Nucleic acid detection method

A method for detecting the presence of a target nucleic acid of defined sequence in a sample which method comprises the steps of:

• a) contacting said sample with:
• i. a first oligonucleotide probe (P1);
• ii. a polymerase;
• iii. a first nicking agent;
• and
• iv. one or more modified nucleotides;
• to produce in the presence of the target nucleic acid a double-stranded nucleic acid amplifier comprising a target derived strand containing at least one cleavage site for the first nicking agent and a probe (P1) derived strand;
• wherein the first oligonucleotide probe (P1) comprises a complementarity region capable of sequence specific hybridisation to the target nucleic acid;
• and whereby following production of the double-stranded nucleic acid amplifier:
• (A) the first nicking agent specifically recognises the double-stranded nucleic acid amplifier and cleaves the target derived strand of the double-stranded nucleic acid amplifier at said cleavage site to produce a primer that remains hybridised to the probe (P1) derived strand;
• and (B) the polymerase extends said primer to reproduce said double-stranded nucleic acid amplifier and displaces the target derived strand fragment (F1) that is 3′ of said cleavage site;
• b) contacting the fragment (F1) produced in step a) with:
• i. a second oligonucleotide probe (P2);
• and
• ii. a second nicking agent;
• wherein the second oligonucleotide probe (P2) comprises a complementarity region capable of sequence specific hybridisation to fragment (F1) which following hybridisation to fragment (F1) produces a cleavage site for the second nicking agent;
• whereby the second nicking agent specifically recognises the double-stranded nucleic acid formed when the second oligonucleotide probe (P2) hybridises to fragment (F1) and cleaves said second oligonucleotide probe (P2) to produce a probe fragment (F2);
• and
• c) detecting the presence of the probe fragment (F2) produced in step b) wherein the presence of said detected probe fragment (F2) indicates the presence of the target nucleic acid in said sample;
• wherein the first and/or second nicking agent(s) is a double-strand cleaving agent which functions as a nicking agent due to the double-stranded nucleic acid that is specifically recognised by said double-strand cleaving agent containing one or more modifications that renders one of its strands resistant to cleavage by said double-strand cleaving agent, said modifications being integrated into the probe (P1) derived strand of the double-stranded nucleic acid amplifier and/or into fragment (F1) by the polymerase using the one or more modified nucleotides.