Multiple analysis method for amplicon by using fluorescence-based multiple melting analysis

A method for multiplex analysis of amplification products, the method including steps of:

• a) preparing target-specific fluorescence-based multiple melting analysis (FMMA) probe/primer sets which each include an FMMA probe and a non-labeled primer, said FMMA probe having a linear structure of 5′-R-T-Q-C-3′, wherein R is a fluorescent label, T is a melting temperature control sequence for inducing different temperatures of different amplification products, Q is a quencher, and C is a sequence complementary to a target nucleic acid sequence, and wherein each FMMA probe/primer is designed to produce each amplification product having a different melting temperature;
• b) amplifying multiple target nucleic acids in a sample containing one or more target nucleic acids by a nucleic acid polymerase chain reaction alone using the target-specific FMNMA probe/primer sets obtained in step a) to produce an amplification reaction product comprising a first amplification product with a first melting point and a second amplification product with a second melting point, wherein the first melting point and the second melting point differ from each other;
• and
• c) performing a melting temperature analysis on the first and the second amplification products.