Real-time, sequence-specific detection of nucleic acids during strand displacement amplification

Strand displacement amplification (SDA) is an isothermal nucleic acid ampli fication method based on the primer-directed nicking activity of a restrict ion enzyme and the strand displacement activity of an exonuclease-deficient polymerase. Here we describe fluorogenic reporter probes that permit realt ime, sequence-specific detection of targets amplified during SDA. The new p robes possess the single-strand half of a BsoBI recognition sequence flanke d on opposite sides by a fluorophore and a quencher. The probes also contai n target-binding sequences located 3' to the BsoBI site. Fluorophore and qu encher are maintained in sufficiently close proximity that fluorescence is quenched in the intact single-stranded probe. If target is present during S DA, the probe is converted into a fully double-stranded form and is cleaved by the restriction enzyme BsoBI, which also serves as the nicking agent fo r SDA. Fluorophore and quencher diffuse apart upon probe cleavage, causing increased fluorescence. Target replication may thus be followed in real tim e during the SDA reaction. Probe performance may be enhanced by embedding t he fluorogenic BsoBI site within the loop of a folded hairpin structure. Th e new probe designs permit detection of as few as 10 target copies within 3 0 min in a closed-tube, real-time format, eliminating the possibility of ca rry-over contamination. The probes may be used to detect RNA targets in SDA mixtures containing reverse transcriptase. Furthermore, a two-color compet itive SDA format permits accurate quantification of target levels from the real-time fluorescence data. (C) 1999 Academic Press.