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目的:改进现有的检测表皮生长因子受体(EGFR)基因突变的荧光PCR法并开发出新的试剂盒,将其与直接测序法和ARMS法进行对比,验证该试剂盒用于临床诊断的敏感性、特异性和准确性。方法:收集2013年6月至2015年8月手术确诊的141例非小细胞肺癌(NSCLC)的石蜡包埋组织标本。采用盲法分别使用直接测序法、ARMS法和新试剂盒检测EGFR突变,比较新试剂盒与其他两种检测方法的差异,结果不一致时采用三种方法分别重复检验一次。结果:三种方法检测成功率均为100%,新试剂盒与直接测序法测得结果完全一致的比率达75.9%(107/141),在直接测序法测得的96例突变阳性中,92例在新试剂盒检测中得到验证(95.8%)。而直接测序法显示突变阴性的45例中,新试剂盒检测发现了23例突变阳性,两种检测方法的结果存在统计学差异(x2=40.745,P<0.05)。与直接测序法进行比较,新试剂盒检测EGFR突变的敏感性、特异性分别为95.8%、48.9%,阳性预测值、阴性预测值分别为80.0%、84.6%,检测准确度为80.9%。以ARMS检测法为金标准,新试剂盒测得结果完全一致的比率达84.4%(119/141),两者的一致性比较好(K=0.749,P<0.05),敏感性、特异性分别为94.1%、86.4%。结论:改进后EGFR基因突变检测的试剂盒在技术上较好地控制了检测结果的假阳性和假阴性,该检测方法较直接测序法具有更好的敏感性和准确性,与现有的ARMS法一致性较高。
OBJECTIVE: To improve the existing fluorescent PCR method for detecting epidermal growth factor receptor (EGFR) gene mutation and develop a new kit, which is compared with direct sequencing and ARMS to verify the kit for clinical diagnosis Sensitivity, specificity and accuracy. METHODS: Paraffin-embedded tissue specimens of 141 non-small cell lung cancer (NSCLC) surgically diagnosed between June 2013 and August 2015 were collected. The direct sequencing method, ARMS method and the new kit were used to detect the EGFR mutation. The difference between the new kit and the other two test methods was compared. When the results were inconsistent, three methods were used to test the EGFR mutation respectively. Results: The success rate of the three methods was 100%. The rate of the new kit was 75.9% (107/141), which was the same as that of the direct sequencing method. Of the 96 positive cases tested by direct sequencing, 92 Cases in the new kit test was verified (95.8%). However, in the 45 cases with negative mutation by direct sequencing, 23 cases were found positive by the new kit test. The results of the two methods were statistically different (x2 = 40.745, P <0.05). Compared with direct sequencing, the sensitivity and specificity of the new kit for detecting EGFR mutation were 95.8% and 48.9%, respectively. The positive predictive value and negative predictive value were 80.0% and 84.6%, respectively, and the detection accuracy was 80.9%. The accuracy of the ARMS assay was 84.4% (119/141) with good agreement (K = 0.749, P <0.05). Sensitivity and specificity 94.1%, 86.4%. Conclusion: The improved kit for detecting EGFR gene mutation has better control of false positives and false negatives of the detection results. The detection method has better sensitivity and accuracy than the direct sequencing method. Compared with the existing ARMS Law consistency is high.