Method of detecting amplification product or target nucleic acid

A method of detecting a target nucleic acid, comprising a step of obtaining an amplification product by amplifying a target nucleic acid with primers and a step of detecting the amplification product by hybridizing it with a probe nucleic acid, wherein

• the target nucleic acid comprises F3, F2 and F1 regions in this order from a 5′ terminal side;
• B3c, B2c and B1c regions in this order from a 3′ terminal side;
• an FP region in the region from the F2 to F1 regions and/or a BPc region in the region from the B2c to B1c regions;
• and further comprises at least one of an LF region in the region from the F2 to F1 regions and an LBc region in the region from the B2c to B1c regions;
• the probe nucleic acid comprises a nucleotide sequence complementary to a sequence selected from the group consisting of sequence in the FP and BPc region and sequences complementary thereto;
• the primers include an FIP primer comprising sequence identical to that of the F2 region at the 3′ terminal side and a sequence complementary to that of the F1 region at the 5′ terminal side;
• an F3 primer comprising sequence identical to that of the F3 region;
• a BIP primer comprising sequence complementary to that of the B2c region at the 3′ terminal side and sequence identical to that of the B1c region at the 5′ terminal side;
• a B3 primer comprising sequence complementary to that of the B3c region;
• and at least one of a loop primer LFc comprising sequence complementary to that of the LF region and a loop primer LBc comprising sequence identical to that of the LBc region;
• and
• wherein the FP and F2 regions and/or the BPc and B2c regions have a non-overlapping region of at least 10 bases or more and overlapping regions of 10 bases or less and the FP and LF regions and/or the BPc and LBc region have a non-overlapping region of at least 10 bases or more and overlapping regions of 10 bases or less.

A method comprising detecting a single-nucleotide mutation by amplifying a target nucleic acid having a single-nucleotide mutation with primers and hybridizing the amplification product with a probe nucleic acid, wherein:

• the target nucleic acid comprises F3, F2 and F1 regions in this order from a 5′ terminal side;
• B3c, B2c and B1c regions in this order from a 3′ terminal side;
• an FP region having the single-nucleotide mutation in the region from the F2 to F1 regions and/or a BPc region having the single-nucleotide mutation in the region from the B2c to B1c regions;
• and further comprises at least one of an LF region in the region from the F2 to F1 regions and an LBc region in the region from the B2c to B1c regions;
• the probe nucleic acid comprises a nucleotide sequence complementary to a sequence selected from the group consisting of sequence in the FP and BPc region, and sequences complementary thereto;
• the primers include an FIP primer comprising sequence identical to that of the F2 region at the 3′ terminal side and sequence complementary to that of the F1 region at the 5′ terminal side;
• an F3 primer comprising sequence identical to that of the F3 region;
• a BIP primer comprising sequence complementary to that of the B2c region at the 3′ terminal side and sequence identical to that of the B1c region at the 5′ terminal side;
• a B3 primer comprising sequence complementary to that of the B3c region;
• and at least one of a loop primer LFc comprising sequence complementary to that of the LF region and a loop primer LBc comprising sequence identical to that of the LBc region;
• and
• wherein the primers are designed such that the F2 region does not overlap the single-nucleotide mutation in the FP region and/or the B2c region does not overlap the single-nucleotide mutation in the BPc region;
• and, the LF region does not overlap the single-nucleotide mutation in the FP region and/or the LBc region does not overlap the single-nucleotide mutation in the BPc region.

A method of detecting a single-nucleotide mutation, comprising:

• a step of obtaining an amplification product by amplifying a target nucleic acid having a single-nucleotide mutation with primers;
• and
• a step of detecting the amplification product by hybridizing it with probe nucleic acid, wherein:
• the target nucleic acid comprises F3, F2 and F1 regions in this order from a 5′ terminal side;
• B3c, B2c and B1c regions in this order from a 3′ terminal side;
• an FP region having the single-nucleotide mutation in the region from the F2 to F1 regions and/or a BPc region having the single-nucleotide mutation in the region from the B2c to B1c regions;
• and further comprises at least one of an LF region in the region from the F2 to F1 regions and an LBc region in the region from the B2c to B1c regions;
• the probe nucleic acid comprises nucleotide sequence complementary to a sequence selected from the group consisting of sequence in the FP and BPc region, and sequences complementary thereto;
• and
• the primers include an FIP primer comprising sequence identical to that of the F2 region at the 3′ terminal side and sequence complementary to that of the F1 region at the 5′ terminal side;
• an F3 primer comprising sequence identical to the F3 region;
• a BIP primer comprising sequence complementary to that of the B2c region at the 3′ terminal side and sequence identical to that of the B1c region at the 5′ terminal side;
• a B3 primer comprising sequence complementary to that of the B3c region;
• and at least one of a loop primer LFc comprising sequence complementary to that of the LF region and a loop primer LBc comprising sequence identical to that in the LBc region;
• and
• the F2 region does not overlap the single-nucleotide mutation in the FP region and/or the B2c region does not overlap the single-nucleotide mutation in the BPc region and the LF region does not overlap the single-nucleotide mutation in the FP region and/or the LBc region does not overlap the single-nucleotide mutation in the BPc region.

A method for detecting a target nucleic acid sequence comprising:

• amplifying a target nucleic acid using primers, and
• detecting the amplification product by hybridizing it with a probe nucleic acid;
• wherein:
• the target nucleic acid comprises in order F3, F2 and F1 regions from a 5′ terminal side;
• in order B3c, B2c and B1c regions from a 3′ terminal side;
• an FP region in the region from the F2 to F1 regions and/or a BPc region in the region from the B2c to B1c regions;
• and further comprises at least one of an LF region in the region from F2 to F1 regions and an LBc region in the region from the B2c to B1c regions;
• the primers include an FIP primer comprising sequence identical to that of the F2 region at the 3′ terminal side and sequence complementary to that of the F1 region at the 5′ terminal side;
• an F3 primer comprising sequence identical to that of the F3 region;
• a BIP primer comprising sequence complementary to that of the B2c region at the 3′ terminal side and sequence identical to that of the B1c region at the 5′ terminal side;
• a B3 primer comprising sequence complementary to that of the B3c region;
• and at least one of a loop primer LFc comprising sequence complementary to that of the LF region and a loop primer LBc comprising sequence identical to that of the LBc region;
• and wherein said primers are designed in such a manner that the FP and F2 regions and/or the BPc and B2c regions have a non-overlapping region of at least 10 bases or more and overlapping regions of 10 bases or less and that the FP and LF regions and/or the BPc and LBc regions have a non-overlapping region of at least 10 bases or more and overlapping regions of 10 bases or less;
• and
• the probe nucleic acid comprises a nucleotide sequence complementary to sequence selected from the group consisting of sequence in the FP and BPc region and sequences complementary thereto.