Target detection and signal amplification

A method for detecting the presence or absence of a first target molecule in a sample, the method comprising:

• (a) contacting the sample with at least one molecular complex comprising a blocker oligonucleotide (BL) and a first catalytic nucleic acid enzyme, the BL comprising first and second segments hybridised to the first enzyme by complementary base pairing, and an intermediate segment between the first and second segments comprising a first substrate for a catalytic nucleic acid enzyme, wherein at least a segment of the first substrate is not hybridised to the first enzyme;
• a first initiator enzyme, and a reporter substrate;
• wherein,
• the first initiator enzyme has binding specificity for the first target and the first substrate of said intermediate segment and hybridisation of the target to the first initiator enzyme induces catalytic activity of the first initiator enzyme thereby facilitating cleavage of the first substrate when hybridised to the first initiator enzyme;
• the cleavage of the first substrate causes dissociation of hybridised strands of the blocker oligonucleotide and the first catalytic nucleic acid enzyme of said molecular complex;
• and
• the first enzyme of said molecular complex has binding specificity for the reporter substrate and, after said dissociation, is capable of hybridising to the reporter substrate and cleaving it to thereby provide a detectable signal indicative of the presence of the first target in the sample;
• and
• (b) determining whether a detectable signal is generated by said contacting, wherein detection of the signal indicates the first target molecule is present, and failure to detect the signal indicates the first target molecule is absent.