Nucleic acid determination employing pyryilium dye

A kit for quantifying a target nucleic acid in a sample, comprising:

• a reactor containing a reaction chamber for PCR, in which a required amount of a dye compound represented by the following formula is provided, ##STR92## wherein ##STR93## is (i) a pyrylium ring or (ii) a pyrylium-analog ring of 5 or 6 members where X is O, S, Se, or Te;
• R.sup.1 and R.sup.2 are independently a hydrogen atom, a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted lower aralkyl group, or a substituted or unsubstituted cycloalkyl group;
• R.sup.3 is a group of --A or --L--A wherein L is L--L.sup.1 --, --L.sup.2 --L.sup.3 --, or --L.sup.4 --L.sup.5 --L.sup.6 --, L.sup.1 to L.sup.6 being independently --(CH.dbd.CH )--, a bivalent group derived from a substituted or unsubstituted aryl group, a substituted or unsubstituted alkylene group, or --CH.dbd.R.sup.4 --, wherein R.sup.4 is a cyclic structure having an oxo group;
• A is a substituted or unsubstituted aryl group, or --CH.dbd.R.sup.5, wherein R.sup.5 is a substituted or unsubstituted heterocycle, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted aromatic ring;
• in the pyrylium ring or a pyrylium-analogous ring containing X, the hydrogen atom bonded to the carbon atom which is not linked to R.sup.1, R.sup.2, or R.sup.3 may be substituted by a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted lower aralkyl group;
• and
• Y.sup.- is an anion.

A kit for quantifying a target nucleic acid, comprising

• a reaction chamber for PCR;
• and
• a reagent chamber,
• wherein the reagent chamber contains a dye compound represented by the following formula, and the reagent chamber is provided so that the dye compound in the reagent chamber is transferred to the reaction chamber, ##STR94## wherein ##STR95## is (i) a pyrylium ring or (ii) a pyrylium-analog ring of 5 or 6 members where X is O, S, Se, or Te;
• R.sup.1 and R.sup.2 are independently a hydrogen atom, a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted lower aralkyl group, or a substituted or unsubstituted cycloalkyl;
• R.sup.3 is a group of --A or --L--A wherein L is --L.sup.1 --, --L.sup.2 --L.sup.3 --, or --L.sup.4 --L.sup.5 --L.sup.6 --, L.sup.1 to L.sup.6 being independently --(CH.dbd.CH)--, a bivalent group derived from a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, a substitute or unsubstituted lower aralkyl group, or a substituted or unsubstituted cycloalkyl group;
• R.sup.3 is a group of --A or --L--A wherein L is --L.sup.1 --, L.sup.2 --L.sup.3 --, or --L.sup.4 --L.sup.5 --L.sup.6 --, L.sup.1 to L.sup.6 being independently --(CH.dbd.CH)--, a bivalent group derived from a substituted or unsubstituted aryl group, a substituted or unsubstituted alkylene group, or --CH.dbd.R.sup.4 --, wherein R.sup.4 is a cyclic structure having an oxo group;
• A is a substituted or unsubstituted aryl group, or --CH.dbd.R.sup.5, wherein R.sup.5 is a substituted or unsubstituted heterocycle, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted aromatic ring;
• in the pyrylium ring or a pyrylium-analogous ring containing X, the hydrogen atom bonded to the carbon atom which is not linked to R.sup.1, R.sup.2, or R.sup.3 may be substituted by a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted lower aralkyl group;
• and
• Y.sup.- is an anion.

A method for measuring a number of a target microorganism or target cells, a number of a specific gene, or a copy number of a specific gene, comprising the steps of:

• extracting nucleic acid from a sample containing a microorganism or cells to be detected;
• providing a serial dilution of the extracted nucleic acid;
• conducting PCR with respect to each of the sample dilutions containing extracted nucleic acid with a primer set required for amplificatin of a specific base sequence region of the extracted nucleic acid to provide double-stranded nucleic acid;
• reacting the resulting double-stranded nucleic acid with a dye compound represented by the following formula to bind the dye compound to the double-stranded nucleic acid;
• and
• measuring intensity of the fluorescence from the dye compound inserted into the double-stranded nucleic acid;
• and
• deriving the number of the microorganism or cells,-the specified genes, or copies of the specified genes in the sample on the basis of the dilution rate at which fluorescence is observed, ##STR96## wherein ##STR97## is (i) a pyrylium ring or (ii) a pyrylium-analog ring of 5 or 6 members where X is 0, S, Se, or Te;
• R.sup.1 and R.sup.2 are independently a hydrogen atom, a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted lower aralkyl group, or a substituted or unsubstituted cycloalkyl;
• R.sup.3 is a group of --A or --L--A wherein L is --L.sup.1--, L.sup.2 --L.sup.3 --, or --L.sup.4 --L.sup.5 --L.sup.6 --L.sup.1 to L.sup.6 being independently --(CH.dbd.CH)--, a bivalent group derived from a substituted or unsubstituted aryl group, a substituted or unsubstituted alkylene group, or --CH.dbd.R.sup.4 --, wherein R.sup.4 is a cyclic structure having an oxo group;
• A is a substituted or unsubstituted aryl group, or --CH.dbd.R.sup.5, wherein R.sup.5 is a substituted or unsubstituted heterocycle, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted aromatic ring;
• in the pyrylium ring or a pyrylium-analogous ring containing X, the hydrogen atom bonded to the carbon atom which is not linked to R.sup.1, R.sup.2, or R.sup.3 may be substituted by a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted lower aralkyl group;
• and
• Y.sup.- is an anion.

A kit for measuring a number of a target microorganism or target cells, a number of a specific gene, or a copy number of the specific gene, comprising:

• a reactor comprising a plurality of reaction chambers for serially diluting a nucleic acid extracted from a sample containing microorganisms or cells to be detected, each of the reaction chambers containing a dye compound represented by the following formula, wherein the reaction chambers are employed for the PCR to amplify a specific sequence of the extracted nucleic acid which is peculiar to the microorganisms or cells, ##STR98## wherein ##STR99## is (i) a pyrylium ring or (ii) a pyrylium-analog ring of 5 or 6 members where X is O, S, Se, or Te;
• R.sup.1 and R.sup.2 are independently a hydrogen atom, a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted lower aralkyl group, or a substituted or unsubstituted cycloalkyl;
• R.sup.3 is a group of --A or --L--A wherein L is --L.sup.1 --, L.sup.2 --L.sup.3 --, or --L.sup.4 --L.sup.5 --L.sup.6 --, L.sup.1 to L.sup.6 being independently --(CH.dbd.CH)--, a bivalent group derived from a substituted or unsubstituted aryl group, a substituted or unsubstituted alkylene group, or --CH.dbd.R.sup.4 --, wherein R.sup.4 is a cyclic structure having an oxo group;
• A is a substituted or unsubstituted aryl group, or --CH.dbd.R.sup.5, wherein R.sup.5 is a substituted or unsubstituted heterocycle, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted aromatic ring;
• in the pyrylium ring or a pyrylium-analogous ring containing X, the hydrogen atom bonded to the carbon atom which is not linked to R.sup.1, R.sup.2, or R.sup.3 may be substituted by a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted lower aralkyl group;
• and
• Y.sup.- is an anion.

A method for quantifying a target nucleic acid in a sample, comprising the steps of:

• subjecting a sample containing a nucleic acid to be detected to MPN-PCR with a primer set required for amplification of a specific base sequence region of the target nucleic acid to provide a double-stranded nucleic acid;
• reacting the double-stranded nucleic acid with a dye compound represented by the following formula to bind the dye compound to the double-stranded nucleic acid;
• and
• measuring intensity of the fluorescence of the dye compound inserted into the double-stranded nucleic acid to quantify the target nucleic acid in the sample, ##STR100## wherein ##STR101## is (i) a pyrylium ring or (ii) a pyrylium-analog ring of 5 or 6 members where X is O, S, Se, or Te;
• R.sup.1 and R.sup.2 are independently a hydrogen atom, a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted lower aralkyl group, or a substituted or unsubstituted cycloalkyl;
• R.sup.3 is a group of --A or --L--A wherein L is --L.sup.1 --, L.sup.2 --L.sup.3 --, or --L.sup.4 --L.sup.5 --L.sup.6 --, L.sup.1 to L.sup.6 being independently --(CH.dbd.CH)--, a bivalent group derived from a substituted or unsubstituted aryl group, a substituted or unsubstituted alkylene group, or --CH.dbd.R.sup.4 --, wherein R.sup.4 is a cyclic structure having an oxo group);
• A is a substituted or unsubstituted aryl group, or --CH.dbd.R.sup.5, wherein R.sup.5 is a substituted or unsubstituted heterocycle, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted aromatic ring;
• in the pyrylium ring or a pyrylium-analogous ring containing X, the hydrogen atom bonded to the carbon atom which is not linked to R.sup.1, R.sup.2, or R.sup.3 may be substituted by a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted lower aralkyl group;
• and
• Y.sup.- is an anion.

A kit for quantifying a target nucleic acid in a sample, comprising:

• a reactor comprising reaction chambers for MPN-PCR, wherein in each of the reaction chambers a required amount of a dye compound represented by the following formula is contained, ##STR102## wherein ##STR103## is (i) a pyrylium ring or (ii) a pyrylium-analog ring of 5 or 6 members where X is O, S, Se, or Te;
• R.sup.1 and R.sup.2 are independently a hydrogen atom, a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted lower aralkyl group, or a substituted or unsubstituted cycloalkyl;
• R.sup.3 is a group of --A or --L--A wherein L is --L.sup.1 --, --L.sup.2 --L.sup.3 --, or --L.sup.4 --L.sup.5 --L.sup.6 --, L.sup.1 to L.sup.6 being independently --(CH.dbd.CH)--, a bivalent group derived from a substituted or unsubstituted aryl group, a substituted or unsubstituted alkylene group, or --CH.dbd.R.sup.4 --, wherein R.sup.4 is a cyclic structure having an oxo group;
• A is a substituted or unsubstituted aryl group, or --CH.dbd.R.sup.5, wherein R.sup.5 is a substituted or unsubstituted heterocycle, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted aromatic ring;
• in the pyrylium ring or a pyrylium-analogous ring containing X, the hydrogen atom bonded to the carbon atom which is not linked to R.sup.1, R.sup.2, or R.sup.3 may be substituted by a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or substituted lower alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted lower aralkyl group;
• and
• Y.sup.- is an anion.

A kit for quantifying a target nucleic acid in a sample comprising

• a reaction chamber for MPN-PCR and a reagent chamber, wherein
• the reagent chamber contains a required amount of a dye compound represented by the following formula, and the reagent chamber is provided so that the dye compound in the reagent chamber is transferred to the reaction chamber, ##STR104## wherein ##STR105## is (i) a pyrylium ring or (ii) a pyrylium-analog ring of 5 or 6 members where X is O, S, Se, or Te;
• R.sup.1 and R.sup.2 are independently a hydrogen atom, a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted lower aralkyl group, or a substituted or unsubstituted cycloalkyl;
• R.sup.3 is a group of --A or --L--A wherein L is --L.sup.1 --, --L.sup.2 --L.sup.3 --, or --L.sup.4 --L.sup.5 --L.sup.6 --, L.sup.1 to L.sup.6 being independently --(CH.dbd.CH)--, a bivalent group derived from a substituted or unsubstituted aryl group, a substituted or unsubstituted alkylene group, or --CH.dbd.R.sup.4 --, wherein R.sup.4 is a cyclic structure having an oxo group;
• A is a substituted or unsubstituted aryl group, or --CH.dbd.R.sup.5, wherein R.sup.5 is a substituted or unsubstituted heterocycle, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted aromatic ring;
• in the pyrylium ring or a pyrylium-analogous ring containing X, the hydrogen atom bonded to the carbon atom which is not linked to R.sup.1, R.sup.2, or R.sup.3 may be substituted by a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted lower aralkyl group;
• and
• Y.sup.- is an anion.

A method for measuring a number of a target microorganism or target cells, a number of a specific gene, or a copy number of a specific gene, comprising the steps of:

• extracting nucleic acid from a sample containing a microorganism or cells to be detected;
• providing a serial dilution of the extracted nucleic acid;
• conducting MPN-PCR with respect to each of the diluted samples containing extracted nucleic acid with a primer set required for amplification of a specific base sequence region of the extracted nucleic acid to provide a double-stranded nucleic acid;
• reacting the double-stranded nucleic acid with a dye compound represented by the following formula to bind the dye compound to the double-stranded nucleic acid;
• measuring intensity of the fluorescence from the dye compound inserted into the double-stranded nucleic acid;
• and
• deriving the number of the microorganisms or cells, the specified genes, or copies of the specified genes in the sample on the basis of the dilution rate at which fluorescence is still observed, ##STR106## wherein ##STR107## is (i) a pyrylium ring or (ii) a pyrylium-analog ring of 5 or 6 members where X is O, S, Se, or Te;
• R.sup.1 and R.sup.2 are independently a hydrogen atom, a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted lower aralkyl group, or a substituted or unsubstituted cycloalkyl;
• R.sup.3 is a group of --A or --L--A wherein L is --L.sup.1 --, L.sup.2 --L.sup.3 --, or L.sup.4 --L.sup.5 --L.sup.6 --, L.sup.1 to L.sup.6 being independently --(CH.dbd.CH)--, a bivalent group derived from a substituted or unsubstituted aryl group, a substituted or unsubstituted alkylene group, or --CH=R.sup.4--, wherein R.sup.4 is a cyclic structure having an oxo group;
• A is a substituted or unsubstituted aryl group, or --CH.dbd.R.sup.5, wherein R.sup.5 is a substituted or unsubstituted heterocycle, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted aromatic ring;
• in the pyrylium ring or a pyrylium-analogous ring containing X, the hydrogen atom bonded to the carbon atom which is not linked to R.sup.1, R.sup.2, or R.sup.3 may be substituted by a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted lower aralkyl group;
• and
• Y.sup.- is an anion.

A kit for measuring a number of a target microorganism or target cells, a number of a specific genes, or a copy number of the specific gene, comprising

• a reactor comprising a plurality of reaction chambers for serially diluting a nucleic acid extracted from a sample containing a microorganism or cells to be detected, each of the reaction chambers containing a dye compound represented by the following formula, wherein the reaction chambers are employed for the MPN-PCR to amplify a specific sequence of the extracted nucleic acid which is peculiar to the microorganisms of cells, ##STR108## wherein ##STR109## is (i) a pyrylium ring or (ii) a pyrylium-analog ring of 5 or 6 members where X is O, S, Se, or Te;
• R.sup.1 and R.sup.2 are independently a hydrogen atom, a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted lower aralkyl group, or a substituted or unsubstituted cycloalkyl;
• R.sup.3 is a group of --A or --L--A wherein L is --L.sup.1 --, L.sup.2 --L.sup.3 --, or --L.sup.4 --L.sup.5 --L.sup.6 --, L.sup.1 to L.sup.6 being independently --(CH.dbd.CH)--, a bivalent group derived from a substituted or unsubstituted aryl group, a substituted or unsubstituted alkylene group, or --CH.dbd.R.sup.4 --, wherein R.sup.4 is a cyclic structure having an oxo group;
• A is a substituted or unsubstituted aryl group, or --CH.dbd.R.sup.5, wherein R.sup.5 is a substituted or unsubstituted heterocycle, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted aromatic ring;
• in the pyrylium ring or a pyrylium-analogous ring containing X, the hydrogen atom bonded to the carbon atom which is not linked to R.sup.1, R.sup.2, or R.sup.3 may be substituted by a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted lower aralkyl group;
• and
• Y.sup.- is an anion.

A method for quantifying a target nucleic acid, comprising the steps of:

• conducting PCR of a nucleic acid sample with a primer set required for amplification of a specific base sequence region of the target nucleic acid to provide a double-stranded nucleic acid in the presence of a dye compound represented by the following formula;
• and
• measuring intensity of the fluorescence from the dye compound to quantitatively determine the target nucleic acid in the sample, ##STR110## wherein ##STR111## is (i) a pyrylium ring or (ii) a pyrylium-analog ring of 5 or 6 members where X is O, S, Se, or Te;
• R.sup.1 and R.sup.2 are independently a hydrogen atom, a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted lower aralkyl group, or a substituted or unsubstituted cycloalkyl;
• R.sup.3 is a group of --A or --L--A wherein L is --L.sup.1 --, L.sup.2 --L.sup.3 --, or --L.sup.4 --L.sup.5 --L.sup.6 --, L.sup.1 to L.sup.6 being independently --(CH.dbd.CH)--, a bivalent group derived from a substituted or unsubstituted aryl group, a substituted or unsubstituted alkylene group, or --CH.dbd.R.sup.4 --, wherein R.sup.4 is a cyclic structure having an oxo group;
• A is a substituted or unsubstituted aryl group, or --CH.dbd.R.sup.5 wherein R.sup.5 is a substituted or unsubstituted heterocycle, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted aromatic ring;
• in the pyrylium ring or a pyrylium-analogous ring containing X, the hydrogen atom bonded to the carbon atom which is not linked to R.sup.1, R.sup.2, or R.sup.3 may be substituted by a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted lower aralkyl group;
• and
• Y.sup.- is an anion.

A method for quantifying a target nucleic acid, comprising the steps of:

• conducting PCR of a nucleic acid sample with a primer set required for amplification of a specific base sequence region of the target nucleic acid to provide a PCR amplified product containing a double-stranded nucleic acid;
• adding a dye compound represented by the following formula to the PCR-amplified product;
• and
• measuring intensity of the fluorescence from the dye compound in the PCR-amplified product to quantitatively determine the target nucleic acid in the sample, ##STR112## wherein ##STR113## is (i) a pyrylium ring or (ii) a pyrylium-analog ring of 5 or 6 members where X is O, S, Se, or Te;
• R.sup.1 and R.sup.2 are independently a hydrogen atom, a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted lower aralkyl group, or a substituted or unsubstituted cycloalkyl;
• R.sup.3 is a group of --A or --L--A wherein L is --L.sup.1 --, L.sup.2 --L.sup.3 --, or --L.sup.4 --L.sup.5 --L.sup.6 --, L.sup.1 to L.sup.6 being independently --(CH.dbd.CH)--, a bivalent group derived from a substituted or unsubstituted aryl group, a substituted or unsubstituted alkylene group, or --CH.dbd.R.sup.4 --, wherein R.sup.4 is a cyclic structure having an oxo group;
• A is a substituted or unsubstituted aryl group, or --CH.dbd.R.sup.5, wherein R.sup.5 is a substituted or unsubstituted heterocycle, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted aromatic ring;
• in the pyrylium ring or a pyrylium-analogous ring containing X, the hydrogen atom bonded to the carbon atom which is not linked to R.sup.1, R.sup.2, or R.sup.3 may be substituted by a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted lower aralkyl group;
• and
• Y.sup.- is an anion.