CN102277443A - Kit for detecting tacrolimus and cyclosporine A individual medicine administration associated single nucleotide polymorphism (SNP) loci and amplification method and detection method - Google Patents
Kit for detecting tacrolimus and cyclosporine A individual medicine administration associated single nucleotide polymorphism (SNP) loci and amplification method and detection method Download PDFInfo
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Abstract
The invention discloses a detection kit and a detection method for detecting a tacrolimus and cyclosporine A individual medicine administration associated SNP loci by combining multiple polymerase chain reaction (PCR) technology and SNP sensitivity molecular switch technology. The kit can be used for genotyping of three SNP loci associated with tacrolimus and cyclosporine A medicine administration, wherein the three SNP loci include a rs2242480 SNP locus in a CYP3A4 gene, a rs776746 SNP locus in a CYP3A5 gene and a rs1045642 SNP locus in a MDR1 gene. The kit comprises wild type 2* amplification buffer solution, mutant type 2* amplification buffer solution and polymerase, wherein the two kinds of buffer solution contain corresponding SNP wide type and mutant type phenotypic sequence specific primers and endogenous reference primers respectively; the genotyping of the three SNP loci can be accomplished in two multiple PCR reactions; and thus, a molecular biological basis is provided for the reasonable administration of tacrolimus and cyclosporine A.
Description
Technical field
The present invention relates to the test kit and the pcr amplification method thereof in a kind of SNP of detection site, especially utilize multiplex PCR binding molecule switching technique to detect test kit and the multiplex PCR amplification method and the detection method in tacrolimus and ciclosporin A personalized medicine related SNP site.
Background technology
Tacrolimus have another name called FK506 be a kind of from streptomyces (streptomyces tsukubaensis) isolated tunning, its chemical structure belongs to 23 membered macrolide class microbiotic.Neotype immunosuppressant for a kind of brute force.To the selective restraining effect of T cell, mainly combine and suppress the Th cell and discharge IL-2, IL-3, IFN-γ and suppress IL-2R and express by having a liking for plain FK conjugated protein (FKBP) with intracellular immunity.In recent years, as a line medication of liver, renal transplantation, in 14 country's listings such as Japan, the U.S..Clinical experiment shows that it is used better curative effect in transplanting such as the heart, lung, intestines, marrow.Tacrolimus is also being brought into play positive effect in autoimmune disorders such as treatment atopic dermatitis (AD), systemic lupus erythematous (SLE), autoimmunity illness in eye simultaneously.Ciclosporin A (cyclosprine, CyA) ring type polypeptide of forming by 11 amino acid, the active metabolite .1978 Britain that is a kind of fungi in the soil is applied to CyA clinical renal transplantation first, after this CyA is used for liver again, the heart, lung, pancreas, the transplanting of organs such as marrow, all obtain gratifying effect, the survival rate .1984 ciclosporin A that obviously improves patient enters China, three immunosuppressant schemes of " ciclosporin A+azathioprine+hormone " have been formed, improved the transplanting survival rate greatly, simultaneously also make the incidence of post-transplantation acute rejection decline to a great extent .CyA as a kind of potent immunosuppressor, people are more and more to its research in recent years, find that it also can be used to treat autoimmune disorder, hemopathy and parasiticide disease etc.The effective therapeutic domain of tacrolimus and ciclosporin A is very narrow, underdosage or Plasma Concentration are crossed to hang down and may be caused the rejection of graft, and a series of untoward reactions are induced in the too high meeting of dosage, comprise renal toxicity, neurotoxicity, transplantability diabetes, susceptibility to infections increase, canceration, hypertension and gastrointestinal dysfunction.The bioavailability of tacrolimus and ciclosporin A is very big at interindividual variation, vast amount of clinical shows, except being subjected to clinical factors such as patient age, race, body weight, diet to influence that the difference on the gene level is the major cause that causes drug metabolism difference between individuality, the SNP polymorphism is the key factor that influences tacrolimus and ciclosporin A drug metabolism on CYP3A4, CYP3A5 and the MDR1 gene.
Ciclosporin and tacrolimus mainly at liver and gi tract via the hydroxylation of CYP3A4 and CYP3A5 isozyme and demethylation effect and metabolism.The metabolism of these medicines almost is completely, only has the prototype medicine less than 1% can appear in urine or the ight soil.The meta-bolites of ciclosporin and tacrolimus is discharged through urine less than 5% product by discharging in the bile.CYP3A4*1G rs2242480 is that a highest site of mutation frequency is discovered in present all CYP3A4 single nucleotide polymorphism, the CYP3A4*1G transgenation may improve the CYP3A4 enzymic activity, Plasma Concentration/the dosage that influences the patient is than (C/D), when the patient carries this sudden change, then the C/D value reduces, the patient is different acute rejection (AR) to occur, and adverse drug reaction (ADR) occurrence probability is lower, should increase dosage; When the patient is wild homozygote, then should reduce dosage.Chinese CYP3A5*3 rs776746 mutation frequency is higher, and sudden change can cause the Yeast Nucleic Acid insertion portion introne 3 of transcribing, and occurs terminator codon in advance in 109 sites, translates no function protein fragments.When the patient suddenlyd change homozygote for CYP3A5*3, patient C/D value was higher, should reduce drug dose.P-gp encoding gene MDR1 has a plurality of pleomorphism sites, and the expression of enteron aisle P-gp has individual difference.P-gp is the cell traffic albumen that a kind of Triphosaden relies on, and medicine in the cell or in the double-layer of lipoid and poisonous substance can be pumped the extracellular, reduces the absorption of medicine and the drainage of increase medicine.Tacrolimus and ciclosporin A are the substrate of P-gp, are again inhibitor, and the MDR1 gene pleiomorphism can influence pharmacokinetic parameter by the activity that changes P-gp.MDR1 C3435Trs1045642 is the higher SNP of mutation frequency site on the MDR1 gene, individual its enteron aisle P-gp of MDR1 3435 TT genotype expression level is starkly lower than the 3435CT/CC individuality, studies have shown that TT type patient is than the less drug dose of TC/CC type needs of patients.
Summary of the invention
The gene genetic polymorphism of metabolic enzyme CYP3A4, the CYP3A5 of ciclosporin A and tacrolimus and transhipment enzyme MDR1 directly or indirectly influences two kinds of medicines transhipment and metabolic process in vivo.The invention provides a kind of test kit and multiplex PCR amplification method and detection method of utilizing multiplex PCR binding molecule switching technique to detect ciclosporin A and tacrolimus personalized medicine related SNP site.
The technical solution used in the present invention: a kind of test kit that detects ciclosporin A and tacrolimus personalized medicine genes involved mononucleotide polymorphism site, described test kit can be simultaneously detects CYP3A4 gene rs2242480 SNP site, CYP3A5 gene rs776746 SNP site and the MDR1 gene rs1045642SNP site somatotype that increases, and is that confidential reference items detect amplification condition with β-Actin gene; Wild-type 2 * buffering comprises the wild-type forward sequence specific primers in above-mentioned 3 SNP sites, shared reverse primer and confidential reference items sequence upstream and downstream primer, by the wild-type phenotype that judges whether to carry corresponding SNP site that has or not of the back DNA electrophoretic band that increases; Mutant 2 * damping fluid comprises above-mentioned 3 SNP site mutation type forward sequence specific primerses, shared reverse primer and confidential reference items sequence upstream and downstream primers, and having or not of back DNA electrophoretic band judges whether to carry corresponding SNP site mutation type phenotype by increasing; Pairing fragment length band only draws positive findings in wild-type amplification when one of them SNP site, interpretation was wild homozygous when mutant amplification correspondence position band was negative, opposite homozygous for suddenling change, it is heterozygous that its result of positive band all appears in wild-type and mutant amplification, confirms experimenter's genotype through dna gel electrophoresis analysis-by-synthesis by the specific amplification of wild-type and mutant.
Two forward primers in affiliated detection CYP3A4 gene rs2242480 SNP site and the base sequence of a reverse primer are as follows:
Forward wild-type primer: TTTACCCAATAAGGTGAGTGGATGG
Forward mutation type primer: TTTACCCAATAAGGTGAGTGGATGA
Reverse general primer: CCTACATAGAGTCAGTGAAAGAATCAGTGA
Two forward primers in affiliated detection CYP3A5 gene rs776746 SNP site and the base sequence of a reverse primer are as follows:
Forward wild-type primer: ATGTGGTCCAAACAGGGAAGAGATAT
Forward mutation type primer: ATGTGGTCCAAACAGGGAAGAGATAC
Reverse general primer: AGGCAACATGACTTAGTAGACAGATGACA
Two forward primers in affiliated detection MDR1 gene rs1045642 SNP site and the base sequence of a reverse primer are as follows:
Forward wild-type primer: CGGGTGGTGTCACAGGAAGAGATT
Forward mutation type primer: CGGGTGGTGTCACAGGAAGAGATC
Reverse general primer: GGGAGAGACAGTCATGCCTACTTCCT
The component and the content of test kit of the present invention comprise:
Wild-type 2 * amplification buffer mixture, mutant 2 * amplification buffer mixture, high-fidelity polysaccharase.
This test kit totally 40 person-portions detects application, and the storage temperature of test kit is-20 ℃.
The present invention compared with prior art has following advantage and effect:
(1) the present invention adopts the multiplex PCR amplification technique, in a PCR reaction 3 dna fragmentation and 1 confidential reference items fragments that comprise the SNP site is increased simultaneously, improves detection efficiency and reduces the detection cost.
(2) the present invention adopts SNP susceptibility molecular switch technology, and 3 ' end can not be stopped with the non-maturity of the amplification that template complementary primer is caused, and can't form product, avoids the generation of false positive results.
(3) introduce confidential reference items, negative result's interpretation provides foundation, avoids the generation of false negative result.
(4) only need after the present invention increases to finish detection, need not acquire valuable equipment, fast by dna gel electrophoresis and gel imaging system, accurately, sensitivity, special, good reproducibility has significantly reduced detection cost and complicated operation degree, is fit to clinical and the scientific research use.
(5) need not DNA extraction, only need the cracking complete blood cell, the suspension after the lysis is added reaction system can finish amplification, remove the step and the cost of DNA extraction from and avoid aerosol to pollute.
(6) test kit of the present invention provides 2 * amplification buffer of wild-type and mutant respectively, and the user has reduced operation steps as long as whole blood cracking suspension and polysaccharase can increase, and improves the rate of work, can realize that high-level efficiency detects.
(7) wild-type that test kit of the present invention provided and mutant 2 * amplification buffer have used the amplification indicating dye of different colours, distinguish when making things convenient for application of sample, avoid mistake.Can directly carry out gel electrophoresis after the amplification, need not to add loading buffer, convenient to operation.
Embodiment
The primer in the primer in CYP3A4 rs2242480 SNP site, the primer that detects gene C YP3A5 rs776746 SNP site and detection gene M DR1 rs1045642 SNP site.
Below in conjunction with specific embodiment the present invention is described in further detail.
The present invention detects the test kit in tacrolimus and ciclosporin A personalized medicine related SNP site, and described test kit comprises the primer in the primer that detects gene C YP3A4 rs2242480 SNP site, the primer that detects gene C YP3A5rs776746 SNP site and detection gene M DR1 rs1045642 SNP site.
Described test kit comprises two forward primers detecting gene C YP3A4 rs2242480 SNP site and reverse primer, detect two forward primers and reverse primers in gene C YP3A5 rs776746 SNP site and detect two forward primers and a reverse primer in gene M DR1 rs1045642 SNP site.
The primer base sequence (SEQ IDNO.1-9) as follows in described detection gene C YP3A4 rs2242480 site:
Forward wild-type primer: TTTACCCAATAAGGTGAGTGGATGG
Forward mutation type primer: TTTACCCAATAAGGTGAGTGGATGA
Reverse general primer: CCTACATAGAGTCAGTGAAAGAATCAGTGA
The primer base sequence in described detection gene C YP3A5 rs776746 site is as follows:
Forward wild-type primer: ATGTGGTCCAAACAGGGAAGAGATAT
Forward mutation type primer: ATGTGGTCCAAACAGGGAAGAGATAC
Reverse general primer: AGGCAACATGACTTAGTAGACAGATGACA
The primer base sequence in described detection gene M DR1 rs1045642 site is as follows:
Forward wild-type primer: CGGGTGGTGTCACAGGAAGAGATT
Forward mutation type primer: CGGGTGGTGTCACAGGAAGAGATC
Reverse general primer: GGGAGAGACAGTCATGCCTACTTCCT
The detection method that test kit adopted in described detection warfarin personalized medicine related SNP site: examined samples DNA is increased with wild-type and sudden change amplification buffer respectively, in each anti-amplified reaction, simultaneously 3 purpose fragments and 1 confidential reference items fragment are increased, amplified fragments is descending to be successively: internal reference 733bp, MDR1 rs1045642 518bp, CYP3A5 rs776746 303bp, CYP3A4 rs2242480 139bp; Amplification is after gel electrophoresis can be seen the product band that distinguishes according to clip size under UV-light, according to the genotype of judging 3 SNP sites that has or not of band.
The test kit multiplex PCR amplification method in described detection warfarin personalized medicine related SNP site:
Pre-sex change is made of 1 circulation, and its condition is: temperature is 94 ℃, and the time is 5 minutes;
Pcr amplification is made of 30 circulations, and its condition is:
Sex change: temperature is 94 ℃, and the time is 30 seconds;
Annealing: temperature is 58 ℃, and the time is 30 seconds;
Extend: temperature is 72 ℃, and the time is 90 seconds;
Amplification finishes the back and is saved to electrophoresis detection in 4 ℃.
The test method of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
Embodiment one
Step 1: the preparation of complete blood cell lysate
Get person under inspection's peripheric venous blood 300 μ l, add 700 μ l cell pyrolysis liquids, put upside down mixing 5 times, centrifugal 1 minute of 12000rpm, remove supernatant, and centrifuge tube is upside down on the clean thieving paper stopped 2 minutes, guarantee to be deposited in the pipe, add 300 μ l distilled waters, the vortex concussion is mixed into the lysis suspension.
Step 2:PCR amplified reaction
1, disposes the wild-type reaction system: add lysis suspension 4.8 μ l, adding 5 μ l 2 * wild-type amplification damping fluids and 0.2 μ l polysaccharase (2.5U/ μ l) mixing in the PCR pipe and constitute independently reaction system, abundant mixing behind the application of sample, of short duration centrifugal, specifically see the following form:
| 2 * wild-type amplification buffered soln | 5μl |
| The lysis suspension | 4.8μl |
| Polysaccharase (2.5U/ μ l) | 0.2μl |
2, dispose the mutant reaction system: add lysis suspension 4.8 μ l, adding 5 μ l2 * mutant amplification buffer and 0.2 polysaccharase (2.5U/ μ l) mixing in the PCR pipe and constitute independently reaction system, abundant mixing behind the application of sample, of short duration centrifugal, specifically see the following form:
| 2 * mutant amplification buffered soln | 5μl |
| The lysis suspension | 4.8μl |
| Polysaccharase (2.5U/ μ l) | 0.2μl |
3, above-mentioned two reaction systems are carried out the PCR reaction simultaneously, the PCR program sees the following form:
Step 3: amplified production gel electrophoresis
Dispose 1.5% agarose electrophoresis gel, get amplified production 6-8 μ l and directly add in the well, it is that electrophoresis is carried out in the molecular weight contrast that every row alternative one hole adds 100bp Ladder Marker, and deposition condition is that voltage stabilizing 6V/cm glue is long, 40 minutes time.
Step 4: observed result
Use the ultraviolet imagery systematic observation electrophoretic band position that has that it's too late.
Si Tiao is with in the district and descendingly is successively: internal reference 733bp, MDR1 rs1045642 518bp, CYP3A5rs776746 303bp, CYP3A4 rs2242480 139bp; If positive band appears in the 139bp position in the wild-type amplified production, mutant amplified production correspondence position does not have band, and then interpretation is a CYP3A4 wild-type individuality; Otherwise be CYP3A4*1G mutant homozygote individuality; If positive band all appears in the 139bp position in wild-type and the mutant amplified production, then be CYP3A4 heterozygous individuality.
If positive band appears in the 303bp position in the wild-type amplified production, mutant amplified production correspondence position does not have band, and then interpretation is a CYP3A5 wild-type individuality; Otherwise be CYP3A5*3 mutant homozygote individuality; If positive band all appears in the 290bp position in wild-type and the mutant amplified production, then be CYP3A5*3 heterozygous individuality.
If positive band appears in the 518bp position in the wild-type amplified production, mutant amplified production correspondence position does not have band, and then interpretation is a MDR1 wild-type individuality; Otherwise be MDR1 mutant homozygote individuality; If positive band all appears in the 518bp position in wild-type and the mutant amplified production, then be MDR1 heterozygous individuality.
In sum, content of the present invention is not limited in the above-described embodiment, and the knowledgeable people in the same area can propose other embodiment easily within technical director's thought of the present invention, but this embodiment comprises within the scope of the present invention.
Embodiment two
Step 1: the extraction of Whole Blood Genomic DNA and dilution
Get person under inspection's peripheric venous blood 300 μ l, extract the explanation of test kit according to Whole Blood Genomic DNA and extract Whole Blood Genomic DNA.With the concentration of spectrophotometer measurement DNA, and be diluted to 15-20ng/ μ l.
Step 2:PCR amplified reaction
1, disposes the wild-type reaction system: add lysis suspension 4.8 μ l, adding 5 μ l2 * wild-type amplification damping fluid and 0.2 μ l polysaccharase (2.5U/ μ l) mixing in the PCR pipe and constitute independently reaction system, abundant mixing behind the application of sample, of short duration centrifugal, specifically see the following form:
| 2 * wild-type amplification buffered soln | 5μl |
| The lysis suspension | 4.8μl |
| Polysaccharase (2.5U/ μ l) | 0.2μl |
2, dispose the mutant reaction system: add lysis suspension 4.8 μ l, adding 5 μ l2 * mutant amplification buffer and 0.2 polysaccharase (2.5U/ μ l) mixing in the PCR pipe and constitute independently reaction system, abundant mixing behind the application of sample, of short duration centrifugal, specifically see the following form:
| 2 * mutant amplification buffered soln | 5μl |
[0080]?
| The lysis suspension | 4.8μl |
| Polysaccharase (2.5U/ μ l) | 0.2μl |
3, above-mentioned two reaction systems are carried out the PCR reaction simultaneously, the PCR program sees the following form:
Step 3: amplified production gel electrophoresis
Dispose 1.5% agarose electrophoresis gel, get amplified production 6-8 μ l and directly add in the well, it is that electrophoresis is carried out in the molecular weight contrast that every row alternative one hole adds 100bp Ladder Marker, and deposition condition is that voltage stabilizing 6V/cm glue is long, 40 minutes time.
Step 4: observed result
Use the ultraviolet imagery systematic observation electrophoretic band position that has that it's too late.
Si Tiao is with in the district and descendingly is successively: internal reference 733bp, MDR1 rs1045642 518bp, CYP3A5rs776746 303bp, CYP3A4 rs2242480 139bp; If positive band appears in the 139bp position in the wild-type amplified production, mutant amplified production correspondence position does not have band, and then interpretation is a CYP3A4 wild-type individuality; Otherwise be CYP3A4*1G mutant homozygote individuality; If positive band all appears in the 139bp position in wild-type and the mutant amplified production, then be CYP3A4 heterozygous individuality.
If positive band appears in the 303bp position in the wild-type amplified production, mutant amplified production correspondence position does not have band, and then interpretation is a CYP3A5 wild-type individuality; Otherwise be CYP3A5*3 mutant homozygote individuality; If positive band all appears in the 290bp position in wild-type and the mutant amplified production, then be CYP3A5*3 heterozygous individuality.
If positive band appears in the 518bp position in the wild-type amplified production, mutant amplified production correspondence position does not have band, and then interpretation is a MDR1 wild-type individuality; Otherwise be MDR1 mutant homozygote individuality; If positive band all appears in the 518bp position in wild-type and the mutant amplified production, then be MDR1 heterozygous individuality.
In sum, content of the present invention is not limited in the above-described embodiment, and the knowledgeable people in the same area can propose other embodiment easily within technical director's thought of the present invention, but this embodiment comprises within the scope of the present invention.
Claims (5)
1. test kit that detects tacrolimus and ciclosporin A personalized medicine related SNP site, it is characterized in that described test kit comprises the primer in the primer that detects gene C YP3A4 rs2242480 SNP site, the primer that detects gene C YP3A5 rs776746 SNP site and detection gene M DR1 rs1045642 SNP site.
2. the test kit in detection tacrolimus according to claim 1 and ciclosporin A personalized medicine related SNP site, it is characterized in that described test kit comprises two forward primers detecting gene C YP3A4 rs2242480 SNP site and reverse primer, detect two forward primers and reverse primers in gene C YP3A5 rs776746 SNP site and detect two forward primers and a reverse primer in gene M DR1 rs1045642 SNP site.
3. the test kit in detection tacrolimus according to claim 2 and ciclosporin A personalized medicine related SNP site is characterized in that, the primer base sequence in described detection gene C YP3A4 rs2242480 site is as follows:
Forward wild-type primer: tttacccaat aaggtgagtg gatgg
Forward mutation type primer: tttacccaat aaggtgagtg gatga
Reverse general primer: cctacataga gtcagtgaaa gaatcagtga
The primer base sequence in described detection gene cyp3a5 rs776746 site is as follows:
Forward wild-type primer: atgtggtcca aacagggaag agatat
Forward mutation type primer: atgtggtcca aacagggaag agatac
Reverse general primer: aggcaacatg acttagtaga cagatgaca
The primer base sequence in described detection gene mdr1 rs1045642 site is as follows:
Forward wild-type primer: cgggtggtgt cacaggaaga gatt
Forward mutation type primer: cgggtggtgt cacaggaaga gatc
Reverse general primer: gggagagaca gtcatgccta cttcct
4. the detection method that test kit adopted in described detection tacrolimus of claim 1 and ciclosporin A personalized medicine related SNP site, it is characterized in that, examined samples DNA is increased with wild-type and sudden change amplification buffer respectively, in each anti-amplified reaction, simultaneously 3 purpose fragments and 1 confidential reference items fragment are increased, amplified fragments is descending to be successively: internal reference 733bp, MDR1rs1045642 518bp, CYP3A5 rs776746 303bp, CYP3A4 rs2242480 139bp; Amplification is after gel electrophoresis can be seen the product band that distinguishes according to clip size under UV-light, according to the genotype of judging 3 SNP sites that has or not of band.
5. a test kit multiplex PCR amplification method that adopts the described detection warfarin of claim 1 personalized medicine related SNP site is characterized in that,
Pre-sex change is made of 1 circulation, and its condition is: temperature is 94 ℃, and the time is 5 minutes;
Pcr amplification is made of 30 circulations, and its condition is:
Sex change: temperature is 94 ℃, and the time is 30 seconds;
Annealing: temperature is 58 ℃, and the time is 30 seconds;
Extend: temperature is 72 ℃, and the time is 90 seconds;
Amplification finishes the back and is saved to electrophoresis detection in 4 ℃.
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| CN106086192A (en) * | 2016-06-27 | 2016-11-09 | 上海泽因生物科技有限公司 | The parting detecting reagent of tacrolimus personalized medicine related gene |
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