JP2002142780A - Method for assaying enzyme involved in the first phase reaction of drug metabolism, probe and kit therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new method for assaying an enzyme involved in the first phase reaction of drug metabolism by PCR, especially establishing a real time detection method, and a probe and a primer pair therefor. SOLUTION: The method for assaying the enzyme comprises a probe containing an oligonucleotide hybridizing to a specific region of the enzyme involved in the first phase reaction of drug metabolism, a kit for assaying the enzyme containing combination of the probe and the specific primer pair, performing a polymerase chain reaction using the kit, and measuring presence and absence of hydrolysis reaction of the used probe.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring an enzyme involved in a phase I reaction of drug metabolism, and a probe and a kit therefor.

[0002]

2. Description of the Related Art Drug metabolism reactions in humans can be roughly classified into phase I oxidation, reduction and hydrolysis reactions and phase II conjugation reactions.
Enzymes (drug-metabolizin) involved in this drug metabolism reaction
g enzymes) are known to account for about 10% of about 1500 known enzymes.

[0003] In particular, as the enzymes involved in the phase I reaction of drug metabolism, for example, each enzyme (family) shown in Table 1 below and their molecular species are known.

[0004]

【table 1】

[0005] In developing a new drug, it is particularly important to understand the function (increase / decrease in expression level) of an enzyme involved in the phase I reaction of drug metabolism when the new drug is administered. Because if the fluctuation of the enzymes involved in this phase I reaction is not known, the efficacy and side effect of the concomitant drug used in combination with the new drug or the efficacy of the new drug due to the concomitant drug and other ingestion, the increase and decrease of the side effect, etc. This is because it cannot be predicted and the safety of the new drug cannot be confirmed.

Accordingly, in fields such as new drug development,
There is a need for the development of information relating to enzymes involved in the phase I reaction of drug metabolism, in particular, a measurement technique capable of separately measuring the enzymes and their respective molecular species. If such a technology can be developed, for example, it is possible to easily understand the interaction of the new drug with other drugs, the effects during a special disease state, and obtain useful information on the side effects of the new drug, thus ensuring the safety of the new drug. can do.

On the other hand, polymerase chain reaction
(PCR) is widely known as a method for amplifying a nucleic acid, and, for example, RT-PCR (Reverse Transcription-PCR), competitive RT-PCR, and the like are exerting their power in detecting and quantifying a small amount of mRNA.

In recent years, a real-time quantitative detection method using PCR has been established (Taq Man PCR (Genome Res., 6 (10),
986 (1996)), ABI PRISM TM Sequence Detection Syste
m (Applied Biosystems)). This detection method is a method of measuring a nucleic acid using a specific fluorescently labeled probe (TaqMan probe), and its principle is as follows. That is, for example, a probe labeled with a reporter dye at the 5 ′ end and a quencher dye at the 3 ′ end and fluorescently labeled is used as the target DNA.
When a normal PCR reaction is carried out in the state of annealing, the probe is hydrolyzed from the 5 ′ end by the 5′-3 ′ exonuclease activity of the DNA polymerase as the extension reaction proceeds. As a result, the reporter dye at the 5 'end was separated from the quencher dye at the 3' end, thereby maintaining a certain spatial distance initially.
The FRET (Fluoresence Resonance Energy Transfer, a phenomenon of a decrease in the fluorescence intensity based on a decrease in the energy order of the reporter dye due to the resonance of both fluorescent dyes) disappears, and the fluorescence intensity of the reporter dye controlled by the quencher dye increases. By measuring the increase in the fluorescence intensity,
The target nucleic acid can be selectively quantitatively detected in real time.

[0009] This method has an advantage that a complicated process such as analyzing an electrophoresis pattern after a PCR reaction by, for example, amplifying an agarose gel and analyzing an electrophoresis pattern is not required, and it is possible to quantify various kinds of samples in a short time and simultaneously. .

In general, when conducting a clinical test at a clinical test center or the like, it is necessary to test a very large number of samples within a limited time, and there is a demand for the development of a method capable of performing the test efficiently. The real-time detection method described above can be expected to meet this demand.

The present inventors have paid attention to the real-time detection method using PCR, and have conceived its application to the detection of an enzyme involved in the phase I reaction of drug metabolism. In other words, it was thought that such an application would allow simultaneous testing of a large number of specimens under the same PCR conditions using the same apparatus, and that this would allow the measurement and detection of the molecular species of each enzyme as well.

However, currently known drug metabolism
As described above, the enzymes involved in the phase I reaction are extremely numerous, and although the sequences of the respective mRNAs are known, they have no target sequence and do not have overlapping sequence portions and are identical. It was considered that it was difficult to search for an oligonucleotide as each primer pair that could be sufficiently amplified under PCR conditions and that could hybridize to a specific position of the target gene.

[0013] In addition, the primer can hybridize to a specific region sandwiched by such a primer pair faster than these primers under the same PCR conditions, and is specific for each enzyme involved in the phase I reaction of drug metabolism. Construction of a suitable probe was also considered difficult.

In particular, the ease of hybridization between two nucleic acids whose nucleotide sequences are known can be estimated to some extent by calculating the melting point (Tm), but the combination of the primer and probe selected based on this estimation is It is known that good results are not necessarily obtained in DNA measurement, and it is possible to simultaneously distinguish and measure various enzymes involved in the currently known phase I reaction of drug metabolism by the above-mentioned PCR. It was expected that selection of a combination of each primer pair and probe for real-time detection would require a great deal of experimentation by trial and error of a skilled person.

[0015]

SUMMARY OF THE INVENTION An object of the present invention is to establish a method for measuring enzymes involved in the phase I reaction of drug metabolism by PCR, in particular, to establish a real-time detection method, and to provide a probe and primer pair therefor. It is in.

[0016]

Means for Solving the Problems The present inventor repeatedly conducted extensive experiments and studies on various enzymes involved in the phase I reaction of drug metabolism for the above purpose, and as a result, 39 of the above enzymes were identified. The present invention succeeded in providing a target primer pair and a probe combination for the present invention, and thereby completed the present invention.

The present invention relates to a probe used for measuring an enzyme involved in a phase I reaction of drug metabolism.
A probe selected from the group consisting of oligonucleotides that hybridize to any of the regions shown in (53);
In particular, the probe in which a reporter dye and a quencher dye are bound; the probe having a base sequence length in the range of 20 to 40; the probe including any of the sequences shown in SEQ ID NOS: 1 to 53 ; And SEQ ID NOS: 1 to 5
3. The probe is provided which is any of the sequences shown in 3. (1) 222-248 region of EPHX1 gene (2) 174-203 region of EPHX2 gene (3) 767-796 region of PIG3 gene (4) 50-77 region of NMOR2 gene (5) 853-881 region of MAOA gene (6) MAOB gene 1491-1519 region (7) PTGS1 gene 1088-1115 region (8) PTGS2 gene 830-855 region (9) DPYD gene 2345-2370 region (10) AOX1 gene 3500-3527 region (11) XDH gene 3504-3533 region (12) CEL gene 468-493 region (13) CES1 gene 976-1005 region (14) CES2 gene 328-354 region (15) AADAC gene 86-113 region (16) GZMA gene 326-353 region (17) GZMB gene 276-301 region (18) IL17 gene 99-126 region (19) LIPA gene 253-282 region (20) NTE gene 3670-3697 region (21) 255-280 region of UCHL1 gene (22) 105-134 region of UCHL3 gene (23) 728-755 region of FMO1 gene (24) 727-750 region of FMO2 gene (25) 1117-1143 region of FMO2 gene (26) FMO3 gene 735-763 region (27) FMO4 gene 1326-1351 region (28) FMO5 gene 1111-1138 region (29) ALDH1 gene 381-407 region (3 0) ALDH2 gene 1204-1228 region (31) ALDH3 gene 461-488 region (32) ALDH3 gene 1094-1121 region (33) ALDH4 gene 823-848 region (34) ALDH5 gene 1212-1236 region ( (35) ALDH6 gene 1413-1439 region (36) ALDH7 gene 790-816 region (37) ALDH8 gene 95-122 region (38) ALDH9 gene 481-504 region (39) ALDH10 gene 563-588 region ( 40) 168-197 region of ADH1 gene (41) 298-333 region of ADH1 gene (42) 144-173 region of ADH2 gene (43) 438-467 region of ADH2 gene (44) 102-131 region of ADH3 gene ( 45) ADH3 gene 939-966 region (46) ADH4 gene 927-956 region (47) ADH5 gene 322-351 region (48) ADH5 gene 616-641 region (49) ADH6 gene 912-939 region (50) Region 745-772 of the ADH7 gene (51) Region 451-476 of the ESD gene (Genebank accession number M
(52) The region of 483-509 of the HADH2 gene (53) The region of 610-584 of the HEP27 gene Also, the present invention shows any of the following (1) to (53) A kit for measuring an enzyme involved in the phase I reaction of drug metabolism, comprising a combination of a forward primer, a probe and a reverse primer consisting of an oligonucleotide hybridizing to each region to be treated; in particular, the probe further comprises a reporter dye and a quencher dye. The present invention provides the above-mentioned measurement kit which is bound. (1) forward primer; 200-220 region of EPHX1 gene, probe; 222-248 region and reverse primer of the gene; 302-281 region of the gene; (2) forward primer; 145-165 region of EPHX2 gene, probe 174-203 region and reverse primer of the same gene; 237-217 region of the same gene; (3) forward primer; 743-763 region of PIG3 gene;
Probe; 767-796 region and reverse primer of the same gene; 858-838 region of the same gene; (4) forward primer; 26-46 region of the NMOR2 gene;
Probe; 50-77 region and reverse primer of the same gene; 101-80 region of the same gene; (5) forward primer; 827-849 region of the MAOA gene;
Probe; 853-881 region and reverse primer of the same gene; 919-898 region of the same gene; (6) forward primer; 1440-1461 region of the MAOB gene; probe; 1491-1519 region of the same gene and reverse primer; 1555-1535 region of (7) forward primer; 1066-1086 region of PTGS1 gene, probe; 1088-1115 region and reverse primer of the same gene; 1187-1167 region of the same gene; (8) forward primer; PTGS2 gene 792-813 region, probe; 830-855 region and reverse primer of the same gene; 928-908 region of the same gene; (9) forward primer; 2321-2341 region of the DPYD gene, probe; Reverse primer; 2399-2379 region of the same gene; (10) forward primer; AOX1 gene 3464-3484 region; probe; 3500-3527 region of the same gene and reverse Limer; 3585-3564 region of the same gene; (11) forward primer; 3446-3468 region of the XDH gene; probe; 3504-3533 region of the same gene and reverse primer; 3562-3540 region of the same gene; (12) forward primer 429-451 region of the CEL gene;
Probe; 468-493 region and reverse primer of the same gene; 555-533 region of the same gene; (13) forward primer; 935-956 region of the CES1 gene; probe; 976-1005 region of the same gene and reverse primer; the same gene 1061-1041 region of (14) forward primer; 298-319 region of CES2 gene, probe; 328-354 region and reverse primer of the same gene; 383-362 region of the same gene; (15) forward primer; 63-83 region,
Probe; 86-113 region and reverse primer of the same gene; 206-185 region of the same gene; (16) forward primer; 304-324 region of the GZMA gene; probe; 326-353 region and reverse primer of the same gene; 433-413 region of (17) forward primer; 253-274 region of GZMB gene, probe; 276-301 region and reverse primer of the same gene; 350-328 region of the same gene; (18) forward primer; 68-88 area,
Probe; 99-126 region and reverse primer of the same gene; 163-142 region of the same gene; (19) forward primer; 226-248 region of the LIPA gene; probe; 253-282 region of the same gene and reverse primer; 347-327 region of (20) forward primer; 3634-3655 region of NTE gene; probe; 3670-3697 region of gene and reverse primer; 3747-3725 region of same gene; (21) forward primer; of UCHL1 gene 233-253 region, probe; 255-280 region and reverse primer of the same gene; 306-285 region of the same gene; (22) forward primer; 76-97 region of the UCHL3 gene;
Probe; 105-134 region and reverse primer of the same gene; 164-140 region of the same gene; (23) forward primer; 696-716 region of the FMO1 gene; probe; 728-755 region of the same gene and reverse primer; the same gene 790-769 region of (24) forward primer; 705-725 region of FMO2 gene, probe; 727-750 region of reverse gene and reverse primer; 826-806 region of same gene; (25) forward primer; of FMO2 gene 1077-1097 region, probe; 1117-1143 region and reverse primer of the same gene; 1165-1145 region of the same gene; (26) forward primer; 706-726 region of the FMO3 gene, probe; 735-763 region of the same gene and Reverse primer; 844-823 region of the gene; (27) forward primer; 1297-1317 region of FMO4 gene, probe; 1326-1351 region of the gene and reverse ply -; 1422-1401 region of the gene; (28) forward primer; 1063-1084 region of the FMO5 gene, probe; 1111-1138 region and reverse primer of the gene; 1162-1141 region of the gene; (29) forward primer Region 359-379 of the ALDH1 gene, probe; region 381-407 of the gene and a reverse primer; region 494-472 of the gene; (30) forward primer; region 1179-1199 of the ALDH2 gene, probe; 1204 of the gene -1228 region and reverse primer; 1307-1285 region of the same gene; (31) forward primer; 422-442 region of the ALDH3 gene; probe; 461-488 region of the same gene and reverse primer; 549-528 region of the same gene; (32) forward primer; 1058-1078 region of the ALDH3 gene, probe; 1094-1121 region and reverse primer of the gene; 1194-1174 region of the gene; (33) forward primer Primer: 768-788 region of the ALDH4 gene, probe; 823-848 region and reverse primer of the gene; 967-947 region of the gene; (34) forward primer; 1183-1206 region of the ALDH5 gene, probe; 1212-1236 region and reverse primer; 1261-1241 region of the same gene; (35) forward primer; 1382-1404 region of ALDH6 gene; probe; 1413-1439 region of the same gene and reverse primer; 1466-1445 region of the same gene (36) forward primer; 743-764 region of the ALDH7 gene, probe; 790-816 region and reverse primer of the same gene; 878-858 region of the same gene; (37) forward primer; 55-75 region of the ALDH8 gene;
Probe; 95-122 region and reverse primer of the same gene; 182-162 region of the same gene; (38) forward primer; 459-479 region of the ALDH9 gene; probe; 481-504 region and reverse primer of the same gene; 534-513 region, (39) forward primer; 525-546 region of the ALDH10 gene, probe; 563-588 region and reverse primer of the gene; 644-623 region of the gene; (40) forward primer; 94-115 region,
Probe; 168-197 region and reverse primer of the same gene; 365-345 region of the same gene; (41) forward primer; 261-281 region of the ADH1 gene; probe; 298-333 region of the same gene and reverse primer; the same gene 365-345 region of (42) forward primer; 94-115 region of ADH2 gene;
Probe; 144-173 region and reverse primer of the same gene; 431-411 region of the same gene; (43) forward primer; 411-431 region of the ADH2 gene; probe; 438-467 region of the same gene and reverse primer; 518-496 region of (44) forward primer; 72-93 region of ADH3 gene;
Probe; 102-131 region and reverse primer of the same gene; 167-144 region of the same gene; (45) forward primer; 743-763 region of the ADH3 gene; probe; 939-966 region of the same gene and reverse primer; 1113-1091 region of (46) forward primer; 903-924 region of ADH4 gene, probe; 927-956 region of the same gene and reverse primer; 984-964 region of the same gene; (47) forward primer; of ADH5 gene 289-310 region, probe; 322-351 region and reverse primer of the same gene; 495-474 region of the same gene; (48) forward primer; 564-585 region of the ADH5 gene, probe; 616-641 region of the same gene Reverse primer; 676-656 region of the same gene. (49) forward primer; region 889-910 of the ADH6 gene, probe; region 912-939 of the gene and a reverse primer; region 987-967 of the gene. (50) forward primer; 722-743 region of the ADH7 gene, probe; 745-772 region and reverse primer of the gene; 802-781 region of the gene. (51) forward primer; 427-447 region of the ESD gene,
Probe; 451-476 region of the gene and reverse primer; 519-497 region of the gene (Genebank accession number)
Based on the sequence of M13450, the same applies hereinafter). (52) Forward primer; 451-471 region of HADH2 gene, probe; 483-509 region and reverse primer of the gene; 539-519 region of the gene. (53) forward primer; 505-526 region of the HEP27 gene, probe; 610-584 region and reverse primer of the gene; 635-615 region of the gene.

Preferred combinations of the forward primer, probe and reverse primer constituting the above-described assay kit include combinations of oligonucleotides containing the sequences represented by the respective SEQ ID NOs described in the following (1) to (53). Preferably, a combination of oligonucleotides having the sequence represented by each SEQ ID NO can be mentioned. (1) forward primer; SEQ ID NO: 54, probe; SEQ ID NO: 1 and reverse primer; SEQ ID NO: 55; (2) forward primer; SEQ ID NO: 56, probe; SEQ ID NO: 2 and reverse primer; SEQ ID NO: 57, (3) forward SEQ ID NO: 58, probe; SEQ ID NO: 3 and reverse primer; SEQ ID NO: 59, (4) forward primer; SEQ ID NO: 60, probe; SEQ ID NO: 4 and reverse primer; SEQ ID NO: 61, (5) forward primer; 62, probe; SEQ ID NO: 5 and reverse primer; SEQ ID NO: 63, (6) forward primer; SEQ ID NO: 64, probe; SEQ ID NO: 6 and reverse primer; SEQ ID NO: 65, (7) forward primer; SEQ ID NO: 66, probe; SEQ ID NO: 7 and reverse primer; SEQ ID NO: 67, (8) forward primer SEQ ID NO: 68, probe; SEQ ID NO: 8 and reverse primer; SEQ ID NO: 69 (9) forward primer; SEQ ID NO: 70, probe; SEQ ID NO: 9 and reverse primer; SEQ ID NO: 71, (10) forward primer; SEQ ID NO: 72, probe SEQ ID NO: 10 and reverse primer; SEQ ID NO: 73, (11) forward primer; SEQ ID NO: 74, probe; SEQ ID NO: 11 and reverse primer; SEQ ID NO: 75, (12) forward primer; SEQ ID NO: 76, probe; SEQ ID NO: 12 SEQ ID NO: 77, (13) forward primer; SEQ ID NO: 78, probe; SEQ ID NO: 13 and reverse primer; SEQ ID NO: 79, (14) forward primer; SEQ ID NO: 80, probe; SEQ ID NO: 14 and reverse primer; SEQ ID NO: 81, (15) forward primer; SEQ ID NO: 82, professional SEQ ID NO: 15 and reverse primer; SEQ ID NO: 83, (16) forward primer; SEQ ID NO: 84, probe; SEQ ID NO: 16 and reverse primer; SEQ ID NO: 85, (17) forward primer; SEQ ID NO: 86, probe; SEQ ID NO: SEQ ID NO: 87, (18) Forward primer; SEQ ID NO: 88, probe; SEQ ID NO: 18 and reverse primer; SEQ ID NO: 89, (19) Forward primer; SEQ ID NO: 90, probe; SEQ ID NO: 19 and reverse primer SEQ ID NO: 91, (20) forward primer; SEQ ID NO: 92, probe; SEQ ID NO: 20 and reverse primer; SEQ ID NO: 93, (21) forward primer; SEQ ID NO: 94, probe; SEQ ID NO: 21 and reverse primer; SEQ ID NO: 95 (22) forward primer; SEQ ID NO: 96, probe; SEQ ID NO: SEQ ID NO: 97, (23) forward primer; SEQ ID NO: 98, probe; SEQ ID NO: 23 and reverse primer; SEQ ID NO: 99, (24) forward primer; SEQ ID NO: 100, probe;
SEQ ID NO: 24 and reverse primer; SEQ ID NO: 101; (25) forward primer; SEQ ID NO: 102, probe;
SEQ ID NO: 25 and reverse primer; SEQ ID NO: 103; (26) forward primer; SEQ ID NO: 104, probe;
SEQ ID NO: 26 and reverse primer; SEQ ID NO: 105; (27) forward primer; SEQ ID NO: 106, probe;
SEQ ID NO: 27 and reverse primer; SEQ ID NO: 107, (28) forward primer; SEQ ID NO: 108, probe;
SEQ ID NO: 28 and reverse primer; SEQ ID NO: 109; (29) forward primer; SEQ ID NO: 110, probe;
SEQ ID NO: 29 and reverse primer; SEQ ID NO: 111; (30) forward primer; SEQ ID NO: 112, probe;
SEQ ID NO: 30 and reverse primer; SEQ ID NO: 113, (31) forward primer; SEQ ID NO: 114, probe;
SEQ ID NO: 31 and reverse primer; SEQ ID NO: 115, (32) forward primer; SEQ ID NO: 116, probe;
SEQ ID NO: 32 and reverse primer; SEQ ID NO: 117, (33) forward primer; SEQ ID NO: 118, probe;
SEQ ID NO: 33 and a reverse primer; SEQ ID NO: 119; (34) forward primer; SEQ ID NO: 120, probe;
SEQ ID NO: 34 and reverse primer; SEQ ID NO: 121, (35) forward primer; SEQ ID NO: 122, probe;
SEQ ID NO: 35 and reverse primer; SEQ ID NO: 123, (36) forward primer; SEQ ID NO: 124, probe;
SEQ ID NO: 36 and reverse primer; SEQ ID NO: 125, (37) forward primer; SEQ ID NO: 126, probe;
SEQ ID NO: 37 and reverse primer; SEQ ID NO: 127, (38) forward primer; SEQ ID NO: 128, probe;
SEQ ID NO: 38 and reverse primer; SEQ ID NO: 129, (39) forward primer; SEQ ID NO: 130, probe;
SEQ ID NO: 39 and reverse primer; SEQ ID NO: 131; (40) forward primer; SEQ ID NO: 132, probe;
SEQ ID NO: 40 and reverse primer; SEQ ID NO: 133, (41) forward primer; SEQ ID NO: 134, probe;
SEQ ID NO: 41 and reverse primer; SEQ ID NO: 135; (42) forward primer; SEQ ID NO: 136, probe;
SEQ ID NO: 42 and reverse primer; SEQ ID NO: 137; (43) forward primer; SEQ ID NO: 138, probe;
SEQ ID NO: 43 and reverse primer; SEQ ID NO: 139, (44) forward primer; SEQ ID NO: 140, probe;
SEQ ID NO: 44 and reverse primer; SEQ ID NO: 141, (45) forward primer; SEQ ID NO: 142, probe;
SEQ ID NO: 45 and reverse primer; SEQ ID NO: 143, (46) forward primer; SEQ ID NO: 144, probe;
SEQ ID NO: 46 and reverse primer; SEQ ID NO: 145; (47) forward primer; SEQ ID NO: 146, probe;
SEQ ID NO: 47 and a reverse primer; SEQ ID NO: 147, (48) forward primer; SEQ ID NO: 148, probe;
SEQ ID NO: 48 and reverse primer; SEQ ID NO: 149; (49) forward primer; SEQ ID NO: 150, probe;
SEQ ID NO: 49 and reverse primer; SEQ ID NO: 151, (50) forward primer; SEQ ID NO: 152, probe;
SEQ ID NO: 50 and reverse primer; SEQ ID NO: 153; (51) forward primer; SEQ ID NO: 154, probe;
SEQ ID NO: 51 and reverse primer; SEQ ID NO: 155; (52) forward primer; SEQ ID NO: 156, probe;
SEQ ID NO: 52 and a reverse primer; SEQ ID NO: 157; (53) forward primer; SEQ ID NO: 158, probe;
SEQ ID NO: 53 and reverse primer; SEQ ID NO: 159.

Each of the above combinations is used for measuring an enzyme involved in the following phase I reaction of drug metabolism. Combination of (1); for EPHX1 measurement (2) combination; for EPHX2 measurement (3) combination; for PIG3 measurement (4) combination; for NMOR2 measurement (5) combination; for MAOA measurement (6) combination For MAOB measurement (7) combination; For PTGS1 measurement (8) combination; For PTGS2 measurement (9) combination; For DPYD measurement (10) combination; For AOX1 measurement (11) combination; For XDH measurement ( Combination of 12); CEL measurement (13) combination; CES1 measurement (14) combination; CES2 measurement (15) combination; AADAC measurement (16) combination; GZMA measurement (17) combination; GZMB measurement combination (18) combination; IL17 measurement combination (19) combination; LIPA measurement combination (20) combination; NTE measurement combination (21) combination; UCHL1 measurement combination (22) combination; UCHL3 measurement combination (23 For FMO1 measurement Combination (24); For FMO2 measurement (25) combination; For FMO2 measurement (26) combination; For FMO3 measurement (27) combination; For FMO4 measurement (28) combination; FMO5 For measurement (29) combination; For ALDH1 measurement (30) combination; For ALDH2 measurement (31) combination Combination of (32) for ALDH3 measurement; Combination of (33) for measurement of ALDH3; Combination of (34) for measurement of ALDH4; Combination of (35) for measurement of ALDH5; Combination of (36) for measurement of ALDH6; Combination of (37); Combination of ALDH8 (38); Combination of ALDH9 (39); Combination of ALDH10 (40); Combination of ADH1 (41); Combination of ADH1 (42) For ADH2 measurement (43) combination; for ADH2 measurement (44) combination; for ADH3 measurement (45) combination; for ADH3 measurement (46) combination; for ADH4 measurement (47) combination; for ADH5 measurement ( 48) combination; for ADH5 measurement (49) combination; for ADH6 measurement (50) combination; for ADH7 measurement (51) combination; for ESD measurement (52) combination; for HADH2 measurement (53) combination; For HEP27 measurement.

In the present invention, a sample containing a gene of an enzyme involved in the phase I reaction of drug metabolism is further subjected to polymerase chain reaction (PCR) using the assay kit of the present invention described above. A method for measuring an enzyme involved in the phase I reaction of drug metabolism, characterized by measuring the presence or absence of hydrolysis of a probe, and in particular, the presence or absence of the hydrolysis is determined by the presence or absence of fluorescence by excitation light irradiation. Provide a measurement method.

[0021] More preferably, the measuring method is such that a primer pair of a forward primer and a reverse primer, and a reporter dye and a quencher dye are bound to each other and hybridized with a template nucleic acid in a region sandwiched between both primers. 5'-3 '
It is performed by a real-time detection method in which a polymerase chain reaction (PCR) is performed by a DNA polymerase having exonuclease activity.

The real-time detection method according to the present invention is capable of separating and quantifying various enzymes easily and quickly in real time under the same PCR or RT-PCR reaction conditions.
Its establishment is very useful in the clinical and pharmaceutical fields.

In other words, according to the method, the I-position of drug metabolism, which is important in kinetic studies in drug development,
The expression of mRNA in a human sample of an enzyme involved in the phase reaction can be easily quantified, and thereby the change of each enzyme involved in the phase I reaction when exposed to a chemical substance such as a pharmaceutical product. You can know. In addition, according to the method, it is possible to process a variety of samples quickly and simultaneously, and it is also possible to use as a sample total RNA extracted from a small amount of tissue pieces, for example, tissues removed by surgery or tissues removed by biopsy This method is suitable as a method for detecting and quantifying the mRNA expression of an enzyme involved in the phase I reaction.

[0024] Furthermore, in the enzymes involved in specific phase I reactions, changes in the expression level due to enzyme induction in the liver, kidney, etc., are caused by changes in the values in blood (cells having nuclei contained in blood). Although there is a possibility of correlation, the expression level in this blood is small, and a large amount of blood is required for measurement.
However, according to the method of the present invention using a specific primer pair and a probe, the expression level in the blood can be easily measured with a small amount of a blood sample using an existing measuring instrument.

In addition, in the enzymes involved in a specific phase I reaction, changes in the expression level due to enzyme induction in the liver, kidney and the like are caused by changes in the value of cells having nuclei contained in secretions such as saliva. Although it is possible to correlate with the change, the expression level in cells having nuclei contained in secretions such as saliva is also small. According to the method of the present invention, the amount of expression in cells having nuclei contained in the secretion fluid can be easily measured with a small sample amount using existing equipment, using the secretion fluid such as saliva as a sample.

Hereinafter, the probe and primer pair used in the method of the present invention will be described in detail.

As described above, each of these probes and primers is selected from oligonucleotides that hybridize to a specific region of a gene of an enzyme involved in the phase I reaction of drug metabolism.

Here, "to hybridize" means to hybridize under the conditions of PCR (RT-PCR) described later.

The common requirements of the probes and primers according to the present invention are that the amplification product is about 50-400 base pairs in length, that the primer and the probe are as close as possible, and that they are included in the sequence. The ratio of G (guanine) and C (cytosine) is about 50% as much as possible, and four or more G (guanine) are not continuous. Other requirements include that the probe of the present invention has a Tm value of about 70 ° C., and the primer of the present invention has a Tm value of about 60 ° C.

M of each enzyme involved in the phase I reaction of drug metabolism
RNA sequences are known, and each gene bank (GenBank)
nk) with the following registration number. (1) EPHX1 gene; NM # 000120 (2) EPHX2 gene; NM # 001979 (3) PIG3 gene; NM # 004881 (4) NMOR2 gene; NM # 000904 (5) MAOA gene; NM # 000240 (6) MAOB gene NM # 000898 (7) PTGS1 gene; M59979 (8) PTGS2 gene; NM # 000963 (9) DPYD gene; NM # 000110 (10) AOX1 gene; NM # 001159 (11) XDH gene; NM # 000379 (12) CEL gene; NM # 001807 (13) CES1 gene; NM # 001266 (14) CES2 gene; NM # 003869 (15) AADAC gene; NM # 001086 (16) GZMA gene; NM # 006144 (17) GZMB gene; NM # 004131 (18) IL17 gene; NM # 002190 (19) LIPA gene; NM # 000235 (20) NTE gene; NM # 006702 (21) UCHL1 gene; NM # 004181 (22) UCHL3 gene; NM # 006002 (23) FMO1 NM # 002021 (24) FMO2 gene; NM # 001460 (25) FMO2 gene; NM # 001460 (26) FMO3 gene; NM # 006894 (27) FMO4 gene; NM # 002022 (28) FMO5 gene; NM # 001461 (29) ALDH1 gene; AF003341 (30) ALDH2 gene; NM # 000690 (31) ALDH3 gene; NM # 000691 (32) ALDH3 gene; NM # 000691 (33) ALDH4 gene; NM # 003748 (34) ALDH5 NM # 000692 (35) ALDH6 gene; NM # 000693 (36) ALDH7 gene; NM # 000694 (37) ALDH8 gene; NM # 000695 (38) ALDH9 gene; NM # 000696 (39) ALDH10 gene; NM # 000382 (40) ADH1 gene; NM # 000667 (41) ADH1 gene; NM # 000667 (42) ADH2 gene; NM # 000668 (43) ADH2 gene; NM # 000668 (44) ADH3 gene; NM # 000669 (45) ADH3 gene NM # 000669 (46) ADH4 gene; NM # 000670 (47) ADH5 gene; NM # 000671 (48) ADH5 gene; NM # 000671 (49) ADH6 gene; NM # 000672 (50) ADH7 gene; NM # 000673 ( 51) ESD gene; M13450 (52) HADH2 gene; NM # 004493 (53) HEP27 gene; NM # 005794.

In the present specification, the designation of the specific region of each enzyme is in accordance with the sequence of the gene registered with each of the above-mentioned registration numbers.

The number of nucleotides of the oligonucleotides for each primer and probe according to the present invention is at least 15, usually 15 to 50, and preferably 20 to 40. If the number of nucleotides of these primers and probes becomes too large, the single-stranded D
It is difficult to hybridize to NA, and if too small, the specificity of hybridization is reduced.

Examples of preferred specific sequences of the specific nucleotide sequences of the primers and the probes are as described above, and according to each molecular species, SEQ ID NOS: 1 to 53 are respectively used.
(For probes) and SEQ ID NOs: 54-159 (for primers).

It is known that a primer or probe consisting of, for example, 20 nucleotides hybridizes even if a small number of mismatches exist with the template strand, and can function as a PCR primer or a detection probe. I have. Accordingly, the primers and probes of the present invention are also not limited to those having the specific nucleotide sequence described above, but include sequences containing the sequence as a part thereof, and, for example, two or less, particularly one nucleotide in this sequence. Replacement,
It may be a sequence modified by deletion and / or addition.

Each oligonucleotide as the probe and the primer of the present invention can be easily synthesized according to a conventional method using an automatic synthesizer, for example, a DNA synthesizer (Perkin Elmer). The oligonucleotide thus obtained can be further purified, if necessary, using a commercially available purification cartridge or the like.

The probe for real-time detection of the present invention comprises:
A reporter dye is attached at one end, eg, the 5′-end, and a quencher dye is attached at the other end, eg, the 3′-end. The reporter dye is a substance that emits fluorescence when irradiated with excitation light, for example, and the quencher is
When present in close proximity to the reporter dye, it can act on the reporter dye to eliminate its fluorescence. Examples of such reporter dyes include, for example, 6-carboxy-fluorescein (F
AM), tetrachloro-6-carboxyfluorescein (TE
T), 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein (JOE), hexochloro-6-carboxyfluorescein (HEX) and the like. Examples of quencher dyes include, for example, 6-carboxy-tetramethyl-rhodamine (TAMRA).

The probe of the present invention can be prepared by binding a reporter dye and a quencher dye to the probe oligonucleotide having the specific sequence. For example, a few methylene chains are usually used as a linker on the 5 'side of the probe, and a FAM molecule can be bound to a terminal phosphate group in the form of a phosphate ester. Further, a TAMRA molecule can be bound to the 3 ′ side by an amide bond via a structural unit shown below.

[0038]

[Formula 1]

Hereinafter, a PCR detection method and a real-time detection method using the primer pair and the probe of the present invention will be described in detail.

The method of the present invention requires the use of the above-described primer pair and probe of the present invention. Other methods are well-known PCR methods (see, for example, Science, 230, 1350 (1985)), and RT-PCR (Genome Res. ., 6 (10), 986 (1996))
Especially real-time detection methods (eg, TaqMan PCR, AB
I PRISMTM 7700 SEQUENCE DETECTION SYSTEM, Applied
Biosystems, Ver1, June 1996).

This method is particularly useful when measuring mRNA as a method for measuring the expression level of each enzyme involved in the phase I reaction of drug metabolism targeted by the present invention. In this case, a living tissue expressing the specific enzyme is collected, total RNA is extracted according to a conventional method, and cDNA complementary thereto is synthesized according to a conventional method. And PCR may be performed. Also, according to the standard method, total RNA
After extraction, RT-PCR can also be directly performed using the primer and the probe of the present invention.

The PCR reaction and the RT-PCR reaction can basically follow a known method. The reaction conditions can also be appropriately determined according to known methods, and are not particularly different from these methods. Specifically, the conditions described in the following examples can be preferably adopted.

The detection by the real-time detection method can also be performed basically according to a conventional method, for example, by irradiating the PCR reaction solution with an argon laser beam and detecting the emitted fluorescence using a CCD camera.

Thus, by carrying out the method of the present invention, each enzyme involved in the intended phase I reaction of drug metabolism can be measured and detected easily and quickly, and separately for each enzyme.

The present invention also provides a kit for performing the above method. This kit comprises the primer pair of the present invention and a probe. The kit of the present invention can further include a known nucleic acid that can be used as a control, and a primer pair and a probe for measuring the nucleic acid.

According to the method of the present invention, the genes of the enzymes involved in the phase I reaction of drug metabolism can be measured and detected separately for each enzyme. In addition, the enzyme can be quantified quickly and accurately. Thus, basic data relating to the interaction of the new drug with other drugs, contraindications, and the like can be efficiently obtained.

[0047]

EXAMPLES Hereinafter, Test Examples and Examples will be given to explain the present invention in more detail.

[0048]

[Test Example 1] Preparation of calibration curve by real-time detection method (1) Test method The real-time one-step RT-PCR method adopted in this test was performed simultaneously in a maximum of 96 different reactions using 96 wells. It can measure up to 96 samples at a time. Further, the RT reaction and the PCR reaction can be performed in one tube.

The principle of this quantification is to use two types of fluorescent dyes adjacent to each other and to determine the wavelength range between the fluorescence wavelength of one dye (reporter dye) and the excitation light wavelength of the other dye (quencher dye). Utilizes the phenomenon of FRET that occurs when overlaps occur. More specifically, this principle can be explained as follows.
That is, a probe (TaqMan probe) in which two types of fluorescent dyes generating FRET are bound to both ends is a cDNA derived from an enzyme species involved in a phase I reaction of a specific drug metabolism amplified by PCR.
And elongation reaction of PCR starts in this state. When Taq DNA polymerase is present in this reaction system, the TaqMan probe is hydrolyzed by the 5'-3 'endonuclease activity of the polymerase, and the reporter dye bound thereto is eliminated. The elimination of the dye causes a physical distance from the quencher dye, and increases the fluorescence intensity of the reporter dye suppressed by FRET. This increase in fluorescence intensity
Is proportional to the amount of increase of the amplification product. Increase the fluorescence intensity to PC
If measurement is performed for each R reaction, desired quantification is possible.

In this test, a TaqMan probe having FAM bound to the 5 'end of the probe as a reporter dye and TAMRA bound to the 3' end as a quencher dye was used. The binding of each of these dyes and the production of a TaqMan probe by this are performed by the method described in the literature (Genome Res., 6 (10), 986 (1992).
6)).

Also, oligonucleotides as primers and probes are used as automatic synthesizers for AB.
Using a DNA / RNA synthesizer manufactured by Company I, synthesis was performed using a substrate (dNTP) and a specified reagent.

As the sample RNA, total RNA purified from adult brain, adult kidney, adult lung, adult small intestine, fetal liver and adult liver pool was used. still,
All of these RNAs were obtained from Clontech.
Laboratories, Inc.).

Total RNA purified from adult brain, adult kidney, adult lung, adult small intestine, fetal liver and adult liver pool was diluted with RNase-free water and adjusted to 20 μg / mL. Thereafter, these six types were mixed in equal amounts to prepare a calibration curve. Thereafter, 50 μg / mL yeast tRNA (Yeas
(t tRNA, manufactured by GIBCO) at a 5-fold common ratio. 5 μL was used for the measurement.

The RT-PCR reaction was performed using a kit (TaqMan One-Step RT-PCR Master) containing 300 nM forward primer, 900 nM reverse primer and 200 nM TaqMan probe.
Using the Mix Reagents Kit, PE Applied Biosystems), the sequence detection system (ABI PR
ISM ^TM 7700 Sequence Detection System, PE Applied B
iosystems).

The temperature conditions were 48 ° C. for 30 minutes and 95 ° C. for 10 minutes.
After incubating for 1 minute, the cycle of 95 ° C. for 15 seconds and 60 ° C. for 1 minute was performed 50 times, and the fluorescence intensity was measured for each cycle.

Tables 4 and 5 show the results (calibration curves) of the above-mentioned tests performed on each enzyme shown in Tables 2 and 3 below using a primer pair and a probe having the sequence shown in each SEQ ID NO.

[0057]

[Table 2]

[0058]

[Table 3]

[0059]

[Table 4]

[0060]

[Table 5]

From the results shown in Tables 4 and 5, 100,000 pg
When a calibration curve diluted 5 times the common ratio was prepared from the total RNA / 50 μL reaction volume, for EPHX1, 6.4 pg total RNA / 50 μL
It has quantitative properties up to the volume of the reaction solution,
The correlation coefficient (r) of the calibration curve using 0.256 pg to 4000 pg total RNA as the quantification limit was 0.99 or more. However, IL3 is 0.93, ALD
H7 was 0.97.

[0062]

[Sequence List] SEQUENCE LISTING <110> Ostuka Pharmaceutical Factory Inc. <120> A method of detecting human phase I enzymes of drug-metabolizing and a probe and a kit therefor <130> 2FD01JP <150> JP 2000-267163 <151> 2000-09-04 <160> 159 <170> PatentIn Ver. 2.0 <210> 1 <211> 27 <212> DNA <213> human EPHX1 gene <400> 1 acctttggag gacagctgct tccacta 27 <210> 2 <211> 30 <212> DNA <213> human EPHX2 gene <400> 2 cacactttcc cagtggatac cactcatgga 30 <210> 3 <211> 30 <212> DNA <213> human PIG3 gene <400> 3 ttaagcgagg aagtctgatc accagtttgc 30 <210> 4 <211 > 28 <212> DNA <213> human NMOR2 gene <400> 4 ctttcaacgg atccttgaag aatgtggc 28 <210> 5 <211> 29 <212> DNA <213> human MAOA gene <400> 5 ccagagcttc cagcagagag aaaccagtt 29 <210> 6 <211> 29 <212> DNA <213> human MAOB gene <400> 6 attgaccacc atcttttcag caacggctc 29 <210> 7 <211> 28 <212> DNA <213> human PTGS1 gene <400> 7 agctgctgtt cggtgtccag ttccaata 28 <210 > 8 <211> 26 <212> DNA <213> human PTGS2 gene <400> 8 tctttggtct ggtgcctggt ctgatg 26 <2 10> 9 <211> 26 <212> DNA <213> human DPYD gene <400> 9 ctcgtgctct gcctggattt cccatt 26 <210> 10 <211> 28 <212> DNA <213> human AOX1 gene <400> 10 cctgttccga ggttgaaata gactgcct 28 <210> 11 <211> 30 <212> DNA <213> human XDH gene <400> 11 aacaggagat cataagaacc tccgcacaga 30 <210> 12 <211> 26 <212> DNA <213> human CEL gene <400> 12 cggaaacgtc atcgtggtca ccttca 26 <210> 13 <211> 30 <212> DNA <213> human CES1 gene <400> 13 atgctgctgc tgaaaacacc tgaagagctt 30 <210> 14 <211> 27 <212> DNA <213> human CES2 gene <400> 14 ttcaacatga ccttcccttc cgactcc 27 <210> 15 <211> 28 <212> DNA <213> human AADAC gene <400> 15 agccatggag aatgatgtgg ataaacgc 28 <210> 16 <211> 28 <212> DNA <213> human GZMA gene < 400> 16 ccacacgcga aggtgacctt aaactttt 28 <210> 17 <211> 26 <212> DNA <213> human GZMB gene <400> 17 aagacccatc ccccatccag cctata 26 <210> 18 <211> 28 <212> DNA <213> human IL17 gene <400> 18 cccaaattct gaggacaaga acttcccc 28 <210> 19 <211> 30 <212> DNA <213> human LIPA gene <400> 19 caacatggct tgctggca ga ttctagtaac 30 <210> 20 <211> 28 <212> DNA <213> human NTE gene <400> 20 tctacagacc ttaatgagag ccgccgtg 28 <210> 21 <211> 26 <212> DNA <213> human UCHL1 gene <400> 21 tcacgcagtg gccaataatc aagaca 26 <210> 22 <211> 30 <212> DNA <213> human UCHL3 gene <400> 22 tggaatggat cctgaactcc ttagcatggt 30 <210> 23 <211> 28 <212> DNA <213> human FMO1 gene < 400> 23 tgagaaattc cctcccaacc ccaattgt 28 <210> 24 <211> 24 <212> DNA <213> human FMO2 gene <400> 24 ctccgcaatg tactgccacg aaca 24 <210> 25 <211> 27 <212> DNA <213> human FMO2 gene <400> 25 cagcccctag gttccatttt cccaact 27 <210> 26 <211> 29 <212> DNA <213> human FMO3 gene <400> 26 caatttaccg acagccatct ctgactggt 29 <210> 27 <211> 26 <212> DNA <213> human FMO4 gene <400> 27 cacaaagccc agcatcccac ttctgt 26 <210> 28 <211> 28 <212> DNA <213> human FMO5 gene <400> 28 ttgattcagc ccttaggagc cattatgc 28 <210> 29 <211> 27 <212> DNA < 213> human ALDH1 gene <400> 29 caaaacattg cgctactgtg caggttg 27 <210> 30 <211> 25 <212> DNA <213> human ALDH2 gene <400> 3 0 tttggagatg tgcaggatgg catga 25 <210> 31 <211> 28 <212> DNA <213> human ALDH3 gene <400> 31 tcccccagta cctggacaag gatctgta 28 <210> 32 <211> 28 <212> DNA <213> human ALDH3 gene < 400> 32 acatgttctc cagcaacgac aaggtgat 28 <210> 33 <211> 26 <212> DNA <213> human ALDH4 gene <400> 33 agctcagagc acctctgtgg catcaa 26 <210> 34 <211> 25 <212> DNA <213> human ALDH5 gene <400> 34 tggcgtgcag gatgacatga gaatt 25 <210> 35 <211> 27 <212> DNA <213> human ALDH6 gene <400> 35 ctatgcacag gctccatttg gtggctt 27 <210> 36 <211> 27 <212> DNA <213> human ALDH7 gene <400> 36 ctgcagagca ccatcacccg tttctat 27 <210> 37 <211> 28 <212> DNA <213> human ALDH8 gene <400> 37 cctggaacta cccattgaac ctgaccct 28 <210> 38 <211> 24 <212> DNA < 213> human ALDH9 gene <400> 38 attgcctctt ggaagtcggc tcca 24 <210> 39 <211> 26 <212> DNA <213> human ALDH10 gene <400> 39 cggttggcaa aattgtcatg gaagct 26 <210> 40 <211> 30 <212> DNA <213> human ADH1 gene <400> 40 taccatggtg accccacttc ctgtgatttt 30 <210> 41 <211> 36 <212> DNA <213> huma n ADH1 gene <400> 41 aaatgcagaa tttgtaaaaa cccggagagc aactac 36 <210> 42 <211> 30 <212> DNA <213> human ADH2 gene <400> 42 cacagatgac cacgtggtta gtggcaaact 30 <210> 43 <211> 30 <212> DNA <213> human ADH2 gene <400> 43 cttctcccag tacacggtgg tggatgagaa 30 <210> 44 <211> 30 <212> DNA <213> human ADH3 gene <400> 44 tcatgaagtt cgcattaaga tggtggctgc 30 <210> 45 <211> 28 <212 > DNA <213> human ADH3 gene <400> 45 cacgtggaaa ggagctattt ttggaggc 28 <210> 46 <211> 30 <212> DNA <213> human ADH4 gene <400> 46 ttttccagag gagctaataa tcggccgtac 30 <210> 47 <211> 30 <212> DNA <213> human ADH5 gene <400> 47 atcccacttt acatcccaca gtgtggagaa 30 <210> 48 <211> 26 <212> DNA <213> human ADH5 gene <400> 48 aagttggagc ctggctctgt ttgtgc 26 <210> 49 <211 > 28 <212> DNA <213> human ADH6 gene <400> 49 cagtggccag ttgttcttct caggacgt 28 <210> 50 <211> 28 <212> DNA <213> human ADH7 gene <400> 50 cccatcagtg aggtgctgtc agaaatga 28 <210> 51 <211> 26 <212> DNA <213> human ESD gene <400> 51 tttccagtgg atccccaaag gatgtc 26 < 210> 52 <211> 27 <212> DNA <213> human HADH2 gene <400> 52 tcaggttgga caagctgcat actctgc 27 <210> 53 <211> 27 <212> DNA <213> human HEP27 gene <400> 53 ccagctccaa tgccagtgtt ctagtga 27 <210> 54 <211> 21 <212> DNA <213> human EPHX1 gene <400> 54 tcgataagtt ccgtttcacc c 21 <210> 55 <211> 22 <212> DNA <213> human EPHX1 gene <400> 55 aattcattcc gccagtagga ga 22 <210> 56 <211> 21 <212> DNA <213> human EPHX2 gene <400> 56 gccactaccc ggcttatgaa a 21 <210> 57 <211> 21 <212> DNA <213> human EPHX2 gene <400> 57 tttagcggtc tcggagcact t 21 <210> 58 <211> 21 <212> DNA <213> human PIG3 gene <400> 58 ggcccctgtt ttcaaagcta c 21 <210> 59 <211> 21 <212> DNA <213> human PIG3 gene < 400> 59 cagaatttgc tccgtgaaag c 21 <210> 60 <211> 21 <212> DNA <213> human NMOR2 gene <400> 60 tctatgcaca ccaggaaccc a 21 <210> 61 <211> 22 <212> DNA <213> human NMOR2 gene <400> 61 ccctgcctgc tcagttcatc ta 22 <210> 62 <211> 23 <212> DNA <213> human MAOA gene <400> 62 ccttgactgc caagattcac ttc 23 <210> 63 <211> 22 <212> DNA <21 3> human MAOA gene <400> 63 tgcacttaat gacagctccc at 22 <210> 64 <211> 22 <212> DNA <213> human MAOB gene <400> 64 ctttttggag agacatttgc cc 22 <210> 65 <211> 21 <212> DNA <213> human MAOB gene <400> 65 tcacaagtag cccccttttg t 21 <210> 66 <211> 21 <212> DNA <213> human PTGS1 gene <400> 66 ctgcagctga aatttgaccc a 21 <210> 67 <211> 21 < 212> DNA <213> human PTGS1 gene <400> 67 accttgaagg agtcaggcat g 21 <210> 68 <211> 22 <212> DNA <213> human PTGS2 gene <400> 68 agtccctgag catctacggt tt 22 <210> 69 <211> 21 <212> DNA <213> human PTGS2 gene <400> 69 cccattcagg atgctcctgt t 21 <210> 70 <211> 21 <212> DNA <213> human DPYD gene <400> 70 ctttgagagc tgtgacctcc a 21 <210> 71 < 211> 21 <212> DNA <213> human DPYD gene <400> 71 tcagcagagt caattccacc a 21 <210> 72 <211> 21 <212> DNA <213> human AOX1 gene <400> 72 aaggccagcc cttcgaatac t 21 <210> 73 <211> 22 <212> DNA <213> human AOX1 gene <400> 73 tatactgcag ccaacatcca tg 22 <210> 74 <211> 23 <212> DNA <213> human XDH gene <400> 74 accgcttcca ctacttca gc tat 23 <210> 75 <211> 23 <212> DNA <213> human XDH gene <400> 75 ttagactgga gccaacatcc atg 23 <210> 76 <211> 23 <212> DNA <213> human CEL gene <400> 76 caacaactac ctgtatgacg gcg 23 <210> 77 <211> 23 <212> DNA <213> human CEL gene <400> 77 atagttacct ggcagattgg cgt 23 <210> 78 <211> 22 <212> DNA <213> human CES1 gene < 400> 78 ccagagagag tcaacccctt ct 22 <210> 79 <211> 21 <212> DNA <213> human CES1 gene <400> 79 tcctgcttgt taattccgac c 21 <210> 80 <211> 22 <212> DNA <213> human CES2 gene <400> 80 accgcagtgg agtcagagtt tc 22 <210> 81 <211> 22 <212> DNA <213> human CES2 gene <400> 81 atgctgaggt acaggcagtc ct 22 <210> 82 <211> 21 <212> DNA <213> human AADAC gene <400> 82 gcctctccca gataacgttg a 21 <210> 83 <211> 22 <212> DNA <213> human AADAC gene <400> 83 tcaaagctcc cgacaacctt aa 22 <210> 84 <211> 21 <212> DNA < 213> human GZMA gene <400> 84 ccctatccat gctatgaccc a 21 <210> 85 <211> 21 <212> DNA <213> human GZMA gene <400> 85 ctggtttcac atcatccccc t 21 <210> 86 <211> 22 <212> DNA <213> h uman GZMB gene <400> 86 acccagcagt ttatccctgt ga 22 <210> 87 <211> 23 <212> DNA <213> human GZMB gene <400> 87 tttctctcca gctgcagtag cat 23 <210> 88 <211> 21 <212> DNA < 213> human IL17 gene <400> 88 caggaatcac aatcccacga a 21 <210> 89 <211> 22 <212> DNA <213> human IL17 gene <400> 89 tccggttatg gatgttcagg tt 22 <210> 90 <211> 23 <212> DNA <213> human LIPA gene <400> 90 aaaggtccca aaccagttgt ctt 23 <210> 91 <211> 21 <212> DNA <213> human LIPA gene <400> 91 cacacgtcaa aaccagcatc a 21 <210> 92 <211> 22 < 212> DNA <213> human NTE gene <400> 92 ggcaacgtca ttgagaaaat gc 22 <210> 93 <211> 23 <212> DNA <213> human NTE gene <400> 93 aatctctgcc aagtcagtga agc 23 <210> 94 <211> 21 <212> DNA <213> human UCHL1 gene <400> 94 cctgtggcac aatcggactt a 21 <210> 95 <211> 22 <212> DNA <213> human UCHL1 gene <400> 95 aactgatcca tcctcaaatc cc 22 <210> 96 < 211> 22 <212> DNA <213> human UCHL3 gene <400> 96 catcctaact ggcaattcgt tg 22 <210> 97 <211> 25 <212> DNA <213> human UCHL3 gene <400> 97 agaagtaaga ctgcac agac tggtc 25 <210> 98 <211> 21 <212> DNA <213> human FMO1 gene <400> 98 catggtgttc atgacacgct t 21 <210> 99 <211> 22 <212> DNA <213> human FMO1 gene <400> 99 gccagttgtt tatctttcgc tc 22 <210> 100 <211> 21 <212> DNA <213> human FMO2 gene <400> 100 ccacacccgg tttcgttcta t 21 <210> 101 <211> 21 <212> DNA <213> human FMO2 gene < 400> 101 tgttttgagg ctcaaggcca t 21 <210> 102 <211> 21 <212> DNA <213> human FMO2 gene <400> 102 tcacctggac aagtcaaccc t 21 <210> 103 <211> 21 <212> DNA <213> human FMO2 gene <400> 103 cccaacgagc ttgaagttca g 21 <210> 104 <211> 21 <212> DNA <213> human FMO3 gene <400> 104 gtcactcgat tcggaacctt c 21 <210> 105 <211> 22 <212> DNA <213> human FMO3 gene <400> 105 gctctttcct caggactcca tt 22 <210> 106 <211> 21 <212> DNA <213> human FMO4 gene <400> 106 tacatggatg atatcgctgc c 21 <210> 107 <211> 22 <212> DNA < 213> human FMO4 gene <400> 107 catgaggcgg tactgataag ga 22 <210> 108 <211> 22 <212> DNA <213> human FMO5 gene <400> 108 aaggtcttcc ctcctaacct gg 22 <210> 109 <211> 22 <212> DNA <213> human FMO5 gene <400> 109 agcgtccttg gagctctgaa at 22 <210> 110 <211> 21 <212> DNA <213> human ALDH1 gene <400> 110 tgagtgattt agcaggctgc a 21 <210> 111 <211 > 23 <212> DNA <213> human ALDH1 gene <400> 111 tggccacata caccaatagg ttc 23 <210> 112 <211> 21 <212> DNA <213> human ALDH2 gene <400> 112 tggttacttc atccagccca c 21 <210> 113 <211> 23 <212> DNA <213> human ALDH2 gene <400> 113 gctctcccaa caacctcctc tat 23 <210> 114 <211> 21 <212> DNA <213> human ALDH3 gene <400> 114 agctgagtga gaacatggcg a 21 <210 > 115 <211> 22 <212> DNA <213> human ALDH3 gene <400> 115 atggtcgaac ctctccttga gc 22 <210> 116 <211> 21 <212> DNA <213> human ALDH3 gene <400> 116 agttcatcaa ccagcgtgag a 21 <210> 117 <211> 21 <212> DNA <213> human ALDH3 gene <400> 117 gtgcaaggtg atgtggacga t 21 <210> 118 <211> 21 <212> DNA <213> human ALDH4 gene <400> 118 caacatcatc cagtttgtgc c 21 <210> 119 <211> 21 <212> DNA <213> human ALDH4 gene <400> 119 cgaagtggaa gttctttccg c 21 <210> 120 <211> 24 <212> DNA <213> human ALDH5 gene <400> 120 ggtttcttca tcaagcctac tgtc 24 <210> 121 <211> 21 <212> DNA <213> human ALDH5 gene <400> 121 caggcccaaa gatctcctct t 21 <210> 122 <211> 23 <212> DNA < 213> human ALDH6 gene <400> 122 gaacggtctg gatcaactgc tac 23 <210> 123 <211> 22 <212> DNA <213> human ALDH6 gene <400> 123 agttctctgc catttcctga ca 22 <210> 124 <211> 22 <212> DNA <213> human ALDH7 gene <400> 124 actacgtcct atgcagccct ga 22 <210> 125 <211> 21 <212> DNA <213> human ALDH7 gene <400> 125 cgctggaact gtttctggtt g 21 <210> 126 <211> 21 < 212> DNA <213> human ALDH8 gene <400> 126 atctggaagg aaccctttgg c 21 <210> 127 <211> 21 <212> DNA <213> human ALDH8 gene <400> 127 atttctgacg gcttcagcac c 21 <210> 128 <211> 21 <212> DNA <213> human ALDH9 gene <400> 128 agcatggaac tacccctttc a 21 <210> 129 <211> 22 <212> DNA <213> human ALDH9 gene <400> 129 aaagaccatg gcattaccac ag 22 <210> 130 < 211> 22 <212> DNA <213> human ALDH10 gene <400> 130 gcagcgattt gaccacattt tc 22 <210> 131 <211> 22 <212> DNA <213> human ALDH 10 gene <400> 131 taacatggac ttttccctcc ca 22 <210> 132 <211> 22 <212> DNA <213> human ADH1 gene <400> 132 cctaaggccc atgaagttcg ta 22 <210> 133 <211> 21 <212> DNA <213 > human ADH1 gene <400> 133 ccctgaggat tgcttacatc g 21 <210> 134 <211> 21 <212> DNA <213> human ADH1 gene <400> 134 tgataaagtc atcccactcg c 21 <210> 135 <211> 21 <212> DNA <213> human ADH1 gene <400> 135 ccctgaggat tgcttacatc g 21 <210> 136 <211> 22 <212> DNA <213> human ADH2 gene <400> 136 cctaaggctt atgaagttcg ca 22 <210> 137 <211> 21 <212 > DNA <213> human ADH2 gene <400> 137 gtgccaagga agtggtgaat g 21 <210> 138 <211> 21 <212> DNA <213> human ADH2 gene <400> 138 cattcaccac ttccttggca c 21 <210> 139 <211> 23 <212> DNA <213> human ADH2 gene <400> 139 atgaggcaga ctttctccag ttt 23 <210> 140 <211> 22 <212> DNA <213> human ADH3 gene <400> 140 tgaggaggta gaggttgcac ct 22 <210> 141 <211 > 24 <212> DNA <213> human ADH3 gene <400> 141 ccactaacca catgctcatc tgaa 24 <210> 142 <211> 21 <212> DNA <213> human ADH3 gene <400> 142 agaaa cccat tcaggaagtg c 21 <210> 143 <211> 23 <212> DNA <213> human ADH3 gene <400> 143 ggtacggata ctctttccag agc 23 <210> 144 <211> 22 <212> DNA <213> human ADH4 gene <400 > 144 tgctggtagc aaaggattga ct 22 <210> 145 <211> 21 <212> DNA <213> human ADH4 gene <400> 145 ccaaccacca aagaatgttc c 21 <210> 146 <211> 22 <212> DNA <213> human ADH5 gene <400> 146 ggagttacta agctgaaggc gg 22 <210> 147 <211> 22 <212> DNA <213> human ADH5 gene <400> 147 agaaaatgtg ctggttccca tg 22 <210> 148 <211> 22 <212> DNA <213> human ADH5 gene <400> 148 ccttctaggt tgtggcattt ca 22 <210> 149 <211> 21 <212> DNA <213> human ADH5 gene <400> 149 taactgccaa tccgactcct c 21 <210> 150 <211> 22 <212> DNA <213 > human ADH6 gene <400> 150 cctgccagtg ttcaactcaa aa 22 <210> 151 <211> 21 <212> DNA <213> human ADH6 gene <400> 151 gatgtgctgt ctgctcttcc a 21 <210> 152 <211> 22 <212> DNA <213> human ADH7 gene <400> 152 tcagtcccaa ggactctacc aa 22 <210> 153 <211> 22 <212> DNA <213> human ADH7 gene <400> 153 cttcaaaggt gtatcccacg tt 22 < 210> 154 <211> 21 <212> DNA <213> human ESD gene <400> 154 cttccccaac tcataaatgc c 21 <210> 155 <211> 23 <212> DNA <213> human ESD gene <400> 155 acagatcaga gctccatgac ctc 23 <210> 156 <211> 21 <212> DNA <213> human HADH2 gene <400> 156 atcaacactg ccagtgtggc t 21 <210> 157 <211> 21 <212> DNA <213> human HADH2 gene <400> 157 agtgtcatgc ccactattcc c 21 <210> 158 <211> 22 <212> DNA <213> human HEP27 gene <400> 158 ctggtctctt ccattgcagc tt 22 <210> 159 <211> 21 <212> DNA <213> human HEP27 gene <400> 159 cagtttaccc ggatgtcctt g 21

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B024 AA11 CA09 CA20 HA14 4B063 QA01 QA13 QA18 QQ53 QR08 QR55 QR56 QR62 QS03 QS25 QS34

Claims (12)

[Claims] Claims: 1. A probe used for measuring an enzyme involved in a phase I reaction of drug metabolism, comprising a group consisting of an oligonucleotide hybridizing to any of the following regions (1) to (53): Probe selected from: (1) 222-248 region of EPHX1 gene (2) 174-203 region of EPHX2 gene (3) 767-796 region of PIG3 gene (4) 50-77 region of NMOR2 gene (5) 853-881 region of MAOA gene (6) MAOB gene 1491-1519 region (7) PTGS1 gene 1088-1115 region (8) PTGS2 gene 830-855 region (9) DPYD gene 2345-2370 region (10) AOX1 gene 3500-3527 region (11) XDH gene 3504-3533 region (12) CEL gene 468-493 region (13) CES1 gene 976-1005 region (14) CES2 gene 328-354 region (15) AADAC gene 86-113 region (16) GZMA gene 326-353 region (17) GZMB gene 276-301 region (18) IL17 gene 99-126 region (19) LIPA gene 253-282 region (20) NTE gene 3670-3697 region (21) 255-280 region of UCHL1 gene (22) 105-134 region of UCHL3 gene (23) 728-755 region of FMO1 gene (24) 727-750 region of FMO2 gene (25) 1117-1143 region of FMO2 gene (26) FMO3 gene 735-763 region (27) FMO4 gene 1326-1351 region (28) FMO5 gene 1111-1138 region (29) ALDH1 gene 381-407 region (3 0) ALDH2 gene 1204-1228 region (31) ALDH3 gene 461-488 region (32) ALDH3 gene 1094-1121 region (33) ALDH4 gene 823-848 region (34) ALDH5 gene 1212-1236 region ( (35) ALDH6 gene 1413-1439 region (36) ALDH7 gene 790-816 region (37) ALDH8 gene 95-122 region (38) ALDH9 gene 481-504 region (39) ALDH10 gene 563-588 region ( 40) 168-197 region of ADH1 gene (41) 298-333 region of ADH1 gene (42) 144-173 region of ADH2 gene (43) 438-467 region of ADH2 gene (44) 102-131 region of ADH3 gene ( 45) ADH3 gene 939-966 region (46) ADH4 gene 927-956 region (47) ADH5 gene 322-351 region (48) ADH5 gene 616-641 region (49) ADH6 gene 912-939 region (50) ADH7 gene 745-772 region (51) ESD gene 451-476 region (52) HADH2 gene 483-509 region (53) HEP27 gene 610-584 region 2. The probe according to claim 1, further comprising a reporter dye and a quencher dye. 3. The method according to claim 1, wherein the length of the base sequence is in the range of 20-40.
The probe according to 1. 4. The probe according to claim 1, which comprises any one of the sequences shown in SEQ ID NOs: 1 to 53. 5. The probe according to claim 1, which is any one of the sequences shown in SEQ ID NOs: 1 to 53. 6. It is involved in a phase I reaction of drug metabolism, including a combination of a forward primer, a probe and a reverse primer comprising an oligonucleotide hybridizing to each of the following regions (1) to (53): A kit for measuring enzymes. (1) forward primer; 200-220 region of EPHX1 gene, probe; 222-248 region and reverse primer of the gene; 302-281 region of the gene; (2) forward primer; 145-165 region of EPHX2 gene, probe 174-203 region and reverse primer of the same gene; 237-217 region of the same gene; (3) forward primer; 743-763 region of PIG3 gene;
Probe; 767-796 region and reverse primer of the same gene; 858-838 region of the same gene; (4) forward primer; 26-46 region of the NMOR2 gene;
Probe; 50-77 region and reverse primer of the same gene; 101-80 region of the same gene; (5) forward primer; 827-849 region of the MAOA gene;
Probe; 853-881 region and reverse primer of the same gene; 919-898 region of the same gene; (6) forward primer; 1440-1461 region of the MAOB gene; probe; 1491-1519 region of the same gene and reverse primer; 1555-1535 region of (7) forward primer; 1066-1086 region of PTGS1 gene, probe; 1088-1115 region of the same gene and reverse primer; 1187-1167 region of the same gene; (8) forward primer; PTGS2 gene 792-813 region, probe; 830-855 region and reverse primer of the same gene; 928-908 region of the same gene; (9) forward primer; 2321-2341 region of the DPYD gene, probe; Reverse primer; 2399-2379 region of the same gene; (10) forward primer; AOX1 gene 3464-3484 region; probe; 3500-3527 region of the same gene and reverse Limer; 3585-3564 region of the same gene; (11) forward primer; 3446-3468 region of the XDH gene; probe; 3504-3533 region of the same gene and reverse primer; 3562-3540 region of the same gene; (12) forward primer 429-451 region of the CEL gene;
Probe; 468-493 region and reverse primer of the same gene; 555-533 region of the same gene; (13) forward primer; 935-956 region of the CES1 gene; probe; 976-1005 region of the same gene and reverse primer; the same gene 1061-1041 region of (14) forward primer; 298-319 region of CES2 gene, probe; 328-354 region and reverse primer of the same gene; 383-362 region of the same gene; (15) forward primer; 63-83 region,
Probe; 86-113 region and reverse primer of the same gene; 206-185 region of the same gene; (16) forward primer; 304-324 region of the GZMA gene; probe; 326-353 region and reverse primer of the same gene; 433-413 region of (17) forward primer; 253-274 region of GZMB gene, probe; 276-301 region and reverse primer of the same gene; 350-328 region of the same gene; (18) forward primer; 68-88 area,
Probe; 99-126 region and reverse primer of the same gene; 163-142 region of the same gene; (19) forward primer; 226-248 region of the LIPA gene; probe; 253-282 region of the same gene and reverse primer; 347-327 region of (20) forward primer; 3634-3655 region of NTE gene; probe; 3670-3697 region of gene and reverse primer; 3747-3725 region of same gene; (21) forward primer; of UCHL1 gene 233-253 region, probe; 255-280 region and reverse primer of the same gene; 306-285 region of the same gene; (22) forward primer; 76-97 region of the UCHL3 gene;
Probe; 105-134 region and reverse primer of the same gene; 164-140 region of the same gene; (23) forward primer; 696-716 region of the FMO1 gene; probe; 728-755 region of the same gene and reverse primer; the same gene 790-769 region of (24) forward primer; 705-725 region of FMO2 gene, probe; 727-750 region of reverse gene and reverse primer; 826-806 region of same gene; (25) forward primer; of FMO2 gene 1077-1097 region, probe; 1117-1143 region and reverse primer of the same gene; 1165-1145 region of the same gene; (26) forward primer; 706-726 region of the FMO3 gene, probe; 735-763 region of the same gene and Reverse primer; 844-823 region of the gene; (27) forward primer; 1297-1317 region of FMO4 gene, probe; 1326-1351 region of the gene and reverse ply -; 1422-1401 region of the gene; (28) forward primer; 1063-1084 region of the FMO5 gene, probe; 1111-1138 region and reverse primer of the gene; 1162-1141 region of the gene; (29) forward primer Region 359-379 of the ALDH1 gene, probe; region 381-407 of the gene and a reverse primer; region 494-472 of the gene; (30) forward primer; region 1179-1199 of the ALDH2 gene, probe; 1204 of the gene -1228 region and reverse primer; 1307-1285 region of the same gene; (31) forward primer; 422-442 region of the ALDH3 gene; probe; 461-488 region of the same gene and reverse primer; 549-528 region of the same gene; (32) forward primer; 1058-1078 region of the ALDH3 gene, probe; 1094-1121 region and reverse primer of the gene; 1194-1174 region of the gene; (33) forward primer Primer: 768-788 region of the ALDH4 gene, probe; 823-848 region and reverse primer of the gene; 967-947 region of the gene; (34) forward primer; 1183-1206 region of the ALDH5 gene, probe; 1212-1236 region and reverse primer; 1261-1241 region of the same gene; (35) forward primer; 1382-1404 region of ALDH6 gene; probe; 1413-1439 region of the same gene and reverse primer; 1466-1445 region of the same gene (36) forward primer; 743-764 region of the ALDH7 gene, probe; 790-816 region and reverse primer of the gene; 878-858 region of the gene; (37) forward primer; 55-75 region of the ALDH8 gene;
Probe; 95-122 region and reverse primer of the same gene; 182-162 region of the same gene; (38) forward primer; 459-479 region of the ALDH9 gene; probe; 481-504 region and reverse primer of the same gene; 534-513 region, (39) forward primer; 525-546 region of the ALDH10 gene, probe; 563-588 region and reverse primer of the gene; 644-623 region of the gene; (40) forward primer; 94-115 region,
Probe; 168-197 region and reverse primer of the same gene; 365-345 region of the same gene; (41) forward primer; 261-281 region of the ADH1 gene; probe; 298-333 region of the same gene and reverse primer; the same gene 365-345 region of (42) forward primer; 94-115 region of ADH2 gene;
Probe; 144-173 region and reverse primer of the same gene; 431-411 region of the same gene; (43) forward primer; 411-431 region of the ADH2 gene; probe; 438-467 region of the same gene and reverse primer; 518-496 region of (44) forward primer; 72-93 region of ADH3 gene;
Probe; 102-131 region and reverse primer of the same gene; 167-144 region of the same gene; (45) forward primer; 743-763 region of the ADH3 gene; probe; 939-966 region of the same gene and reverse primer; 1113-1091 region of (46) forward primer; 903-924 region of ADH4 gene, probe; 927-956 region of the same gene and reverse primer; 984-964 region of the same gene; (47) forward primer; of ADH5 gene 289-310 region, probe; 322-351 region and reverse primer of the same gene; 495-474 region of the same gene; (48) forward primer; 564-585 region of the ADH5 gene, probe; 616-641 region of the same gene Reverse primer; 676-656 region of the same gene. (49) forward primer; region 889-910 of the ADH6 gene, probe; region 912-939 of the gene and a reverse primer; region 987-967 of the gene. (50) forward primer; 722-743 region of the ADH7 gene, probe; 745-772 region and reverse primer of the gene; 802-781 region of the gene. (51) forward primer; 427-447 region of the ESD gene,
Probe; 451-476 region of the same gene and reverse primer; 519-497 region of the same gene. (52) Forward primer; 451-471 region of HADH2 gene, probe; 483-509 region and reverse primer of the gene; 539-519 region of the gene. (53) forward primer; 505-526 region of the HEP27 gene, probe; 610-584 region and reverse primer of the gene; 635-615 region of the gene. 7. The measurement kit according to claim 6, wherein the probe further comprises a reporter dye and a quencher dye. An oligonucleotide comprising a sequence represented by each of SEQ ID NOs in any one of the following (1) to (53):
8. The measurement kit according to claim 7, which is selected from a combination of a forward primer, a probe and a reverse primer. (1) forward primer; SEQ ID NO: 54, probe; SEQ ID NO: 1 and reverse primer; SEQ ID NO: 55; (2) forward primer; SEQ ID NO: 56, probe; SEQ ID NO: 2 and reverse primer; SEQ ID NO: 57, (3) forward SEQ ID NO: 58, probe; SEQ ID NO: 3 and reverse primer; SEQ ID NO: 59, (4) forward primer; SEQ ID NO: 60, probe; SEQ ID NO: 4 and reverse primer; SEQ ID NO: 61, (5) forward primer; 62, probe; SEQ ID NO: 5 and reverse primer; SEQ ID NO: 63, (6) forward primer; SEQ ID NO: 64, probe; SEQ ID NO: 6 and reverse primer; SEQ ID NO: 65, (7) forward primer; SEQ ID NO: 66, probe; SEQ ID NO: 7 and reverse primer; SEQ ID NO: 67, (8) forward primer SEQ ID NO: 68, probe; SEQ ID NO: 8 and reverse primer; SEQ ID NO: 69 (9) forward primer; SEQ ID NO: 70, probe; SEQ ID NO: 9 and reverse primer; SEQ ID NO: 71, (10) forward primer; SEQ ID NO: 72, probe SEQ ID NO: 10 and reverse primer; SEQ ID NO: 73, (11) forward primer; SEQ ID NO: 74, probe; SEQ ID NO: 11 and reverse primer; SEQ ID NO: 75, (12) forward primer; SEQ ID NO: 76, probe; SEQ ID NO: 12 SEQ ID NO: 77, (13) forward primer; SEQ ID NO: 78, probe; SEQ ID NO: 13 and reverse primer; SEQ ID NO: 79, (14) forward primer; SEQ ID NO: 80, probe; SEQ ID NO: 14 and reverse primer; SEQ ID NO: 81, (15) forward primer; SEQ ID NO: 82, professional SEQ ID NO: 15 and reverse primer; SEQ ID NO: 83, (16) forward primer; SEQ ID NO: 84, probe; SEQ ID NO: 16 and reverse primer; SEQ ID NO: 85, (17) forward primer; SEQ ID NO: 86, probe; SEQ ID NO: SEQ ID NO: 87, (18) Forward primer; SEQ ID NO: 88, probe; SEQ ID NO: 18 and reverse primer; SEQ ID NO: 89, (19) Forward primer; SEQ ID NO: 90, probe; SEQ ID NO: 19 and reverse primer SEQ ID NO: 91, (20) forward primer; SEQ ID NO: 92, probe; SEQ ID NO: 20 and reverse primer; SEQ ID NO: 93, (21) forward primer; SEQ ID NO: 94, probe; SEQ ID NO: 21 and reverse primer; SEQ ID NO: 95 (22) forward primer; SEQ ID NO: 96, probe; SEQ ID NO: SEQ ID NO: 97, (23) forward primer; SEQ ID NO: 98, probe; SEQ ID NO: 23 and reverse primer; SEQ ID NO: 99, (24) forward primer; SEQ ID NO: 100, probe;
SEQ ID NO: 24 and reverse primer; SEQ ID NO: 101; (25) forward primer; SEQ ID NO: 102, probe;
SEQ ID NO: 25 and reverse primer; SEQ ID NO: 103; (26) forward primer; SEQ ID NO: 104, probe;
SEQ ID NO: 26 and reverse primer; SEQ ID NO: 105; (27) forward primer; SEQ ID NO: 106, probe;
SEQ ID NO: 27 and reverse primer; SEQ ID NO: 107, (28) forward primer; SEQ ID NO: 108, probe;
SEQ ID NO: 28 and reverse primer; SEQ ID NO: 109; (29) forward primer; SEQ ID NO: 110, probe;
SEQ ID NO: 29 and reverse primer; SEQ ID NO: 111; (30) forward primer; SEQ ID NO: 112, probe;
SEQ ID NO: 30 and reverse primer; SEQ ID NO: 113, (31) forward primer; SEQ ID NO: 114, probe;
SEQ ID NO: 31 and reverse primer; SEQ ID NO: 115, (32) forward primer; SEQ ID NO: 116, probe;
SEQ ID NO: 32 and reverse primer; SEQ ID NO: 117, (33) forward primer; SEQ ID NO: 118, probe;
SEQ ID NO: 33 and a reverse primer; SEQ ID NO: 119; (34) forward primer; SEQ ID NO: 120, probe;
SEQ ID NO: 34 and reverse primer; SEQ ID NO: 121, (35) forward primer; SEQ ID NO: 122, probe;
SEQ ID NO: 35 and reverse primer; SEQ ID NO: 123, (36) forward primer; SEQ ID NO: 124, probe;
SEQ ID NO: 36 and reverse primer; SEQ ID NO: 125, (37) forward primer; SEQ ID NO: 126, probe;
SEQ ID NO: 37 and reverse primer; SEQ ID NO: 127, (38) forward primer; SEQ ID NO: 128, probe;
SEQ ID NO: 38 and reverse primer; SEQ ID NO: 129, (39) forward primer; SEQ ID NO: 130, probe;
SEQ ID NO: 39 and reverse primer; SEQ ID NO: 131. (40) forward primer; SEQ ID NO: 132, probe;
SEQ ID NO: 40 and reverse primer; SEQ ID NO: 133, (41) forward primer; SEQ ID NO: 134, probe;
SEQ ID NO: 41 and reverse primer; SEQ ID NO: 135; (42) forward primer; SEQ ID NO: 136, probe;
SEQ ID NO: 42 and reverse primer; SEQ ID NO: 137; (43) forward primer; SEQ ID NO: 138, probe;
SEQ ID NO: 43 and reverse primer; SEQ ID NO: 139, (44) forward primer; SEQ ID NO: 140, probe;
SEQ ID NO: 44 and reverse primer; SEQ ID NO: 141, (45) forward primer; SEQ ID NO: 142, probe;
SEQ ID NO: 45 and reverse primer; SEQ ID NO: 143, (46) forward primer; SEQ ID NO: 144, probe;
SEQ ID NO: 46 and reverse primer; SEQ ID NO: 145; (47) forward primer; SEQ ID NO: 146, probe;
SEQ ID NO: 47 and a reverse primer; SEQ ID NO: 147, (48) forward primer; SEQ ID NO: 148, probe;
SEQ ID NO: 48 and reverse primer; SEQ ID NO: 149; (49) forward primer; SEQ ID NO: 150, probe;
SEQ ID NO: 49 and reverse primer; SEQ ID NO: 151. (50) forward primer; SEQ ID NO: 152, probe;
SEQ ID NO: 50 and reverse primer; SEQ ID NO: 153; (51) forward primer; SEQ ID NO: 154, probe;
SEQ ID NO: 51 and reverse primer; SEQ ID NO: 155; (52) forward primer; SEQ ID NO: 156, probe;
SEQ ID NO: 52 and a reverse primer; SEQ ID NO: 157; (53) forward primer; SEQ ID NO: 158, probe;
SEQ ID NO: 53 and reverse primer; SEQ ID NO: 159. 9. The measurement kit according to claim 8, wherein (1)
The combination of is for EPHX1 measurement, the combination of (2) is for measurement of EPHX2, the combination of (3) is for measurement of PIG3, and the combination of (4) is
For measurement of NMOR2, combination of (5) is for measurement of MAOA, (6)
The combination of is for MAOB measurement, the combination of (7) is for measurement of PTGS1, the combination of (8) is for measurement of PTGS2, and the combination of (9) is D
For PYD measurement, (10) combination for AOX1 measurement, (11)
The combination of is for XDH measurement, the combination of (12) is for CEL measurement, the combination of (13) is for CES1 measurement, and the combination of (14) is CE
For measurement of S2, combination of (15) is for measurement of AADAC, (16)
The combination of is for GZMA measurement, the combination of (17) is for GZMB measurement, the combination of (18) is for IL17 measurement, and the combination of (19) is
For measurement of LIPA, combination of (20) is for measurement of NTE, (21)
The combination of is for UCHL1 measurement, the combination of (22) is for measurement of UCHL3, the combination of (23) is for measurement of FMO1, the combination of (24) is for measurement of FMO2, and the combination of (25) Is for FMO2 measurement, (2
The combination of (6) is for measurement of FMO3, the combination of (27) is for measurement of FMO4, the combination of (28) is for measurement of FMO5, the combination of (29) is for measurement of ALDH1, and (30) Is for ALDH2 measurement,
The combination of (31) is for ALDH3 measurement, and the combination of (32) is for ALDH3
The combination of (33) is for measurement of ALDH4, the combination of (34) is for measurement of ALDH5, the combination of (35) is for measurement of ALDH6, and the combination of (36) is for measurement of ALDH7 The combination of (37) is for the measurement of ALDH8, the combination of (38) is for the measurement of ALDH9,
The combination of (39) is for ALDH10 measurement and the combination of (40) is ADH1
The combination of (41) is for measuring ADH1, the combination of (42) is for measuring ADH2, the combination of (43) is for measuring ADH2, and the combination of (44) is for measuring ADH3. The combination of (45) is AD
The combination of (46) is for measurement of ADH4, the combination of (47) is for measurement of ADH5, the combination of (48) is for measurement of ADH5, and the combination of (49) is for measurement of ADH6. For measurement, the combination of (50) is AD
A measurement kit for measuring H7, the combination of (51) is for measurement of ESD, the combination of (52) is for measurement of HADH2, and the combination of (53) is for measurement of HEP27. 10. The forward primer, the probe and the reverse primer are selected from oligonucleotides having a sequence represented by each sequence number according to any one of (1) to (53) according to claim 8. 9. The measurement kit according to 9. A sample containing a gene of an enzyme involved in a phase I reaction of drug metabolism is subjected to a polymerase chain reaction using the measurement kit according to any one of claims 6 to 10, and the probe used is hydrolyzed. A method for measuring an enzyme involved in a phase I reaction of drug metabolism in a human, which comprises determining the presence or absence of decomposition. 12. The measuring method according to claim 11, wherein the presence or absence of hydrolysis is determined by the presence or absence of the emission of fluorescence by irradiation with excitation light.