Improved polynucleotide sequence detection method

A method for the detection of a polynucleotide target sequence in a sample, comprising the steps of:

• Introducing a sample comprising one or more nucleic acid analytes to a first reaction mixture comprising:
• a single-stranded probe oligonucleotide A0 including a region complementary to a target nucleic acid sequence;
• a capture oligonucleotide B0 comprising a region complementary to a region adjacent to the target nucleic acid sequence and a capture moiety;
• and
• a solid support,
• allowing A0 and B0 to hybridise to the nucleic acid analyte;
• introducing the hybridised probes A0 to a second reaction mixture comprising a pyrophosphorolysing enzyme and a ligase, wherein A0 is pyrophosphorolysed in the 3′-5′ direction from the 3′ end to create at least a partially digested strand A1 and A1 undergoes ligation to form A2;
• and
• detecting a signal derived from the products of the previous step, wherein the products are A2 or a portion thereof, or multiple copies of A2 or multiple copies of a portion thereof, and inferring therefrom the presence or absence of the polynucleotide target sequence in the analyte.