Real-time multiplexing detection of target nucleic acid sequences with elimination of false signals

A method of a real-time multiplexing detection of at least three types of target nucleic acid sequences in a sample with elimination of false signals in a real-time multiplex PCR (polymerase chain reaction), which comprises the steps of:

• (a) performing a primary multiplex PCR for amplifying the target nucleic acid sequences in a reaction vessel, wherein the primary multiplex PCR is carried out using at least three primer pairs and a template-dependent nucleic acid polymerase under conditions sufficient to amplify the at least three types of target nucleic acid sequences in order to obtain the amplicons of the target nucleic acid sequences in the sample;
• (b) preparing a secondary nested real-time multiplex PCR reaction mixture in a reaction vessel distinctly different from the reaction vessel used in the step (a), wherein the secondary nested real-time multiplex PCR reaction mixture contains (i) the amplicons obtained in the step (a), (ii) at least three primer pairs for secondary the nested real-time multiplex PCR of the amplicons in which at least one primer of each pair of the at least three primer pairs is a nested primer hybridizable with an internal sequence of the corresponding amplicon and (iii) a template-dependent nucleic acid polymerase, wherein each pair of the at least three primer pairs comprises at least one nested primer having a label generating the signal indicative of the presence of the target nucleic acid sequence during the secondary nested real-time multiplex PCR;
• (c) performing the secondary nested real-time multiplex PCR using the reaction mixture of the step (b) by carrying out at least two cycles of primer annealing, primer extension and denaturation, wherein the signal indicative of the presence of the target nucleic acid sequence is generated from each of the labeled primers during the cycles, such that the secondary nested real-time multiplex PCR can be carried out for the number of cycles not to generate the false signals but to generate the signals indicative of the target nucleic acid sequences;
• and
• (d) detecting the signal indicative of the presence of the target nucleic acid sequence, wherein the detection is performed for each cycle of the repetition of step (c), at the end of the repetition of step (c) or at each of predetermined time intervals during the repetition of step (c), whereby the signals indicative of the target nucleic acid sequences are obtained in a real-time manner without the false signals.