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. 2013 Jan;16(1):25-32.
doi: 10.3779/j.issn.1009-3419.2013.01.05.

[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]

[Article in Chinese]
Jing Zhao  1 , Jinyin Zhao, Xiao Zhao, Weijun Chen, Wei Zhong, Li Zhang, Longyun Li, Mengzhao Wang
Affiliations

[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]

[Article in Chinese]
Jing Zhao et al. Zhongguo Fei Ai Za Zhi. 2013 Jan.

Abstract
in English, Chinese

Background: Epidermal growth factor receptor (EGFR) gene mutation is the most important predictor of the efficiency of EGFR-tyrosine kinase inhibitors in the treatment of non-small cell lung cancer (NSCLC). The detection of EGFR 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 of EGFR 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 ΔCt 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 of EGFR 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 as 1% and 0.1%-0.5% in the background of 500 and 5,000 copies/μL 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 ΔCt value was 14.48. Non-specific amplifications were not found when detecting 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%.

Conclusions: 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.

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。

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Figures

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19 Del检测曲线，19 Del突变型质粒浓度梯度依次为：1×105 copies/μL、1×104 copies/μL、1×103 copies/μL、1×102 copies/μL、1×101 copies/μL。 The amplification curves of 19 Del mutation (the concentrations of mutant plasmid was 1×105 copies/μL, 1×104 copies/μL, 1×103 copies/μL, 1×102 copies/μL and 1×101 copies/μL, respectively).
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21 L858R检测曲线，21 L858R突变型质粒浓度梯度依次为：1×105 copies/μL、1×104 copies/μL、1×103 copies/μL、1×102 copies/μL、1×101 copies/μL。 The amplification curves of 21 L858R mutation (the concentrations of mutant plasmid was 1×105 copies/μL, 1×104 copies/μL, 1×103 copies/μL, 1×102 copies/μL and 1×101 copies/μL, respectively).
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19 Del在500 copies/μL野生型质粒背景下，依次存在10%、5%、1%、0.5%、0.1%、0%的突变率，ARMS-Taqman PCR可检出含1%突变率样本。 The 19 Del mutation amplification curves for different concentrations of mutant plasmids (under the background of 500 copies/μL wild-type plasmids) showed the detection resolution was 1%.
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19 Del，在5, 000 copies/μL野生型质粒背景下，依次存在10%、5%、1%、0.5%、0.1%、0%的突变率，ARMS-Taqman PCR可检出含0.5%突变率样本。 The 19 Del mutation amplification curves for different concentrations of mutant plasmids (under the background of 5, 000 copies/μL wild-type plasmids) showed the detection resolution was 0.5%.
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21 L858R，在500 copies/μL野生型质粒背景下，依次存在10%、5%、1%、0.5%、0.1%、0%的突变率，ARMS-Taqman PCR可检出含1%突变率样本。 The 21 L858R mutation amplification curves for different concentrations of mutant plasmids (under the background of 500 copies/μL wild-type plasmids) showed the detection resolution was 1%.
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21 L858R，在5, 000 copies/μL野生型质粒背景下，依次存在10%、5%、1%、0.5%、0.1%、0%的突变率，ARMS-Taqman PCR可检出含0.1%突变率样本。 The 21 L858R mutation amplification curves for different concentrations of mutant plasmids (under the background of 5c000 copies/μL wild-type plasmids) showed the detection resolution was 0.1%.
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样本1 Exon 19 E746_A750 Deletion突变：荧光检测与测序结果 Exon 19 E746_A750 Deletion mutation (sample 1): the results of ARMS-Taqman and sequencing
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样本2 Exon 21 L858R突变：荧光检测与测序结果 Exon 21 L858R mutation (sample 2): the results of ARMS-Taqman and sequencing
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