| Objective: To develop a highly sensitive assay of Fluorescence Polymerase Chain Reaction(PCR) and assess its ability to detect EGFR mutations in non-small cell lung cancer(NSCLC) by comparison with direct sequencing and amplification refractory mutation system(ARMS).Methods: From June 2013 to August 2015,one hundred and forty-one formalin-fixed paraffin-embedded samples of resected NSCLC were collected in Hunan Cancer Hospital. Direct sequencing, amplification refractory mutation system(fluorescence PCR) and the novel improved PCR assay were used to detect epidermal growth factor receptor(EGFR) mutations separately. The differences of these methods were then further analyzed and compared.Results:The detection success rates of all three methods were 100 %. The consistent rate(completely same points and same mutation types) of the novel assay and direct sequencing was 75.9%(107/141). Among 96 samples with EGFR mutations detected via direct sequencing, the same mutations were detected in 92 samples via the novel assay(95.8 %). However, in the other 45 samples without mutations tested by direct sequencing, 23 samples(51.1 %) were found to be EGFR mutation-positive using the novel assay, and there were significantdifferences between these two methods(X2 = 40.745, P < 0.05). Compared with direct sequencing, the sensitivity and specificity of the novel assay were 95.8 % and 48.9 %, respectively, and the positive predictive value and negative predictive value were 80.0 %, and 84.6 %, respectively, with the accuracy of 80.9 %. When compared with direct sequencing as the gold standard, the consistent rate of these two methods reached84.4 %(119/141), with a high consistence(Kappa = 0.749, P < 0.05).And the sensitivity and specificity of the novel assay were 94.1% and 84.6 %, respectively。Conclusions:The improved method of EGFR mutation detection has shown higher sensitivity and accuracy than direct sequencing, and the results are highly consistent with ARMS. |
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