Simplified polynucleotide sequence detection method

A method of detecting a target polynucleotide sequence in a nucleic acid analyte in a sample, the method comprising the steps of:

• (a) introducing at least one nucleic acid analyte to a first reaction mixture comprising:
• i. a single-stranded probe oligonucleotide A0 comprising a 3′ end perfectly complementary to the target polynucleotide sequence;
• ii. a blocking oligonucleotide complementary to a non-target polynucleotide sequence;
• iii. a pyrophosphorolysing enzyme;
• and
• iv. a ligase;
• wherein the first reaction mixture also comprises the non-target polynucleotide sequence and the blocking oligonucleotide anneals to the non-target polynucleotide sequence to promote annealing of A0 to the analyte, wherein the analyte anneals to A0 to create a first intermediate product which is at least partially double-stranded and in which a 3′ end of A0 forms a double-stranded complex, and wherein A0 of the first intermediate product is pyrophosphorolysed in a 3′-5′ direction from the 3′ end to create at least a partially digested strand A1;
• and
• wherein A1 undergoes ligation using a splint to form A2, wherein the splint includes the target polynucleotide sequence or an oligonucleotide D, and wherein undergoing ligation includes:
• ligation of a 3′ end of A1 to a 5′ end of A1 to form a circular construct;
• or
• ligation of a 3′ end of A1 to a 5′ end of a ligation probe oligonucleotide C;
• and
• (b) detecting the presence or absence of a signal derived from the products of the ligation of the previous step, wherein the products comprise A2 or a portion thereof, or multiple copies of A2 or multiple copies of a portion thereof, and determining from the presence or absence of the signal a presence or absence of the polynucleotide target sequence in the analyte.