CN105002281A - CYP2C9 and VKORC1 gene typing detection reagent kit - Google Patents
CYP2C9 and VKORC1 gene typing detection reagent kit Download PDFInfo
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- CN105002281A CN105002281A CN201510443858.XA CN201510443858A CN105002281A CN 105002281 A CN105002281 A CN 105002281A CN 201510443858 A CN201510443858 A CN 201510443858A CN 105002281 A CN105002281 A CN 105002281A
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- cyp2c9
- dna
- seq
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- C—CHEMISTRY; METALLURGY
- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
Abstract
The invention discloses a CYP2C9 and VKORC1 gene typing detection reagent kit. The CYP2C9 and VKORC1 gene typing detection reagent kit comprises a gene chip and various primers. The gene chip is provided with 4 mutation sites of CYP2C9 and VKORC1 genes and nucleotide sequence probes in normal contrast and complementation with the mutation sites, wherein the probes have the sequences shown in SEQ ID Nos. 1-8 or sequences complementary to the sequences shown in SEQ ID Nos. 1-8; the gene chip is further provided with an DNA sequence marked with biotin points, wherein the DNA sequence is shown in SEQ ID No.9. The primers have the sequences shown in SEQ ID Nos. 10-17. The CYP2C9 and VKORC1 gene typing detection reagent kit provides a CYP2C9 and VKORC1 gene typing joint detection platform, can achieve synchronous joint detection, improve detection specificity, lower the cost and shorten detection time, and has great significance in carrying out CYP2C9 and VKORC1 gene detection and mass screening.
Description
Technical field
The present invention relates to a kind of test kit, being specifically related to a kind of for diagnosing the detection kit of CYP2C9 and VKORC1 gene.
Background technology
Cytochrome P450 (CYP) is the isozyme of one group of structurally and functionally related superfamily genes encoding.In mammalian tissues, P450 is play an important role in the process of bioactive molecules at the metabolism of medicine and abnormal shape biology, steroid hormone synthesis, liposoluble vitamin metabolism and many unsaturations convert fatty acids.Because it plays a very important role in the bio-transformation of exogenous compounds (comprising medicine and poisonous substance), so its activity determines the metabolic rate of medicine, having direct relation with the clearance rate of medicine, is the phasel enzyme of drug metabolism, thus also known as drug metabolism enzyme.P450 enzyme system composition is complicated, is controlled, be called P450 gene superfamilies by gene diversity.In P450 superfamily, mankind's encodes cytochrome P450 gene belongs to 42 families of 17 gene families.What wherein relate to most drug metabolism in body mainly contains 3 gene families (CYP1, CYP2, CYP3).
In CYP450 superfamily, CYP2 is maximum family, there are 15 subfamilies, wherein CYP2A-2E is present in Mammals, CYP2C is wherein maximum subfamily, in this subfamily CYP2C9,2C19 and drug metabolism in close relations, they are to the many distributions in two condition of the katalysis of drug metabolism, and namely the drug metabolism of fast metabolizer and poor metabolizer exists larger difference.Difference should be had to fast metabolism and poor metabolizer's dosage.Warfarin (Warfarin) belongs to coumarins oral anticoagulant, is the first-line drug being used for the treatment of blood embolism diseases (as Cardiac valve replacement, atrial fibrillation, venous thrombosis and lung infraction etc.).CYP2C9 is the main metabolic enzyme of warfarin, and some genotype of its SNP can cause the decline alive of the enzyme of CYP2C9, so that warfarin removing is in vivo slowed down.Warfarin is by suppressing VITAMIN epoxide reductase (VKOR), the thrombogen that vitamin K is participated in, VII, Ⅸ, Ⅹ biosynthesis block, thus performance anticoagulation, therefore the SNP of an important subunit gene (VKORC1) of VKOR also can cause individuality to there is notable difference to the susceptibility of warfarin.In addition the treatment window of warfarin is narrower, and little doses change also may cause thrombus or hemorrhage, and therefore in clinical application, the single nucleotide polymorphism of genes involved detects the administration foundation that can be used as personalized treatment, has important clinical meaning to treatment plan.
At present, the detection analytical technique of gene pleiomorphism is mainly comprised: ARMS fluorescent PCR method, the methods such as gene sequencing, restriction fragment length polymorphism method (RFLP).These methods have respective advantage and defect, still have much room for improvement and stdn.
Therefore a kind of high specificity of Study and Development, susceptibility are high and quick, economical, CYP2C9 and the VKORC1 gene tester of multiple site mutation can be checked simultaneously to seem particularly important, play an important role to CYP2C9 and VKORC1 detection in Gene Mutation.
Summary of the invention
The object of the invention mainly in order to make up the deficiency of existing CYP2C9 and VKORC1 gene test in somatotype, provides a kind of detection kit that simultaneously can detect multiple CYP2C9 and VKORC1 gene mutation site.
A kind of CYP2C9 and VKORC1 gene parting detecting reagent of the present invention, comprising:
(1) gene chip, on it with:
4 mutational sites of (i) CYP2C9 and VKORC1 gene and the nucleotide sequence probe (totally 8 probes) of normal control complementation thereof, its middle probe is SEQ ID Nos:1-8 or the sequence with SEQ ID Nos:1-8 complementation, and concrete sequence is as follows:
Title sequence number sequence
C9*2PN SEQ ID NO.1 TCTTGAACACGGTCCT
C9*2PM SEQ ID NO.2 TCTTGAACACAGTCCT
C9*3PN SEQ ID NO.3 CCAGAGATACATTGACCT
C9*3PM SEQ ID NO.4 CCAGAGATACCTTGACCT
VK1639PN SEQ ID NO.5 ATTGGCCGGGTGCGGT
VK1639PM SEQ ID NO.6 ATTGGCCAGGTGCGGT
VK1173PN SEQ ID NO.7 TCATCGACCCTTGGACTAG
VK1173PM SEQ ID NO.8 TCATCGACTCTTGGACTAG
(ii) be marked with the DNA sequence dna (Bio) of biological vegetarian refreshments, whether successful for monitoring hybridization, sequence is SEQ ID No.9:
Bio CGTCCAAGGGGAAACTGATCT SEQ ID No.9；
(2) various primer, for the DNA sequence dna increased in clinical sample, the DNA sequence dna of its specificity amplification primer is SEQ ID Nos.10-17, and concrete sequence is as follows:
Title sequence number sequence
C19*2-F SEQ ID NO.10 AGTGTCAGCTTCCTCTTTCT
C19*2-R SEQ ID NO.11 CAGTAAGGTCAGTGATATGGAGT
C19*3-F SEQ ID NO.12 GTCCAGGAAGAGATTGAACG
C19*3-R SEQ ID NO.13 CATGGAGTTGCAGTGTAGGA
VK1639-F SEQ ID NO.14 GGTAGGTGCAACAGTAAGG
VK1639-R SEQ ID NO.15 CAGGGTTCAAGTGGTTCTC
VK1173-F SEQ ID NO.16 AGGGTCAGTGACATGGAAT
VK1173-R SEQ ID NO.17 GGTGGAACCAGGTTAGGA
The present invention is directed to the length of various probe generally about 14-25 base, probe 5 ' or 3 ' end carry out aminated process.The probe of the present invention's design has rational base content, Tm value close, is conducive to the synchronism under same hybridization temperature, can not affects the result of hybridization because of the problem of temperature, the accuracy of inspection is increased greatly.
The test kit that the present invention is above-mentioned, described gene chip comprises the position mark for position probe.
The test kit that the present invention is above-mentioned, the probe of described gene chip is fixed on nylon membrane.
The test kit that the present invention is above-mentioned, 5 ' end of described primer is marked with vitamin H.
The preparation method of gene chip of the present invention, comprises the steps:
(1) preparation of DNA probe: 5 ' or 3 ' end of synthesis and the complementation of CYP2C9 and VKORC1 gene DNA is through aminated DNA fragmentation
From DNA, select specific sequence, then according to base complementrity feature, design and synthesize 5 ' or 3 ' end through aminated DNA fragmentation;
(2) stationary probe: above-mentioned probe is fixed on the good nylon membrane of activation treatment
First activation treatment is carried out to nylon membrane, then by probe points on film, form covalent attachment by the amino of probe end and the carboxyl on activation film, securely probe be fixed on film;
(3) gene chip is prepared: utilize sodium hydroxide stopped reaction, obtained gene chip.
The method utilizing test kit of the present invention to detect CYP2C9 and VKORC1 gene type comprises the following steps: utilize in test kit containing the DNA in the primer PCR amplification clinical sample of particular sequence; DNA after amplification is hybridized; Detect the DNA that gene chip combines on the surface.
Concrete steps are as follows:
The first step: amplification sample
To extract DNA carries out pcr amplification by Auele Specific Primer of the present invention in measuring samples, the primer 5 ' holds mark vitamin H, and amplification obtains 5 ' end band biotin labeled DNA product;
Second step: hybridization
Use the hybridization technique in nucleic acid flow hybridization technology platform or any type of nucleic acid level, on the DNA obtain above-mentioned amplification and gene chip, the nucleotide probe of EDS maps reacts;
3rd step: colour developing
Under the effect of alkaline phosphatase AP enzyme, be beneficial to the colour developing of NBT/BCIP system, direct visual perception result or utilize corresponding instrument to read signal analyzing.
In gene test, due to the numerous Suo of operation steps, misoperation is difficult to avoid, the present invention employs the DNA sequence dna being marked with biological vegetarian refreshments in test kit, whether successful for monitoring hybridization, whether convenient operation personnel, when testing process is made mistakes, distinguish fast and make mistakes when hybridizing, to find out rapidly the reason made a mistake, shorten detection time.In addition, in the prior art, due to probe carrier many employings glass etc., permeability is poor, conventional hybridization method generally can only be used to hybridize, operate and comparatively bother, detection time is long, and generally at least all more than 6 hours, and the background of colour developing result is unclean; The present invention uses nylon membrane as probe carrier, because nylon membrane has good permeability compared with other solid support (as glass etc.), not only be beneficial to the application of flow hybridization technology, and there is good elasticity, be convenient to produce and operation, shorten detection time, only need 3.5 hours, the clean background of colour developing result.
CYP2C9 and VKORC1 gene parting detecting reagent of the present invention provides and detects CYP2C9 and VKORC1 gene type joint-detection platform, synchronization combining can be realized detect, improve detection specificity, reduce costs, shorten detection time, to carrying out CYP2C9 and VKORC1 gene test and Mass screening is significant.
Accompanying drawing explanation
Be below the description of the drawings, be convenient to object and the specific features of understanding foregoing invention.
Fig. 1 represents the type of probe and control point on gene chip and the picture of concrete distributing position, and each position represents different CYP2C9 and VKORC1 gene types, and " Bio " representative is marked with the DNA sequence dna of biological vegetarian refreshments.
Fig. 2 .1 represents the picture of C9*2 homozygous mutation DNA analysis result.
Fig. 2 .2 represents the picture of C9*2 heterozygous mutant DNA analysis result.
Fig. 2 .3 represents the picture of C9*3 homozygous mutation DNA analysis result.
Fig. 2 .4 represents the picture of C9*3 heterozygous mutant DNA analysis result.
Fig. 2 .5 represents the picture of VK1639 homozygous mutation DNA analysis result.
Fig. 2 .6 represents the picture of VK1639 heterozygous mutant DNA analysis result.
Fig. 2 .7 represents the picture of VK1173 homozygous mutation DNA analysis result.
Fig. 2 .8 represents the picture of VK1173 heterozygous mutant DNA analysis result.
Fig. 2 .9 represents the detected result picture of wild-type.
Embodiment
The present invention will be described in detail by the following examples.
In embodiment, the EDAC solution of 20%, 0.1% SDS, 0.25% skim-milk, 0.05% Thiomersalate, 0.05% sodium azide all refer to mass ratio, and 0.1% Tween 20 refers to volume ratio.
Step 1 is for detecting the preparation of CYP2C9 and VKORC1 gene chip
The design of step 1-1 probe
4 kinds of different genes type sites (C9*2, C9*3, VK1639, VK1173) are selected according to CYP2C9 and VKORC1 gene pleiomorphism, devise 8 DNA probes (SEQ ID Nos:1-8) altogether, the length of probe is generally about 14-25 base, probe 5 ' or 3 ' end carry out aminated process, and prepare Bio(SEQ ID No.9), for CYP2C9 and VKORC1 genetic polymorphism detection.
The point sample of step 1-2 DNA probe is with fixing
1) point sample of probe and arrangement
Direct oligonucleotide DNA probe fixing in, first by the DNA probe probe dilution liquid (0.5MNa of pH8.4
2cO
3and 0.5MNaHCO
3solution) mixing, carry out point sample.After completing the synthesis of DNA probe, start DNA spot sample device, under the control of chip manufacturing program, carry out the printing of DNA probe.Get a DNA probe by DNA print needle at every turn, DNA probe is delivered to the point sample position of specifying by three-dimensional fixed point transfer device.Complete after once printing, the cleaning of lotus sample print needle, drying, carry out the point sample of next round probe, the like, until all DNA probes transmission point sample is complete.
2) fixing means of DNA probe
Nylon membrane is processed, first put into 0.1M HCl solution and soaked for 30 seconds, then the film removing residual solution put into 20% EDAC solution soak 15 minutes, finally be placed on to wash in membranous disc and rinsed for 10 seconds by the purified water of 200mL, this step repeats 3 times, then is put on thieving paper and removes unnecessary raffinate.Proceed to that temperature is 20 DEG C, humidity be 45% drying baker in dry 12 hours.The nylon membrane Kimwipes paper of having dried is separated, proceeds in sealing film bag, put in a little intermembranous refrigerator, under being housed in the temperature of 4 DEG C, for subsequent use.
By micropipette equipment, the above-mentioned probe prepared is put respectively on above-mentioned nylon membrane, often drip 0.4 μ L.After some film terminates, film is positioned over room temperature 15 minutes, reacts.Then film is proceeded in 0.1M NaOH solution and soak 10 minutes, stopped reaction.Washed film is proceeded to temperature is 20 DEG C, humidity be 45% drying baker in dry 12 hours, i.e. obtained gene chip.
Step 2 is for the design of the Auele Specific Primer of DNA in the measuring samples that increases and detection method
Step 2-1 is used for design and the pcr amplification of the Specific PCR primers of DNA in measuring samples
According to CYP2C9 and VKORC1 gene order Data Design, 4 pairs of primers (SEQ ID Nos:10-17) are for detecting.PCR reaction system is the sample DNA that 50 μ L reaction systems include 46.5 μ l PCR MIX, 0.5 μ l DNA enzymatic polysaccharase, 3 μ l handle well.Reaction conditions is: 95 ° of C 9min, and 95 ° of C sex change 30 s, 55 ° of C annealing 30s, 72 ° of C extend 40s, totally 40 circulations, and final step 72 ° of C extend 5min.Obtain DNA sample to be detected.
Step 2-2 utilizes DNA chip to detect
Detected by the gene chip that the DNA sample to be detected obtained in step 2-1 is prepared in step 1, the probe reaction in DNA sample and gene chip on nylon membrane, finally judges according to colour developing situation.
By the amplified production sex change 5 minutes under 95 ° of C obtained, transfer to rapidly in mixture of ice and water, place 2 minutes.Then temperature bath is in advance joined in the 0.8 mL hybridization solution (2 × SSC/0.1%SDS) of 45 ° of C, add after mixing in the reacting hole of hybridization instrument, be applied to the gene chip that step 1 is obtained, 45 ° of C hybridize 20 minutes, then solution W B1(0.5 × SSC/0.1%SDS is used, 42 ° of C temperature baths) clean 3 times.Add 0.5mL to blockade liquid (0.25% skim-milk, 0.05% Thiomersalate), 25 ° of C close 5 minutes.The enzyme mark liquid (the AP enzyme with marked by streptavidin dissolved in TBS) of 0.5mL is added, enzyme mark 5 minutes after draining.Clean 4 times by 0.8mL solution A (TBS, 0.1% Tween20 and 0.05% sodium azide), then add the nitrite ion (NBT/BCIP) of 0.5mL, lucifuge develops the color 5 minutes.Finally rinse 3 times by solution B, dry, analyze colour developing situation, sentence read result.
Sequence table
<110> Chaozhou Kaipu Biochemistry Co., Ltd.
<120> CYP2C9 and VKORC1 gene parting detecting reagent
<160> 17
<210> 1
<211> 16
<212> DNA
<213> artificial sequence
<220>
<223> C9*2PN
<400> 1
tcttgaacac ggtcct 16
<210> 2
<211> 16
<212> DNA
<213> artificial sequence
<220>
<223> C9*2PM
<400> 2
tcttgaacac agtcct 16
<210> 3
<211> 18
<212> DNA
<213> artificial sequence
<220>
<223> C9*3PN
<400> 3
ccagagatac attgacct 18
<210> 4
<211> 18
<212> DNA
<213> artificial sequence
<220>
<223> C9*3PM
<400> 4
ccagagatac cttgacct 18
<210> 5
<211> 16
<212> DNA
<213> artificial sequence
<220>
<223> VK1639PN
<400> 5
attggccggg tgcggt 16
<210> 6
<211> 16
<212> DNA
<213> artificial sequence
<220>
<223> VK1639PM
<400> 6
attggccagg tgcggt 16
<210> 7
<211> 19
<212> DNA
<213> artificial sequence
<220>
<223> VK1173PN
<400> 7
tcatcgaccc ttggactag 19
<210> 8
<211> 19
<212> DNA
<213> artificial sequence
<220>
<223> VK1173PM
<400> 8
tcatcgactc ttggactag 19
<210> 9
<211> 21
<212> DNA
<213> artificial sequence
<220>
<223> Bio
<400> 9
cgtccaaggg gaaactgatc t 21
<210> 10
<211> 20
<212> DNA
<213> artificial sequence
<220>
<223> C19*2-F
<400> 10
agtgtcagct tcctctttct 20
<210> 11
<211> 23
<212> DNA
<213> artificial sequence
<220>
<223> C19*2-R
<400> 11
cagtaaggtc agtgatatgg agt 23
<210> 12
<211> 20
<212> DNA
<213> artificial sequence
<220>
<223> C19*3-F
<400> 12
gtccaggaag agattgaacg 20
<210> 13
<211> 20
<212> DNA
<213> artificial sequence
<220>
<223> C19*3-R
<400> 13
catggagttg cagtgtagga 20
<210> 14
<211> 19
<212> DNA
<213> artificial sequence
<220>
<223> VK1639-F
<400> 14
ggtaggtgca acagtaagg 19
<210> 15
<211> 19
<212> DNA
<213> artificial sequence
<220>
<223> VK1639-R
<400> 15
cagggttcaa gtggttctc 19
<210> 16
<211> 19
<212> DNA
<213> artificial sequence
<220>
<223> VK1173-F
<400> 16
agggtcagtg acatggaat 19
<210> 17
<211> 18
<212> DNA
<213> artificial sequence
<220>
<223> VK1173-R
<400> 17
ggtggaacca ggttagga 18
Claims (4)
1. a CYP2C9 and VKORC1 gene parting detecting reagent, comprising:
(1) gene chip, on it with:
4 mutational sites of (i) CYP2C9 and VKORC1 gene and the nucleotide sequence probe of normal control complementation thereof, its middle probe is SEQ ID Nos:1-8 or the sequence with SEQ ID Nos:1-8 complementation;
(ii) be marked with the DNA sequence dna of biological vegetarian refreshments, sequence is SEQ ID No.9;
(2) various primer, sequence is SEQ ID Nos.10-17.
2. test kit described in claim 1, is characterized in that, described gene chip comprises the position mark for position probe.
3. test kit described in claim 1, is characterized in that, the probe of described gene chip is fixed on nylon membrane.
4. test kit described in claim 1, is characterized in that, 5 ' end of described primer is marked with vitamin H.
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| CN201510443858.XA CN105002281A (en) | 2015-07-27 | 2015-07-27 | CYP2C9 and VKORC1 gene typing detection reagent kit |
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|---|---|---|---|
| CN201510443858.XA CN105002281A (en) | 2015-07-27 | 2015-07-27 | CYP2C9 and VKORC1 gene typing detection reagent kit |
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| CN201510443858.XA Pending CN105002281A (en) | 2015-07-27 | 2015-07-27 | CYP2C9 and VKORC1 gene typing detection reagent kit |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107841540A (en) * | 2017-12-13 | 2018-03-27 | 益善生物技术股份有限公司 | A kind of CYP2C9 and/or VKORC1 gene pleiomorphisms quick detection kit |
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| CN107841540A (en) * | 2017-12-13 | 2018-03-27 | 益善生物技术股份有限公司 | A kind of CYP2C9 and/or VKORC1 gene pleiomorphisms quick detection kit |
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Application publication date: 20151028 |