Method for detecting nucleic acids by amplification of a circularized nucleic acid

A method of detecting a target nucleic acid, the method comprising:

• incubating a first polynucleotide and a sample comprising a target nucleic acid, whereby the target nucleic acid and the first polynucleotide hybridize to form a hybridization product,
• wherein the first polynucleotide comprises a first complementary region that hybridizes to a first region of the target nucleic acid;
• and a second complementary region that hybridizes to a second region of the target nucleic acid,
• wherein the second region of the target nucleic acid is located 3′ of the first region on the target nucleic acid, and the first region and second region of the target nucleic acid are separated by about 18 nucleotides to about 10000 nucleotides;
• and the second complementary region is located 3′ of the first complementary region on the first polynucleotide;
• incubating the hybridization product in the presence of a first nucleic acid polymerase to extend the first polynucleotide to prepare an extended product;
• incubating the extended product in the presence of a nucleic acid ligase to prepare a second polynucleotide having a cyclic structure;
• incubating the second polynucleotide having a cyclic structure in the presence of a primer set and a second nucleic acid polymerase to amplify the second polynucleotide having a cyclic structure to prepare an amplification product,
• wherein the primer set comprises
• a forward primer comprising a polynucleotide sequence of 15-30 nt that are identical to continuous nucleotides of a third region of the first polynucleotide located between the first complementary region and the second complementary region of the first polynucleotide, and a reverse primer comprising a polynucleotide sequence of 15-30 nt that are identical to continuous nucleotides located between the first region and the second region of the target nucleic acid, or
• a forward primer comprising a polynucleotide sequence of 15-30 nt that are identical to continuous nucleotides located between the first region of the target nucleic acid and the second region of the target nucleic acid, and a reverse primer comprising a polynucleotide sequence of 15-30 nt that are identical to continuous nucleotides located between the first complementary region of the first polynucleotide and the second complementary region of the first polynucleotide;
• and
• detecting the amplification product to detect the target nucleic acid.