A multiple detection method for distinguishing gene mutations based on melting curves of extended quenching probes

Conventional PCR methods can detect only a few targets simultaneously and do not fulfill most clinical requirements, especially those for detecting plasma circulating DNA. By designing characteristic universal fluorescent probes, combining multiplex PCR with the invasive reaction, and analyzing the resulting differences in the melting curves formed by extension with double-stranded probes, we developed a new method to distinguish between three mutations in the same fluorescent channel and nine mutations in three fluorescent channels in a single tube. After optimization, this method was used to distinguish between 27 mutations using only three reactions, and mutations representing as low as 0.2%-0.5% of DNA could be detected, even when up to nine mutations were present at the same time. Testing of nine clinical samples, including three L858R-positive, four 19 del-positive, and two L861Q-positive samples, showed consistent results with digital PCR tests. Compared with the conventional PCR method, our method expands the capabilities of fluorescence detection by achieving multiplex detection in a single-tube, thereby providing a simple, low-cost tool for clinical applications.