snippet | 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... R734.2; 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... |
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| SubjectTerms | Taqman探针 扩增阻滞突变系统 突变检测 肺肿瘤 表皮生长因子受体 |
| Title | ARMS技术联合Taqman探针检测100例非小细胞肺癌EGFR基因突变 |
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