Method for detecting target nucleic acid sequences

A method for detecting the presence of n target nucleic acid sequences in a sample, wherein, n is an integer of ≥2, and, said method comprises the following steps:

• (1) for each target nucleic acid sequence to be detected, providing at least one upstream oligonucleotide sequence and at least one mediator probe;
• wherein, said upstream oligonucleotide sequence comprises a sequence complementary to said target nucleic acid sequence;
• and, said mediator probe comprises in 5′ to 3′ direction, a mediator sequence and a target specific sequence, said mediator sequence comprises a sequence not complementary to said target nucleic acid sequence, and, said target specific sequence comprises a sequence complementary to said target nucleic acid sequence;
• and, when hybridizing with said target nucleic acid sequence, said upstream oligonucleotide sequence is located upstream to said target specific sequence;
• and, the mediator sequences comprised in all the mediator probes are different from each other;
• and, under a condition that allows nucleic acid hybridization, contacting the sample with the upstream oligonucleotide sequences and the mediator probes as provided;
• (2) under a condition that allows cleavage of mediator probes, contacting the product of Step (1) with an enzyme having 5′ nuclease activity;
• (3) providing m detection probes, and under a condition that allows nucleic acid hybridization, contacting the product of Step (2) with said m detection probes, wherein,
• m is an integer less than n and greater than 0, and
• each detection probe independently comprises in 3′ to 5′ direction, one or more capture sequences complementary to one or more mediator sequences or parts thereof, and a templating sequence;
• and, said m detection probes comprise at least n capture sequences, which are complementary to the mediator sequences or parts thereof of the mediator probes provided in Step (1), respectively;
• and,
• each detection probe is independently labeled with a reporter group and a quencher group, wherein, said reporter group can generate a signal, and, said quencher group can absorb or quench the signal generated by said reporter group;
• and, the signal, as generated by each detection probe when hybridizing with its complementary sequence, is different from the signal, as generated when not hybridizing with its complementary sequence;
• and,
• (4) under a condition that allows extension reaction of a nucleic acid polymerase, contacting the product of Step (3) with a nucleic acid polymerase;
• (5) subjecting the product of Step (4) to melting curve analysis;
• and according to the result of melting curve analysis, determining whether each target nucleic acid sequence is present in said sample;
• optionally, said method further comprises the following steps:
• (6) according to the result of the melting curve analysis, determining the level of the target nucleic acid sequence corresponding to each melting peak.