JP2003310278A - METHOD FOR MEASURING mRNA OF ENZYME ASSOCIATED WITH HUMAN SULFURIC ACID CONJUGATION - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To establish a method for measuring mRNA encoding an enzyme associated with a sulfuric acid conjugation in human by using the PCR, especially a real time detection method, and a probe and primer for the same method. <P>SOLUTION: This method for measuring the mRNA encoding the enzyme associated with the sulfuric acid conjugation in human uses a measurement kit. The measurement kit is selected from combinations of primer pairs of forward primers and reverse primers consisting of probes and oligonucleotides hybridizing with specific domains of a gene. The probes are used for the measurement of the mRNA of the enzyme associated with the sulfuric acid conjugation and consist of oligonucleotides hybridizing with the specific domains of the gene. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring mRNA encoding an enzyme involved in sulfate conjugation in humans, a probe and a kit therefor.

[0002]

2. Description of the Related Art It is well known that sulfate conjugation, which is one of the phase II reactions of drug metabolism, is catalyzed by sulfotransferase.

By the way, in developing a new drug, it is important to understand the movement (increase / decrease in expression level) of an enzyme involved in sulfate conjugation when the new drug is administered. This is because unless the changes in the enzymes involved in sulfate conjugation are known, it is not possible to predict the increase or decrease in the efficacy and side effects of the concomitant drug used in combination with the new drug or the increase or decrease in the efficacy or side effect of the new drug due to the concomitant drug or other ingested substance. , Because the safety of the new drug cannot be confirmed.

Therefore, in the field of new drug development, etc., information relating to mRNAs encoding enzymes involved in sulfate conjugation in humans, especially those capable of differentiating and measuring mRNAs encoding enzymes involved in sulfate conjugation. Development of technology is required. If such a technology can be developed, the safety of the new drug can be secured, for example, because it is possible to easily grasp the interaction with other drug of the new drug and the influence in the special disease state, and to obtain useful information on the side effect of the new drug. be able to.

On the other hand, polymerase chain reaction (PCR) is widely known as a nucleic acid amplification method,
For example, RT-PCR (Reverse Transcription-PCR),
Competitive RT-PCR and the like are effective in detecting and quantifying a small amount of mRNA.

Recently, a real-time quantitative detection method using PCR has been established (Taq Man PCR (Genome Res., 6 (1
0), 986 (1996)), ABI PRISM ^TM Sequence Detection Sy
stem (Applied Biosystems)). This is a method of measuring a nucleic acid using a specific fluorescently labeled probe (TaqMan prove). More specifically, for example, when an ordinary PCR reaction is performed in a state where a fluorescently labeled probe having a reporter dye added to the 5'end and a quencher dye added to the 3'end is annealed to the target DNA, the extension reaction proceeds. Accordingly, the probe is hydrolyzed from the 5'end by the 5'-3 'exonuclease activity of the DNA polymerase. As a result, the reporter dye at the 5'end was separated from the quencher dye at the 3'end, which caused the FR to initially maintain a certain spatial distance.
ET (Fluoresence Resonance Energy Transfer, a phenomenon in which the fluorescence intensity of the reporter dye decreases due to the resonance of both fluorescent dyes lowering the fluorescence intensity), the effect disappears, and the fluorescence intensity of the reporter dye controlled by the quencher dye increases, thus By measuring the increase in the fluorescence intensity, the target nucleic acid can be selectively detected quantitatively in real time. According to this method, P
After the CR reaction, the amplified product is subjected to agarose gel electrophoresis, for example, and complicated steps such as analyzing the migration pattern are not required, and there is an advantage that various samples can be quantified simultaneously in a short time.

However, 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 specimens within a limited time, and it is necessary to develop a method capable of efficiently performing the test. There is a demand, and the above real-time detection method can be expected to meet this demand.

[0008] The present inventors focused their attention on the above-mentioned real-time detection method by PCR, and if this method can be used for detection of mRNA encoding an enzyme involved in human sulfate conjugation, the above-mentioned respective PCR conditions can be simultaneously applied using the same device. Based on the idea that mRNAs can be measured and detected separately, we have conducted intensive research.

[0009] However, the mRNAs encoding the enzymes involved in sulfate conjugation in humans can be sufficiently amplified under the same PCR conditions with the respective genes as target genes, although the sequences of the respective mRNAs are known. It was considered difficult to search for an oligonucleotide as each primer pair capable of hybridizing to a specific position of the target gene. In addition, the specific region sandwiched between such primer pairs can hybridize faster than these primers under the same PCR conditions, and it is considered that the construction of a specific probe in human is similarly difficult. It was In particular, although the easiness of hybridizing two nucleic acids whose nucleotide sequences are known can be estimated to some extent by calculating the melting point (Tm), the combination of the primer and the probe selected by this estimation is not always necessary for DNA measurement. MRNAs encoding enzymes involved in sulfate conjugation in all humans known to date, since they are known not to produce successful results in
The above-mentioned PC that can be measured separately for
It was expected that the selection of the combination of each primer pair and probe for real-time detection by R would require a great deal of experimentation by trial and error by a skilled person.

[0010]

An object of the present invention is to provide an mRNA encoding an enzyme involved in sulfate conjugation in humans.
To establish a measurement method by PCR, particularly a real-time detection method, and to provide a probe and a primer pair therefor.

The present inventors have solved the above problems by using mRNA encoding an enzyme involved in sulfate conjugation in humans.
As a result of repeating a large number of experiments and researches,
MRN encoding three enzymes involved in sulfate conjugation
We have succeeded in providing a target primer pair and probe combination for A, and have completed the present invention described below.

[0012]

According to the present invention, a probe used for the measurement of mRNA of an enzyme involved in sulfate conjugation in human, which hybridizes to each of the following regions (1) to (6): A probe characterized by being selected from soybean-oligonucleotides; in particular, the probe in which a reporter dye and a quencher dye are bound; the probe having a base sequence length of 20-40;
Provided are the probes comprising any of the sequences set forth in SEQ ID NO: 7, as well as the probes which are any of the sequences set forth in SEQ ID NO: 1-SEQ ID NO: 7. (1) 1318 to 1345 region of CHST2 gene (2) 791 to 819 region of SULT1B1 gene or 448 to 479 region (3) 972 to 999 region of TPST1 gene (4) 431 to 452 region of SULT1A1 gene (5) Regions 441 to 465 of SULT1A2 gene (6) Regions 277 to 302 of SULT1A3 gene

According to the present invention, the following (1) to (6)
A primer pair of a forward primer and a reverse primer each consisting of an oligonucleotide that hybridizes to each of the following regions of a gene encoding an enzyme involved in sulfate conjugation in humans, and an oligo that hybridizes to the following region sandwiched between both primers. At least one selected from the combination of probes consisting of nucleotides
A kit for measuring mRNA encoding an enzyme involved in sulfate conjugation in human, which comprises a set, and in particular, the above-mentioned measurement kit in which the probe further binds a reporter dye and a quencher dye. (1) primer pair; 1295-1316 region and 1365-1347 region of CHST2 gene, probe; 1318-1345 region (2) primer pair; 768-786 region and 880-861 region of SULT1B1 gene, probe; 791-819 region , Or the primer pair; 386-404 region and 518 of the SULT1B1 gene
-498 region, probe; 448-479 region (3) primer pair; 950-968 region of TPST1 gene and
1022-1002 region, probe; 972-999 region (4) primer pair; 391-408 region and 510-491 region of SULT1A1 gene, probe; 431-452 region (5) primer pair; 416-438 region of SULT1A2 gene and 513-492 region, probe; 441-465 region (6) primer pair; 248-267 region and 329-309 region of SULT1A3 gene, probe; 277-302 region

Preferred combinations (sets) of the primer pair and the probe that constitute the above kit include those containing a sequence represented by each SEQ ID NO: selected from the following (1) to (6), and more preferably each: The thing of the sequence shown by a sequence number can be mentioned. (1) Primer pair; SEQ ID NOS: 8 and 9, probe; SEQ ID NO: 1 (2) Primer pair; SEQ ID NOS: 10 and 11, probe;
SEQ ID NO: 2 or primer pair; SEQ ID NOS: 12 and 1
3, probe; SEQ ID NO: 3 (3) primer pair; SEQ ID NOS: 14 and 15, probe;
SEQ ID NO: 4 (4) primer pair; SEQ ID NO: 16 and 17, probe;
SEQ ID NO: 5 (5) primer pair; SEQ ID NOS: 18 and 19, probe;
SEQ ID NO: 6 (6) primer pair; SEQ ID NO: 20 and 21, probe;
Sequence number 7

Further, each of the above-mentioned sets can be used for the measurement of mRNA encoding the following enzymes involved in sulfate conjugation. (1) set; CHST2 measurement set (2) set; SULT1B1 measurement set (3) set; TPST1 measurement set (4) set; SULT1A1 measurement set (5) set; SULT1A2 measurement set Set of (6); for measurement of SULT1A3 According to the present invention, a sample containing mRNA encoding an enzyme involved in sulfate conjugation in human is further analyzed by polymerase chain reaction (PCR) using the measurement kit according to any one of the above. , RT-PCR), and the presence or absence of hydrolysis of the probe used is measured, which encodes an enzyme involved in sulfate conjugation in human.
The above-mentioned measurement method, in which the presence or absence of the above-mentioned hydrolysis is determined by the presence or absence of coloration of fluorescence due to irradiation with excitation light, is provided.

More specifically, the primer pair of a forward primer and a reverse primer and a probe having a reporter dye and a quencher dye and hybridizing with a template nucleic acid in a region sandwiched between the both primers are used for 5'- D having 3'exonuclease activity
Polymerase chain reaction (PC
Performing R) provides a real-time detection method for measuring mRNA encoding an enzyme involved in sulfate conjugation in humans.

The method for real-time detection of mRNA encoding an enzyme involved in sulfate conjugation in humans according to the present invention is capable of fractionating and quantifying various enzymes simply and quickly in real time under the same PCR or RT-PCR reaction conditions. Yes, its establishment is very beneficial in the clinical and pharmaceutical fields.

In addition, in view of the expression of mRNA encoding an enzyme involved in sulfate conjugation in the tissue excised by surgery or the tissue excised by biopsy, various samples can be processed rapidly and from a few tissue pieces. It can be detected with the extracted total RNA.

Furthermore, in the mRNA encoding an enzyme involved in a specific sulfate conjugation, the change in the expression level due to the enzyme induction in the liver, kidney, etc. may change the value in blood (cells having a nucleus contained in blood). Although it may be correlated with the change, the expression level in blood is small and a large amount of blood is required for measurement. However, if the primer and probe designed by the present inventor are used, measurement can be performed with a device that can measure a small amount of sample, and it is useful for measuring the expression level in blood. In addition, in mRNA encoding an enzyme involved in specific sulfate conjugation, changes in the expression level in liver, kidney, etc. may be correlated with changes in the value of cells having nuclei contained in secretory fluid such as saliva. However, the expression level in the cells having nuclei contained in the secretory fluid such as saliva is low, and a large amount of secretory fluid such as saliva is required for the measurement. However, if the primer and probe designed by the present inventor are used, measurement can be performed with a device capable of measuring with a small amount of sample, and it is useful for measuring the expression level in saliva or the like.

The probe and primer pairs used in the method of the present invention will be described in detail below. As described above, each probe and primer is an oligonucleotide that hybridizes to a specific region of the gene of the enzyme involved in sulfate conjugation. Selected from.

The term "hybridize" as used herein means to hybridize under the conditions of PCR (RT-PCR) described later.

The requirements common to the probe and the primer according to the present invention are that the amplification product has a length of 50 to 400 base pairs, that the primer and the probe are as close as possible, and that the G contained in the sequence is It can be mentioned that the ratio of (guanine) and C (cytosine) is around 50% as much as possible, and that four or more G (guanine) are not continuous. Further, other requirements found in the probe of the present invention include that the Tm value is around 70 ° C, and similarly, the primer has a Tm value of 60 ° C.
Before and after can be mentioned.

The mRNA sequences of the respective enzymes involved in sulfate conjugation are known and registered in GeneBank under the following registration numbers. (1) CHST2 gene; XM_031551 (2) SULT1B1 gene; NM_014465 (3) TPST1 gene; NM_003596 (4) SULT1A1 gene; NM_001055 (5) SULT1A2 gene; NM_001054 (6) SULT1A3 gene; NM_0031

In the present specification, the designation of the specific region of the mRNA encoding the enzyme involved in sulfate conjugation is based on the sequence from the start codon of the gene registered with each of the above registration numbers.

The number of nucleotides of oligonucleotides for each primer and probe is at least 15,
It is usually in the range of 15 to 50, preferably 20 to 40. If the number of nucleotides in the primers and probes is too large, the single-stranded DN
It becomes difficult to hybridize to A. On the other hand, if it is too small, the specificity of hybridization decreases.

Examples of preferred specific sequences of specific nucleotide sequences of primers and probes are, as described above, SEQ ID NOS: 1 to 7 (in the case of probes) and SEQ ID NOS: 8 to 21 (primers) depending on each molecular species. In the case of)).

It is known that, for example, a primer or probe consisting of 20 nucleotides hybridizes even if there is a small mismatch with the template strand and can function as a PCR primer or a detection probe. There is. Therefore, the primer and probe of the present invention are also not limited to those having the above-mentioned specific nucleotide sequence, and those including this as a part thereof, or substitution, deletion and / or deletion of, for example, 2 or less nucleotides in this sequence. Or, a sequence modified by addition can be included.

Each of the above-mentioned probe of the present invention and each oligonucleotide as a primer is prepared by an automatic synthesizer,
For example, it can be easily synthesized by using a DNA synthesizer (Perkin Elmer Co., Ltd.), and the obtained oligonucleotide can be further purified, if necessary, by using a commercially available purification cartridge or the like.

The real-time detection probe of the present invention comprises:
A reporter dye is attached to one end, for example the 5'-end, and a quencher dye is attached to the other end, for example the 3'-end. The reporter dye is a substance that fluoresces upon irradiation with excitation light, for example, and the quencher has a function of acting on the reporter dye when it exists in close proximity to the reporter dye to eliminate the generation of the fluorescence. Can be one. Examples of the reporter dye include, for example, 6-carboxy-fluorescein (FAM), tetrachloro-6-carboxyfluorescein (TET), 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein. Inn (JOE),
Hexochloro-6-carboxyfluorescein (HE
X) and the like. Examples of quencher dyes include:
For example, 6-carboxy-tetramethyl-rhodamine (T
AMRA) and the like.

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, on the 5'side of the probe, usually several methylene chains are used as a linker, and a FAM molecule can be bound to the terminal phosphate group in the form of phosphate ester. Further, a TAMRA molecule can be bound to the 3'side by an amide bond via the structural unit shown below.

[0031]

[Chemical 1]

The real-time detection method by PCR using the primer pair and probe of the present invention will be described in detail below.

In the method of the present invention, it is essential to use the above-mentioned primer pair and probe of the present invention, and other known PCR methods (see, for example, Science, 230, 1350 (1985)), RT-PCR (Genome Res). ., 6 (10), 986 (1996)
, Etc., especially real-time detection methods (eg TaqMan PC
R, ABI PRISMTM 7700 SEQUENCE DETECTION SYSTEM, App
lied Biosystems, Ver1, June 1996)).

The method is particularly useful for measuring mRNA as a method for measuring the expression level of mRNA encoding an enzyme involved in sulfate conjugation, which is the subject of the present invention. In this case, m that encodes an enzyme involved in a specific sulfate conjugation
Biological tissues expressing RNA are collected, total RNA is extracted according to a conventional method, and then cDNA complementary thereto.
Is synthesized according to a conventional method, and then PCR may be performed using the primer pair of the present invention and the probe. Alternatively, RT-PCR can be directly performed using the primer and probe of the present invention after extracting total RNA according to a conventional method.

The PCR reaction and RT-PCR reaction can basically follow known methods. The reaction conditions can also be appropriately determined according to known methods and are not particularly different. Specifically, the conditions shown in Examples below can be preferably adopted.

The detection can also be carried out 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, mRNA encoding each enzyme involved in the sulphate conjugation involved in the metabolism of a desired drug, environmental hormone, in-vivo substance and the like can be easily and rapidly converted into the sulphate conjugate. It can be measured and detected for each mRNA encoding each involved enzyme.

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

According to the method of the present invention, the mRNA encoding the enzyme involved in sulfate conjugation can be measured and detected for each mRNA encoding the enzyme involved in sulfate conjugation. MR to code
NA can be quantified quickly and accurately.

[0040]

EXAMPLES Hereinafter, test examples and examples will be given in order to explain the present invention in more detail.

[0041]

[Test example] Preparation of calibration curve by real-time detection method (1) Test method Real-time one-step RT-PC adopted in this test
The R method uses 96 wells and can simultaneously perform up to 96 different reactions to measure up to 96 analytes at one time. Moreover, RT reaction and PCR reaction can be performed in one tube. Furthermore, measurement with 384 wells is also possible.

The principle of the present quantification is that, by using two types of fluorescent dyes adjacent to each other, the wavelength region between the fluorescence wavelength of one dye (reporter dye) and the excitation light wavelength of the other dye (quencher dye) is used. It uses FRET that occurs when the two overlap. MRN that encodes an enzyme involved in specific sulfate conjugation amplified by PCR using a probe (TaqMan probe) in which two types of FRET-producing fluorescent dyes are bound to both ends
Hybridization to cDNA derived from A species, in this state, PCR extension reaction starts, TaqMan probe is hydrolyzed by the 5′-3 ′ endonuclease activity of Taq DNA polymerase, the reporter dye is released, and the quencher dye And the fluorescence intensity of the reporter dye that had been suppressed by FRET increased, and this increase in fluorescence intensity was proportional to the increase in PCR amplification product. By doing so, the desired quantification becomes possible.

In this test, FAM was used as a reporter dye on the 5'end side of the probe, and TAMRA was used as a quencher dye on the 3'end side. The binding of each dye and the preparation of the TaqMan probe by this were performed according to the method described in the literature (Genome Res., 6 (10), 986 (1996)).

Further, the oligonucleotides as the primers and the probes are
Using a DNA / RNA synthesizer manufactured by Company I, the substrate (dN
TP) and defined reagents.

As the sample RNA, total RNA purified from an adult liver pool was used. The total RNA was purchased from Clontech Laboratories, Inc.

Total RNA purified from adult liver pool
Was diluted with RNase-free water to 20 μg / mL to prepare a calibration curve. Thereafter, using 50 μg / mL yeast tRNA (Yeast tRNA, manufactured by GIBCO),
Diluted at a 5-fold public ratio. 5 μL was used for the measurement.

The RT-PCR reaction consisted of 300 nM forward primer, 900 nM reverse primer and 2 nM.
TaqMan One-Step RT-PC with 00nM TaqMan probe
R Master Mix Reagents Kit (PE Applied Biosystems)
With 50 μL / tube system using ABI PRI
SM ^™ 7700 Sequence Detection System (PE Appl
ied Biosystems).

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

(2) Test Results The results of the above-mentioned test (calibration performed on the mRNAs encoding the enzymes involved in sulfate conjugation shown in Table 1 below using a primer pair and a probe having the sequences shown by the respective SEQ ID NOs. The line) is shown in Table 2.

[0050]

[Table 1]

[0051]

[Table 2]

From Table 2 above, 100,000 pg total RNA
A calibration curve prepared by diluting the reaction solution volume of 50 μL with a 5-fold common ratio was prepared, and CHST2 had quantitative properties up to 160 pg total RNA / 50 μL reaction solution volume, and S
ULT1B1 has a quantitative property up to 32 pg total RNA / 50 μL reaction solution volume, TPST1 has a quantitative property up to 800 pg total RNA / 50 μL reaction solution volume, and SULT1A1 has 32 pg total RNA volume.
Quantitative up to / 50 μL reaction volume, SULT
1A2 had quantification up to 800 pg total RNA / 50 μL reaction volume, and SULT1A3 had quantification up to 160 pg total RNA / 50 μL reaction volume. The correlation coefficients (r) of the calibration curves were all 0.99 or more.

[0053]

[Sequence list]

SEQUENCE LISTING <110> Otsuka Pharmaceutical Factory, Inc. <120> mRNA of enzymes involved in sulfate conjugation in humans
Method, probe and kit therefor <130> 02P1177 <160> 21 <170> PatentIn Ver. 2.0 <210> 1 <211> 28 <212> DNA <213> human CHST2 gene <400> 1 tacgattttg tgggactgtt ggtgagcc 28 <210> 2 <211> 29 <212> DNA <213> human SULT1B1 gene <400> 2 actggaagaa ttacttcacc gtggcccaa 29 <210> 3 <211> 32 <212> DNA <213> human SULT1B1 gene <400> 3 cagccttttc ctggtacctg ggaagaatat ct 32 <210> 4 <211> 28 <212> DNA <213> human TPST1 gene <400> 4 tggatatgac ccatatgcca acccacct 28 <210> 5 <211> 22 <212> DNA <213> human SULT1A1 gene <400> 5 acatggccaa ggtgcaccct ga 22 <210> 6 <211> 25 <212> DNA <213> human SULT1A2 gene <400> 6 agtgtaccct caccctggga cctgg 25 <210> 7 <211> 26 <212> DNA <213> human SULT1A3 gene <400> 7 ctggagactc tgaaagacac accgcc 26 <210> 8 <211> 22 <212> DNA <213> human CHST2 gene <400> 8 ccgtcaagac actacggaga gt 22 <210> 9 <211> 19 <212> DNA <213> human CHST2 gene <400> 9 ggcaaactgc tccatttcg 19 <210> 10 <211> 19 <212> DNA <213> human SULT1B1 gene <400> 10 tatgcgtaaa ggg acggct 19 <210> 11 <211> 20 <212> DNA <213> human SULT1B1 gene <400> 11 tgcggaattg aagtgcagtt 20 <210> 12 <211> 19 <212> DNA <213> human SULT1B1 gene <400> 12 tggctcgtaa tgccaagga 19 <210> 13 <211> 21 <212> DNA <213> human SULT1B1 gene <400> 13 caggaaccat aggccacttt t 21 <210> 14 <211> 19 <212> DNA <213> human TPST1 gene <400> 14 ttgctcctat gcttgccaa 19 <210> 15 <211> 21 <212> DNA <213> human TPST1 gene <400> 15 ttgggatcag gttttccgta g 21 <210> 16 <211> 18 <212> DNA <213> human SULT1A1 gene <400> 16 aacgcaaagg atgtggca 18 <210> 17 <211> 20 <212> DNA <213> human SULT1A1 gene <400> 17 tccgtaggac acttctccga 20 <210> 18 <211> 23 <212> DNA <213> human SULT1A2 gene <400> 18 actaccactt ctaccacatg gcc 23 <210> 19 <211> 22 <212> DNA <213> human SULT1A2 gene <400> 19 ggacccatag gacacttctc ca 22 <210> 20 <211> 20 <212> DNA <213> human SULT1A3 gene <400> 20 aggtcaatga tccaggggaa 20 <210> 21 <211> 21 <212> DNA <213> human SULT1A3 gene <400> 21 ggcaggtgtg acttgatgag c 21

   ─────────────────────────────────────────────────── ─── Continued front page    F term (reference) 4B024 AA01 AA11 CA12 HA13                 4B063 QA01 QA18 QQ53 QR32 QR36                       QR56 QS25 QS34 QS36 QX02

Claims (12)

[Claims] 1. An enzyme m involved in sulfate conjugation in humans.
A probe used for measuring RNA, comprising the following (1)
A probe selected from oligonucleotides that hybridize to each of the regions (6) to (6). (1) 1318 to 1345 region of CHST2 gene (2) 791 to 819 region of SULT1B1 gene or 448 to 479 region (3) 972 to 999 region of TPST1 gene (4) 431 to 452 region of SULT1A1 gene (5) Regions 441 to 465 of SULT1A2 gene (6) Regions 277 to 302 of SULT1A3 gene 2. The probe according to claim 1, wherein the reporter dye and the quencher dye are bound to each other. 3. The probe according to claim 1, wherein the base sequence has a length of 20-40. 4. The probe according to claim 1, which comprises any of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 7. 5. The probe according to any one of claims 1 to 3, which is one of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 7. 6. A primer pair consisting of a forward primer and a reverse primer, each of which comprises an oligonucleotide that hybridizes to each of the following regions of a gene encoding an enzyme involved in sulfate conjugation in humans according to the following (1) to (6): And a kit for measuring mRNA encoding an enzyme involved in sulfate conjugation in human, comprising at least one set selected from a combination of probes consisting of oligonucleotides hybridizing to the following region sandwiched between both primers. (1) primer pair; 1295-1316 region and 1365-1347 region of CHST2 gene, probe; 1318-1345 region (2) primer pair; 768-786 region and 880-861 region of SULT1B1 gene, probe; 791-819 region , Or the primer pair; 386-404 region and 518 of the SULT1B1 gene
-498 region, probe; 448-479 region (3) primer pair; 950-968 region of TPST1 gene and
1022-1002 region, probe; 972-999 region (4) primer pair; 391-408 region and 510-491 region of SULT1A1 gene, probe; 431-452 region (5) primer pair; 416-438 region of SULT1A2 gene and 513-492 region, probe; 441-465 region (6) primer pair; 248-267 region and 329-309 region of SULT1A3 gene, probe; 277-302 region 7. The measurement kit according to claim 6, wherein the probe further comprises a reporter dye and a quencher dye bound thereto. 8. The measurement according to claim 6 or 7, which is selected from the combination of a primer pair consisting of an oligonucleotide containing the sequence shown by each SEQ ID NO: and a probe in the set shown in (1) to (6) below. kit. (1) Primer pair; SEQ ID NOS: 8 and 9, probe; SEQ ID NO: 1 (2) Primer pair; SEQ ID NOS: 10 and 11, probe;
SEQ ID NO: 2 or primer pair; SEQ ID NOS: 12 and 1
3, probe; SEQ ID NO: 3 (3) primer pair; SEQ ID NOS: 14 and 15, probe;
SEQ ID NO: 4 (4) primer pair; SEQ ID NO: 16 and 17, probe;
SEQ ID NO: 5 (5) primer pair; SEQ ID NOS: 18 and 19, probe;
SEQ ID NO: 6 (6) primer pair; SEQ ID NO: 20 and 21, probe;
Sequence number 7 9. The measurement kit according to claim 8, wherein
The set of (1) is for CHST2 measurement, and the set of (2) is S
For the measurement of ULT1B1, the set of (3) is for the measurement of TPST1, and the set of (4) is for the measurement of SULT1A1,
A measurement kit in which the set of (5) is for measuring SULT1A2 and the set of (6) is for measuring SULT1A3. 10. A combination of a primer pair consisting of an oligonucleotide having a sequence represented by each SEQ ID NO: and a probe in the set represented by (1) to (6) according to claim 8. The measurement kit described in. 11. A sample containing mRNA encoding an enzyme involved in sulfate conjugation in humans, according to any one of claims 6 to 1.
A polymerase chain reaction (PCR) is carried out using the measurement kit according to any one of 0 to 16 above, and the presence or absence of hydrolysis of the probe used is measured to detect mRNA encoding an enzyme involved in sulfate conjugation in humans. Measuring method. 12. The measuring method according to claim 11, wherein the presence or absence of hydrolysis is determined by the presence or absence of coloration of fluorescence upon irradiation with excitation light.