STEP-WISE DETECTION OF MULTIPLE TARGET SEQUENCES IN ISOTHERMAL NUCLEIC ACID AMPLIFICATION REACTIONS

摘要

Compositions and methods useful in nucleic acid assays are provided. The invention permits detection of multiple target sequences and control nucleic acids using isothermal nucleic acid amplification methods and subsequent detection of amplification products at different temperature steps by at least two probes with different annealing temperatures. This method can be used in isothermal nucleic acid amplification reactions to detect multiple targets of interest. In a particular example, cycling hybridization probes with different spectral and hybridization temperatures are used to detect different target sequences. Probes become fluorescent when they are cleaved by a thermostable ribonuclease, which only acts when the probes are hybridized to their respective templates.