CN105861686A - Kit for testing clozapine application effect based on rs2032582 and application method of kit - Google Patents
Kit for testing clozapine application effect based on rs2032582 and application method of kit Download PDFInfo
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- CN105861686A CN105861686A CN201610298347.8A CN201610298347A CN105861686A CN 105861686 A CN105861686 A CN 105861686A CN 201610298347 A CN201610298347 A CN 201610298347A CN 105861686 A CN105861686 A CN 105861686A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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Abstract
The invention relates to a kit for testing clozapine application effect based on rs2032582 and an application method of the kit, and particularly relates to a method for testing single nucleotide polymorphism of ABCB1 (ATP binding cassette subfamily B member 1) NO.8 exon in the field of genes. Sequence of the nucleotide, which is isolated and comprises single nucleotide polymorphism locus, is as shown as SEQ ID NO:1; the single nucleotide polymorphism locus is located at NO.3169, rs2032582 (C->T). The method for testing the nucleotide includes the steps of firstly, testing the nucleotide at the locus NO.3169 of the sequence as shown as SEQ ID NO:1; secondly, testing whether there is of the single nucleotide polymorphism at the locus NO.3169 or not by means of a Taqman-MGB typing method. According to the kit, the application method thereof and the method for testing the nucleotide, a foundation for research on ABCB1 gene polymorphism and clinical medicine safety is laid, a theoretical basis for individualization of clinical medicine is provided, and a guidance for research and development of novel medicines based on pharmacogenomics idea is also provided.
Description
Technical field
The present invention relates to gene technology field, the mononucleotide being specifically related to a kind of ABCB1 gene the 8th exon is many
The detection method of state property.
Background technology
Clozapine (Clozapine) the chloro-11-of chemical entitled 8-(4-methyl isophthalic acid-piperazinyl)-5H-dibenzo [b, e] [1,
4] diazepine, molecular formula C18H19ClN4.Clozapine is the medicine of domestic most common treatment mental sickness, not only to spirit
The sick positive symptom is effective, also has certain effect to negative symptoms.It is applicable to acute and each hypotype chronic schizophrenia, right
Hallucinatory paranoid form, hebephrenictype are effective.The affective symptom relevant with schizophrenia can also be alleviated (such as depression, sense of guilt, Jiao
The symptoms such as worry).But, clozapine drug effect in clinic and drug side effect have obvious individual variation, and reason is its generation
Thank and affected by inherited genetic factors and environmental factors, but inherited genetic factors is topmost reason.Therefore more and chlorine nitrogen is developed
Molecular marked compound that flat metabolism is relevant thus patient is realized individualized treatment and is necessary.
ABCB1 (ATP binding cassette subfamily B member 1) gene is also called multidrug resistance base
Because of (MDR1).ABCB1 gene is positioned on No. 7 chromosome q21.1 of the mankind, and its cDNA total length 4669bp comprises 29 exons,
Transcribe the mRNA of available 4.5kb, the transmembrane glycoprotein (P-gp) of one a length of 170kDa of coding.P-glycoprotein is by 1280
Individual amino acid transporter is constituted, and is an important member of the ATP binding cassette transporter body superfamily of high conservative in heredity, also
It it is the export-oriented transporter of ATP dependence.P-P-glycoprotein expression is in intestinal epithelial cell membrane, official jargon film, stones in intrahepatic bile duct cell membrane, and kidney is near
Convoluted tubule cell membrane, and the blood brain barrier collectively formed with blood capillary epithelium, its major function is to stop medicine and exotic
Matter enters inside body tissue, such as antidepressant drug, antineoplastic agent, glucocorticoid and amyloid etc..Due to P-gp pair
The outer row effect of exogenous material and medicine etc., the difference that ABCB1 gene pleiomorphism and P-gp express, may result in different groups or
Different susceptibilitys is there is in individuality to some disease.Due to the effect in resisting mental disease drug metabolism of the ABCB1 gene and work
Property there is individual variation, the relation therefore finding Metabolism of Clozapine and ABCB1 gene pleiomorphism is that prior art is badly in need of solving
A difficult problem.Retrieval to existing document, applicant finds no ABCB1 gene the 8th exon SNP of the present invention
Rs2032582 is the relevant report of clozapine molecular marked compound.
Summary of the invention
For defect of the prior art, it is an object of the invention to provide the list of a kind of ABCB1 gene the 8th exon
The detection method of nucleotide polymorphisms.The present invention is that the relation of research ABCB1 gene pleiomorphism and clinical drug safety is established
Basis, provides fundamental basis for clinical application individuation, the most also provides for new drug development based on pharmacogenomics theory
Instruct foundation.
The present invention is to be realized by following technical scheme,
First aspect, the present invention provides the nucleotide containing mononucleotide polymorphism site of a kind of separation, described nucleotide
Sequence as shown in SEQ ID NO:1, described mononucleotide polymorphism site is positioned at the 3169th, and rs2032582 is C → T.
Second aspect, the present invention provides a kind of described nucleotide in the test kit of preparation detection clozapine medication effect
Purposes.
The third aspect, the present invention provides the nucleotide of a kind of separation, the sequence of described nucleotide such as SEQ ID NO:1 institute
Show, and the 3169th is C.
Fourth aspect, the present invention provides a kind of test kit detecting clozapine medication effect, and described test kit includes: according to
The primer pair of the mononucleotide polymorphism site rs2032582 design of sequence, probe as shown in SEQ ID NO:1.
Preferably, described primer to for:
Forward primer 5 '-CTGGACAAGCACTGAAAGATAA-3 '
Reverse primer 5 '-AGAGCATAGTAAGCAGTAGGGAGTA-3 '.
Preferably, described probe is:
Forward 5'-FAM-TAGAAGGTCCTGGGAAGGTG-NFQ-MGB-3'
Reversely 5'-VIC-TAGAAGGTTCTGGGAAGGTG-NFQ-MGB-3'.
5th aspect, the present invention provides the non-diagnostic purpose using method of a kind of described test kit, comprises the steps:
Step one, extracts sample DNA;
Step 2, utilizes described primer to expand, obtains amplified production;
Whether step 3, use Taqman-MGB classifying method, bonding probes, exist such as SEQ ID NO in detection sample:
Mononucleotide polymorphism site rs2032582 shown in 1.
Preferably, step 3 comprises the steps: to use ABI7700 quantitative real time PCR Instrument, the site to taken sample
Rs2032582 carries out typing, detects the 3169th and no there is single nucleotide polymorphism.
Preferably, a length of 50-150bp of described amplified production.
Preferably, a length of 15-25bp of described primer.
Preferably, in step 2, the actual conditions of described amplification is as follows:
Reaction system cumulative volume is 5 μ l, wherein,
PCR reaction condition: 94 DEG C of 2mins;40×(94℃30s;58 DEG C of 30s, 72 DEG C of 30s) 72 DEG C of 10mins.
6th aspect, the present invention provides the primer pair of a kind of SNP site for detecting described nucleotide, described primer pair
For:
Forward primer 5 '-CTGGACAAGCACTGAAAGATAA-3 '
Reverse primer 5 '-AGAGCATAGTAAGCAGTAGGGAGTA-3 '.
7th aspect, the present invention provides the probe of a kind of SNP site for detecting nucleotide as claimed in claim 1,
Described probe is:
Forward 5'-FAM-TAGAAGGTCCTGGGAAGGTG-NFQ-MGB-3'
Reversely 5'-VIC-TAGAAGGTTCTGGGAAGGTG-NFQ-MGB-3'.
Compared with prior art, the present invention has a following beneficial effect: the present invention for research ABCB1 gene pleiomorphism with
The relation of clinical drug safety is laid a good foundation, and provides fundamental basis for clinical application individuation, is also based on medicine base simultaneously
Foundation is instructed in the new drug development offer read because of group scientific principle.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are merely to illustrate the present invention
Rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, usual more solito condition,
The molecular cloning of such as Sambrook et al.: laboratory manual (New York:Cold Spring Harbor Laboratory
Press, 1989) condition described in, or according to the condition proposed by manufacturer.
Embodiment
The present invention relates to the detection side of the single nucleotide polymorphism of ABCB1 gene the 8th exon SNP rs2032582
Method, relates to separation nucleic acid and the primer being correlated with simultaneously.The present invention has had been surprisingly found that ABCB1 gene the 8th by association analysis
SNP rs2032582 on exon can be as the molecular marked compound of clozapine, and the specifying information of this ABCB1 gene is
ABCB1 gene LOCUS NC_000007, total length 209461bp, the present invention uses Taqman-MGB probe technique to ABCB1 gene
The 8th exon SNP site and frequency distribution situation detected, find ABCB1 gene the 8th exon
In 27439th C → T polymorphism, i.e. its coded protein sequence, the 893rd amino acids Arg (R) → His (H) is polymorphic,
I.e. SNP rs2032582.ABCB1 gene is at chromosome position: Homo sapiens chromosome7, GRCh38.p2
(87503863..87713323), SNP rs2032582 is at chromosome position Chromosome:7:87531302.In the present invention
Relating to the concrete nucleotide fragments sequence of ABCB1 gene as shown in SEQ ID NO.1, wherein mononucleotide polymorphism site is positioned at
The 3169th of sequence.
The present invention is directed to primer that this SNP rs2032582 relate to be correlated with to and probe.
Specifically, present invention SNP rs2032582 upstream and downstream design on ABCB1 gene the 8th exon
Taqman-MGB primer, amplified production length 50-150bp;Designing Taqman-MGB probe between upstream and downstream primer, probe is long
15-25bp.Taken sample ABCB1 gene SNP site rs2032582 carried out point with ABI7700 quantitative real time PCR Instrument afterwards
Type, finds ABCB1 gene the 8th exon SNP (i.e. the 27439th C → T polymorphism), and frequency is 0.255.Send out through analyzing
Existing, this SNP cause the 893rd amino acids byOwing to this is the amino acid whose displacement of heterogeneity, because of
This can cause the protein active of ABCB1 coded by said gene to change.
The present invention has substantial amounts of analytical technology to can be used for detecting whether site described in ABCB1 gene the 8th exon deposits
In single nucleotide polymorphism.These technology include (but being not limited to): DNA sequencing, sequencing by hybridization;Enzymatic mispairing cutting, allos
RNA polymerase, dot blot, oligonucleotide arrays (DNA chip), Mini-sequencing, molecular beacon etc., denaturing high-performance liquid phase
Chromatography (DHPLC).On the other hand, the detection method of the present invention is used for assessing individual ABCB1 gene the 8th exon SNP
Rs2032582 polymorphism and the relation of Metabolism of Clozapine.Test sample for the present invention is not particularly limited, for detection
For SNP, can be to separate, from blood, tissue etc., DNA or mRNA extracted sample.For ABCB1 gene the 8th extra
For aobvious sub-activity, can any sample containing ABCB1 gene the 8th exon, such as blood etc..
Described " isolated or purification " refers in genomic DNA level, utilizes primer each many to expanding
The gene order in state property site and utilize purification kit that amplified production is purified.
The following is the specific implementation process to the present invention program:
The sample that the present embodiment uses is from 995 schizophrenic's blood samples of Shanghai Mental Health Center
Product, age 18-60 year, all participants are agreed to sign by family members on formal Informed Consent Form.The present embodiment relates to
The cDNA sequence of ABCB1 gene the 8th exon is listed in SEQ ID NO:1, and its coded aminoacid sequence is listed in SEQ ID
NO:2;The genome sequence of ABCB1 gene the 8th exon can obtain from GenBank.
Design of primers: use Primer 5.0 software, with GenBank data base's ABCB1 gene (ID:5243) the 8th extra
Aobvious son and exon are stencil design pair of primers with intron junction sequence, Invitrogen company synthesize.
Primer information:
The DNA sequence primer information of amplification ABCB1 gene the 8th exon SNP site:
Forward primer 5 '-CTGGACAAGCACTGAAAGATAA-3 ' (SEQ ID NO:3)
Reverse primer 5 '-AGAGCATAGTAAGCAGTAGGGAGTA-3 ' (SEQ ID NO:4).
Taqman-MGB probe:
Forward 5'-FAM-TAGAAGGTCCTGGGAAGGTG (SEQ ID NO:5)-NFQ-MGB-3'
Reversely 5'-VIC-TAGAAGGTTCTGGGAAGGTG (SEQ ID NO:6)-NFQ-MGB-3'.
PCR amplification condition: (the every reagent of system is provided by ABI company, easily can obtain from disclosed channel),
Reaction system cumulative volume is 5 μ l, wherein:
PCR reaction condition: 94 DEG C of 2mins;40×(94℃30s;58 DEG C of 30s, 72 DEG C of 30s) 72 DEG C of 10mins.
Above-mentioned PCR reaction is carried out in 96 orifice plates, is placed in ABI7700 quantitative real time PCR Instrument, reads in each sample
ABCB1 gene the 8th exon SNP rs2032582 polymorphism, experimental result is as shown in table 1.
Relatively finding that ABCB1 gene the 8th exon SNP has polymorphism (i.e. the 27439th C → T), frequency is
0.255.Through analyze find, this SNP cause the 893rd amino acids byAssociation analysis finds ABCB1
Significant is had before and after the C genotype of gene the 8th exon SNP rs2032582 and T genotype patient's Clozapine in Treating
Body difference, P value is 0.028.
In sum, the present invention is that the relation of research ABCB1 gene pleiomorphism and clinical drug safety is laid a good foundation, for
Clinical application individuation is provided fundamental basis, and the most also provides guidance to depend on for new drug development based on pharmacogenomics theory
According to.
Above the specific embodiment of the present invention is described.It is to be appreciated that the invention is not limited in above-mentioned
Particular implementation, those skilled in the art can make various deformation or amendment within the scope of the claims, this not shadow
Ring the flesh and blood of the present invention.
Table 1
Claims (13)
1. the nucleotide containing mononucleotide polymorphism site separated, it is characterised in that the sequence of described nucleotide such as SEQ
Shown in ID NO:1, described mononucleotide polymorphism site is positioned at the 3169th, and rs2032582 is C → T.
2. a nucleotide as claimed in claim 1 purposes in the test kit of preparation detection clozapine medication effect.
3. the nucleotide separated, it is characterised in that the sequence of described nucleotide as shown in SEQ ID NO:1, and the 3169th
Position is C.
4. the test kit detecting clozapine medication effect, it is characterised in that described test kit includes: according to such as SEQ ID
The primer pair of the mononucleotide polymorphism site rs2032582 design of sequence shown in NO:1, probe.
Test kit the most according to claim 4, is characterized in that, described primer to for:
Forward primer 5 '-CTGGACAAGCACTGAAAGATAA-3 '
Reverse primer 5 '-AGAGCATAGTAAGCAGTAGGGAGTA-3 '.
Test kit the most according to claim 4, is characterized in that, described probe is:
Forward 5'-FAM-TAGAAGGTCCTGGGAAGGTG-NFQ-MGB-3'
Reversely 5'-VIC-TAGAAGGTTCTGGGAAGGTG-NFQ-MGB-3'.
7. the non-diagnostic purpose using method of a test kit as claimed in claim 4, it is characterised in that include walking as follows
Rapid:
Step one, extracts sample DNA;
Step 2, utilizes described primer to expand, obtains amplified production;
Whether step 3, use Taqman-MGB classifying method, bonding probes, exist such as SEQ ID NO:1 institute in detection sample
The mononucleotide polymorphism site rs2032582 shown.
8. using method as claimed in claim 7, is characterized in that, step 3 comprises the steps: to use ABI7700 fluorescence fixed
Amount PCR instrument, carries out typing to the site rs2032582 of taken sample, detects the 3169th and no there is single nucleotide polymorphism.
9. using method as claimed in claim 7, is characterized in that, a length of 50-150bp of described amplified production.
10. using method as claimed in claim 7, is characterized in that, a length of 15-25bp of described primer.
11. usings method as claimed in claim 7, it is characterised in that in step 2, the actual conditions of described amplification is as follows:
Reaction system cumulative volume is 5 μ l, wherein,
PCR reaction condition: 94 DEG C of 2mins;40×(94℃30s;58 DEG C of 30s, 72 DEG C of 30s) 72 DEG C of 10mins.
12. 1 kinds for detecting the primer pair of the SNP site of nucleotide as claimed in claim 1, it is characterised in that described primer
To for:
Forward primer 5 '-CTGGACAAGCACTGAAAGATAA-3 '
Reverse primer 5 '-AGAGCATAGTAAGCAGTAGGGAGTA-3 '.
13. 1 kinds for detecting the probe of the SNP site of nucleotide as claimed in claim 1, it is characterised in that described probe
For:
Forward 5'-FAM-TAGAAGGTCCTGGGAAGGTG-NFQ-MGB-3'
Reversely 5'-VIC-TAGAAGGTTCTGGGAAGGTG-NFQ-MGB-3'.
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108929913A (en) * | 2018-08-21 | 2018-12-04 | 潍坊德诺泰克生物科技有限公司 | For detecting the primed probe group and its application of rs2032582 |
CN109295229A (en) * | 2018-10-22 | 2019-02-01 | 北京华夏时代生物工程有限公司 | Detection method is sequenced in the SNP fluorescence in situ hybridization of ABCB1 and SLCO1B1 |
CN112195230A (en) * | 2020-09-17 | 2021-01-08 | 青岛大学附属医院 | Gene detection reagent, kit, detection method and application for clozapine personalized medication guidance |
CN112980943A (en) * | 2021-03-31 | 2021-06-18 | 山东英盛生物技术有限公司 | Method for detecting tacrolimus precise medication, primer, PCR reagent and kit |
CN114569731A (en) * | 2022-03-01 | 2022-06-03 | 山东大学 | Molecular beacon modified nano-carrier and application thereof in preparation of anti-tumor products |
-
2016
- 2016-05-06 CN CN201610298347.8A patent/CN105861686A/en active Pending
Non-Patent Citations (3)
Title |
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GIORGIO CONSOLI等: "ABCB1 polymorphisms are associated with clozapine plasma levels in psychotic patient", 《FUTURE MEDICINE》 * |
JIA M等: "homo sapiens ATP-binding cassette, sub-family B(MDR/TAP), member 1(ABCB1), mRNA", 《GENBANK DATABASE》 * |
XU Q等: "Association studies of genomic variants with treatment response to risperidone, clozapine, quetiapine and chlorpromazine in the Chinese Han population", 《THE PHARMACOGENOMICS JOURNAL》 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108929913A (en) * | 2018-08-21 | 2018-12-04 | 潍坊德诺泰克生物科技有限公司 | For detecting the primed probe group and its application of rs2032582 |
CN109295229A (en) * | 2018-10-22 | 2019-02-01 | 北京华夏时代生物工程有限公司 | Detection method is sequenced in the SNP fluorescence in situ hybridization of ABCB1 and SLCO1B1 |
CN112195230A (en) * | 2020-09-17 | 2021-01-08 | 青岛大学附属医院 | Gene detection reagent, kit, detection method and application for clozapine personalized medication guidance |
CN112980943A (en) * | 2021-03-31 | 2021-06-18 | 山东英盛生物技术有限公司 | Method for detecting tacrolimus precise medication, primer, PCR reagent and kit |
CN114569731A (en) * | 2022-03-01 | 2022-06-03 | 山东大学 | Molecular beacon modified nano-carrier and application thereof in preparation of anti-tumor products |
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