Highly sensitive and specific real-time PCR by employing serial invasive reaction as a sequence identifier for quantifying EGFR mutation abundance in cfDNA

Detection of EGFR mutations in circulating cell-free DNA (cfDNA) is beneficial to monitor the therapeutic effect, tumor progression, and drug resistance in real time. However, it requires thatthe mutation detection method has the ability to quantify the mutation abundance accurately. Although the next-generation sequencing (NGS) and digital PCR showed high sensitivity for quantifying mutations in cfDNA, the use of expensive equipment and the high-cost hampered their applications in theclinic. Herein, we propose a highly sensitive and specific real-time PCR by employing serial invasive reaction as a sequence identifier for quantifying EGFR mutation abundance in cfDNA (termed as qPCR-Invader). The mutation abundance can be quantified by using the difference of Ct values between mutant and wild-type targets without the need of making a standard curve.The method can quantify a mutation level as lower as 0.1% (10 copies/tube). Thirty-six tissue samples fromnon-small-cell lung cancer (NSCLC) patients were detected by our method and 14/36 tissues gave EGFR L858R mutation-positive results, whereas ARMS-PCR just identified 12 of L858R mutant samples. The two inconsistent samples were confirmed as L858R mutant by pyrophosphorolysis-activated polymerization method, indicating that qPCR-Invader is more sensitive than ARMS-PCR for mutation detection. The L858R mutation abundances of 19 cfDNA samples detected by qPCR-Invader were close to that from NGS, indicating our method can precisely quantify mutation abundance in cfDNA. The qPCR-Invader just needs a common real-time PCR device to accomplish quantification of EGFR mutations, and the fluorescence probes are universal for any target detection. Therefore, it could be used in most laboratories to analyze mutations in cfDNA.