REAL-TIME MULTIPLEXING DETECTION OF TARGET NUCLEIC ACID SEQUENCES WITH ELIMINATION OF FALSE SIGNALS

摘要

PURPOSE: A method for detecting three or more kinds of target nucleic acids is provided to eliminate a false signal. CONSTITUTION: A method for detecting three or more kinds of target nucleic acids in a sample by real time multiplex PCR without a false signal comprises: a step of performing primary multiplex PCR of the target nucleic acid sequences in a reaction container; a step of preparing a secondary nested real time multiplex PCR mixture in another reaction container; a step of performing primer annealing, primer elongation, and denaturation of the reaction mixture through 2 or more cycles; and a step of detecting a signal which shows the presence of the target nucleic acid sequence. [Reference numerals] (AA) Nested real-time multiplex PCR method; (BB) Primary multiplex PCR; (CC) Forward primer; (DD) Target nucleic acid; (EE) Reverse primer; (FF) Multiplex PCR by repeating denaturation, hybridization, and elongation; (GG) Secondary nested real-time multiplex PCR using labeled nested primer; (HH) Labeled nested primer as forward primer; (II) Amplicon prepared from primary multiplex PCR; (JJ) Reverse primer; (KK) Labeled nested primer which is hybridized on internal sequence of corresponding amplicon prepared from primary multiplex PCR is single- or double-labeled primer such as scorpion, sunrise, lux, plexer, amplifluor, and antiprimer and can be used as forward and/or reverse primer.; (LL) Repeat of denaturation, hybridization, and elongation; (MM) Detecting false positive signal-excluded real-time multiplex target by reaction cycle which generates target signal but does not generate false positive signal; (OO) L indicates one or more probe molecules containing interactive double labeling.