Analysis kit, analyzer, and methods for analyzing template nucleic acid or target substance

A method for analyzing a template nucleic acid, comprising:

• fractionating a sample comprising a template nucleic acid into a plurality of template nucleic acid fractions;
• amplifying a target sequence and its complementary sequence in the template nucleic acid with respect to each of the plurality of template nucleic acid fractions in the presence of a nucleic acid amplification reagent;
• detecting generation or quenching of a signal that shows an amplification of the target sequence or the complementary sequence with respect to each of the plurality of template nucleic acid fractions after the amplification;
• and
• discriminating a template nucleic acid fraction in which the generation or quenching of the signal that shows the amplification has been detected among the plurality of template nucleic acid fractions as an amplified fraction in which the target sequence or the complementary sequence has been amplified,
• wherein the nucleic acid amplification reagent comprises:
• a primer set that amplifies the target sequence and the complementary sequence;
• and
• a signal generating substance that generates or quenches the signal in response to the amplification,
• wherein the signal generating substance is selected from the group consisting of a substance that generates the signal in a state where it is bound sequence-dependently and quenches the signal in a state where it is not bound;
• and a substance that quenches the signal in the state where it is bound sequence-dependently and generates the signal in the state where it is not bound,
• generating and quenching of the signal by the signal generating substance are reversible;
• the signal generating substance includes a fluorogenic probe, which comprises at least two fluorescent atomic groups per molecule that exhibit an exciton effect,
• the fluorogenic probe is selected from the group consisting of a probe that generates the signal in a state where it is bound to the target and quenches the signal in a state where it is dissociated from the target and a probe that quenches the signal in the state where it is bound to the target and generates the signal in the state where it is dissociated from the target,
• the primer set optionally comprises a fluorogenic primer, which comprises at least two fluorescent atomic groups per molecule that exhibit an exciton effect,
• the fluorogenic primer is selected from the group consisting of a primer that generates the signal in the state where it is bound to the target and quenches the signal in the state where it is dissociated from the target and a primer that quenches the signal in the state where it is bound to the target and generates the signal in the state where it is dissociated from the target,
• the at least two fluorescent atomic groups that exhibit the exciton effect in the fluorogenic probe or the primer are included in a base,
• the base comprises a structure represented by the following formulae (16), (16b), (17), or (17b):
• wherein in the formulae (16), (16b), (17), and (17b),
• B is an atomic group having a natural nucleobase (adenine, guanine, cytosine, thymine, or uracil) skeleton or an artificial nucleobase skeleton,
• E is:
• (i) an atomic group having a deoxyribose skeleton, a ribose skeleton, or a structure derived from either one of them, or
• (ii) an atomic group having a peptide structure or a peptoid structure,
• Z11 and Z12 are each an atomic group exhibiting fluorescence, and may be identical to or different from each other,
• L1, L2, and L3 are each a linker (a linking atom or an atomic group), the main chain length (the number of main chain atoms) thereof is arbitrary, L1, L2, and L3 each may or may not contain each of C, N, O, S, P, and Si in the main chain, L1, L2, and L3 each may or may not contain each of a single bond, a double bond, a triple bond, an amide bond, an ester bond, a disulfide bond, an imino group, an ether bond, a thioether bond, and a thioester bond in the main chain, and L1, L2, and L3 may be identical to or different from each other,
• D is CR, N, P, P═O, B, or SiR, where R is a hydrogen atom, an alkyl group, or an arbitrary substituent, and
• b is a single bond, a double bond, or a triple bond, or alternatively,
• in the formulae (16) and (16b), L1 and L2 are each a linker, L3, D, and b may not be present, and L1 and L2 may be bonded directly to B, provided that:
• in the formulae (16), and (17), E is an atomic group described in the item (i), and at least one 0 atom in a phosphoric acid linkage may be substituted with an S atom;
• in the formulae (16b), and (17b), E is an atomic group described in the item (ii);
• and
• in the formulae (17) and (17b), the respective Bs may be identical to or different from each other, and the respective Es may be identical to or different from each other.