Abstract
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.