CN103122389A - Reagent kit and detecting method used for CYP3A5SNP rs776746 typing rapid detection - Google Patents
Reagent kit and detecting method used for CYP3A5SNP rs776746 typing rapid detection Download PDFInfo
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Abstract
The invention relates to a reagent kit used for CYP3A5SNP (rs776746) typing rapid detection, which comprises a kit cover, a kit body, a container which can be inserted into the kit body and which is filled with enzyme reaction mixture, a container which can be inserted into the kit body and which is filled with primer mixture, a container which can be inserted into the kit body and which is filled with positive control and a container which can be inserted into the kit body and which is filled with water. The invention also relates to a detecting method used for CYP3A5SNP (rs776746) typing rapid detection, and the method comprises the following steps: (1) preparing a PCR reaction system by using the reagent kit; (2) conducting PCR reaction to obtain amplified products; (3) conducting agarose gel electrophoresis detection on the amplified products; and (4) after the electrophoresis detection, determining the type to be AA type (*1/*1) when 389bp and 237bp stripes emerge, determining the type to be GG type (*3/*3) when 389bp and 201bp stripes emerge, and determining the type to be AG type (*1/*3) when 389bp, 237bp and 201bp stripes emerge. The detection method used for CYP3A5SNP (rs776746) typing rapid detection disclosed by the invention is short in time and low in cost.
Description
Technical field
The present invention relates to the molecular Biological Detection field, specifically, the present invention relates to a kind of test kit and detection method for CYP3A5SNP (rs776746) somatotype rapid detection.
Background technology
The Major Enzymes of human body metabolism's medicine is Cytochrome P450 superfamily (Cytochrome P450, CYP), and it is that a class mainly is present in the monooxygenase in liver, enteron aisle.CYP3A is the subfamily of Cytochrome P450, mainly is present in liver and small intestine, accounts for 30% of liver CYP total amount, for liver and the abundantest metabolic enzyme of in-vivo content, participates in the medicine of metabolism at known CYP450, approximately has 50% to be the CYP3A mediation.The expression of CYP3A5 is polymorphism and distributes, and its function only exists in the Black American of 10% ~ 20% white man, 33% Oriental and 55%, and drug metabolism and individual difference thereof are had important impact.The metabolism of CYP3A5 catalysis medicine and cholesterol, steroid and other lipids synthetic relates to many reactions.Take potent immunosuppressor tacrolimus as example, the rejection after clinical oral administration is mainly used in preventing to transplant with treating organs.But narrower because of its treatment window, the CYP3A5 gene pleiomorphism affects the expression of CYP3A5 enzyme, causes individual difference large, makes the tacrolimus Plasma Concentration of taking same dose different.Studies show that, be positioned at the A6986G of CYP3A5 gene the 3rd intron, can cause CYP3A5 differential expression between individuality, the individuality of GG type (CYP3A5*3/*3) is modal mutant, can cause the Splicing defect of CYP3A5 protein, thereby lose protein function.This genotypic individuality, because of the disappearance of metabolic enzyme, when taking the tacrolimus of same dose, Plasma Concentration is higher than AA type (CYP3A5*1/*1) or AG type (CYP3A5*1/*3).
Document " different CYP3A5*3 genotype and the relation of Determination of sirolimus in human whole blood in Chinese Kidney Transplantation Recipients " (" Chinese Journal of Pharmaceuticals " 2010,41,9) discloses a kind of four primer PCR methods, but can't reappear according to the method.
Summary of the invention
The object of the present invention is to provide a kind of test kit for CYP3A5SNP (rs776746) somatotype rapid detection.
The present invention also aims to provide a kind of detection method for CYP3A5SNP (rs776746) somatotype rapid detection.
In order to realize method of the present invention, the invention provides a kind of test kit for CYP3A5SNP (rs776746) somatotype rapid detection, comprising: lid, box body, can embed described box body the container that the enzyme reaction mixture is housed, can embed described box body the container that primer mixture is housed, can embed the container that positive control is housed of described box body and can embed the container that water is housed of described box body.
As used in this article, term " can embed box body " and refer to that container can freely insert box body and take out as required from box body.
Preferably, in the described container that primer mixture is housed, peripheral primer 1, peripheral primer 2, inner primer 1 and inner primer 2 are housed, the nucleotide sequence of described peripheral primer 1 is as shown in SEQ ID NO:l, the nucleotide sequence of described peripheral primer 2 is as shown in SEQ ID NO:2, the nucleotide sequence of described inner primer 1 is as shown in SEQ ID NO:3, and the nucleotide sequence of described inner primer 2 is as shown in SEQ ID NO:4.
In the present invention,
The sequence of SEQ ID NO:l is:
5’-CTTGCAGCATTTAGTCCTTGTG-3’
The sequence of SEQ ID NO:2 is:
5’-ACCCAGGAAGCCAGACTTTGAT-3’
The sequence of SEQ ID NO:3 is:
5’-TTTAAAGAGCTCTTTTGTCTTTGAG-3’
The sequence of SEQ ID NO:4 is:
5’-TGTGGTCCAAACAGGGAAGAGATCT-3’
Preferably, the concentration ratio of described peripheral primer and described inner primer is 1:5 ~ 1:7.More preferably, the concentration of described peripheral primer 1 and described peripheral primer 2 is 0.714 μ M, and the concentration of described inner primer 1 and described inner primer 2 is 4.286 μ M.
Preferably, described positive control is the masterplate DNA of GG type (* 3/*3), AA type (* 1/*1), AG type (* 1/*3).
In the present invention, described positive control for example can adopt following manner to obtain:
CYP3A5SNP (rs776746) somatotype of choosing through sequence verification is two poba gene group DNA of AA type (* 1/*1) and GG type (* 3/*3);
Take above-mentioned DNA as masterplate, use peripheral primer to carry out the amplification of PCR product;
After above-mentioned two PCR product purifications, be connected respectively on the T-carrier, and transform intestinal bacteria.
Utilize bacterium colony PCR to choose positive colony, and preserve positive strain;
Inoculate above-mentioned positive strain incubated overnight, and carry out plasmid extraction, obtain being connected with respectively the plasmid DNA of AA type (* 1/*1) and GG type (* 3/*3) purpose fragment.
Preferably, described enzyme reaction mixture comprises archaeal dna polymerase, dNTP and MgCl
2More preferably, described enzyme reaction mixture is the HotStarTaq Plus Master Mix of QIAGEN company.
In addition, the present invention also provides a kind of detection method for CYP3A5SNP (rs776746) somatotype rapid detection, and the method comprises the following steps:
1) prepare the PCR reaction system with test kit of the present invention;
2) carry out the PCR reaction according to following condition, to obtain amplified production:
94 ℃ of-98 ℃ of denaturation 10-20min;
94 ℃ of-98 ℃ of sex change 20-40sec, 54 ℃ of-56 ℃ of renaturation 20-40sec, 68 ℃-72 ℃ are extended 20-40sec, 25-45 circulation;
68 ℃-72 ℃ are extended 5-10min;
3) amplified production is carried out sepharose (2.5%) electrophoresis detection; And
4) after electrophoresis detection, 389bp and 237bp band occurring is AA type (* 1/*1), and 389bp and 201bp band occurring is GG type (* 3/*3), and 389bp, 237bp and 201bp band occurring is AG type (* 1/*3).
Preferably, in described step 2) in carry out PCR reaction according to following condition:
95 ℃ of denaturation 15min;
95 ℃ of sex change 30sec, 55 ℃ of renaturation 30sec, 72 ℃ are extended 30sec, 42 circulations;
72 ℃ are extended 7min.
In addition, the present invention also provides a kind of using method of test kit, and the method comprises the following steps:
1) prepare the PCR reaction system with test kit of the present invention;
2) carry out the PCR reaction according to following condition, to obtain amplified production:
94 ℃ of-98 ℃ of denaturation 10-20min;
94 ℃ of-98 ℃ of sex change 20-40sec, 54 ℃ of-56 ℃ of renaturation 20-40sec, 68 ℃-72 ℃ are extended 20-40sec, 25-45 circulation;
68 ℃-72 ℃ are extended 5-10min;
3) amplified production is carried out sepharose (2.5%) electrophoresis detection;
4) after electrophoresis detection, 389bp and 237bp band occurring is AA type (* 1/*1), and 389bp and 201bp band occurring is GG type (* 3/*3), and 389bp, 237bp and 201bp band occurring is AG type (* 1/*3).
Preferably, in described step 2) in carry out PCR reaction according to following condition:
95 ℃ of denaturation 15min;
95 ℃ of sex change 30sec, 55 ℃ of renaturation 30sec, 72 ℃ are extended 30sec, 42 circulations;
72 ℃ are extended 7min.
CYP3A5SNP of the present invention (rs776746) the somatotype method for quick used time is short, cost is low.
Description of drawings
Fig. 1 is the structural representation of test kit of the present invention;
Fig. 2 is the test-results figure of detection method of the present invention;
Fig. 3 is the test-results figure of detection method of the present invention.
In figure: 1, lid; 2, box body; 3, the container of enzyme reaction mixture is housed; 4, the container of primer mixture is housed; 5, the container of water is housed; 6, the container of GG type (* 3/*3) positive control is housed; 7, the container of AA type (* 1/*1) positive control is housed; 8, the container of AG type (* 1/*3) positive control is housed; 9, AA type (* 1/*1) positive control; 10, AG type (* 1/*3) positive control; 11, GG type (* 3/*3) positive control; 12,100bp DNA Ladder(Takara); 13,100pb DNALadder; 14, test sample; 15, test sample.
Embodiment
Below the invention will be further described for the description by embodiment, but this is not to be limitation of the present invention, those skilled in the art are according to basic thought of the present invention, can make various modifications or improvement, but only otherwise break away from basic thought of the present invention, all within the scope of the present invention.
Embodiment 1 test kit of the present invention
a kind of test kit for CYP3A5SNP (rs776746) somatotype rapid detection, comprise: lid 1, box body 2, can embed the 1.5mL centrifuge tube 3 that the enzyme reaction mixture is housed of described box body 2, can embed the 1.5mL centrifuge tube 4 that primer mixture is housed of described box body 2, can embed the 1.5mL centrifuge tube 5 that aseptic deionized water is housed of described box body 2, can embed the 1.5mL centrifuge tube 6 of the GG type that is equipped with (* 3/*3) positive control of described box body 2, can embed the 1.5mL centrifuge tube 7 of the AA type that is equipped with (* 1/*1) positive control of described box body 2, can embed the 1.5mL centrifuge tube 8 of the AG type that is equipped with (* 1/*3) positive control of described box body 2.Be equipped with in the 1.5mL centrifuge tube 3 of enzyme reaction mixture HotStarTaq Plus Master Mix(QIAGEN is housed).Be equipped with in the 1.5mL centrifuge tube 4 of primer mixture peripheral primer 1, peripheral primer 2, inner primer 1 and inner primer 2 are housed, the nucleotide sequence of described peripheral primer 1 is as shown in SEQ ID NO:l, the nucleotide sequence of described peripheral primer 2 is as shown in SEQ ID NO:2, the nucleotide sequence of described inner primer 1 is as shown in SEQ ID NO:3, and the nucleotide sequence of described inner primer 2 is as shown in SEQ ID NO:4.The concentration of described peripheral primer 1 and described peripheral primer 2 is 0.714 μ M, and the concentration of described inner primer 1 and described inner primer 2 is 4.286 μ M.
Test kit described in design implementation example 1, wherein:
The HotStarTaq Plus Master Mix(QIAGEN that 625 μ L are housed in the 1.5mL centrifuge tube 3 of enzyme reaction mixture is housed), described HotStarTaq Plus Master Mix contains PCR and detects required archaeal dna polymerase, dNTP(dATP, dCTP, dGTP, each 0.4mM of dTTP), MgCl
23mM.
Be equipped with in the 1.5mL centrifuge tube 4 of primer mixture 210 μ L Primer Mix are housed, wherein the concentration of peripheral primer 1, peripheral primer 2 is 0.714 μ M, and the concentration of inner primer 1 and inner primer 2 is 4.286 μ M.
Be equipped with 0.5mL sterilization deionized water is housed in the 1.5mL centrifuge tube 5 of aseptic deionized water.
The masterplate DNA30 μ L that GG type (* 3/*3) is housed in the 1.5mL centrifuge tube 6 of positive control is housed.
The masterplate DNA30 μ L that AA type (* 1/*1) is housed in the 1.5mL centrifuge tube 7 of positive control is housed.
The masterplate DNA30 μ L that AG type (* 1/*3) is housed in the 1.5mL centrifuge tube 8 of positive control is housed.
Prepare 25 μ L CYP3A5SNP (rs776746) somatotype detection reaction systems with the mentioned reagent box: positive control (the masterplate DNA of GG type (* 3/*3), AA type (* 1/*1) or AG type (* 1/*3)) 2 μ L, Master Mix12.5 μ L, Primer Mix4.2 μ L, sterilization deionized water 6.3 μ L.Reaction system with this preparation detects, and comprises the following steps:
Carry out the PCR reaction, wherein the PCR reaction conditions is as follows:
95 ℃ of denaturation 15min;
95 ℃ of sex change 30sec, 55 ℃ of renaturation 30sec, 72 ℃ are extended 30sec, 42 circulations;
72 ℃ are extended 7min.
Carry out sepharose (2.5%) electrophoresis detection with using three kinds of positive control PCR to react the amplified production that obtains, result as shown in Figure 2.
As seen from Figure 2, after electrophoresis detection, 389bp and 237bp band to occur be AA type (* 1/*1), 389bp occurs and the 201bp band is GG type (* 3/*3), 389bp, 237bp occurs and the 201bp band is AG type (* 1/*3).
Reappear this experiment 10 times, find that result is consistent, circulation ratio is very good.
Carry out the human blood extracting genome DNA according to following manner:
Use TIANGEN Biotech's poba gene group to extract test kit (DP-318), press the test kit explanation and extract the human blood genomic dna.Extract two kinds of different human blood genomic dnas.
Be to replace positive control with the human blood genomic dna that extracts according to the PCR reaction system difference for preparing 25 μ L with the similar mode of embodiment 2, specific as follows:
The PCR reaction is carried out in K960 gradient thermal cycler (Hangzhou lattice), and concrete Parameter Conditions is as follows: 95 ℃ of denaturation 15min; 95 ℃ of sex change 30sec, 55 ℃ of renaturation 30sec, 72 ℃ are extended 30sec, 42 circulations; 72 ℃ are extended 7min.
Pcr amplification product is identified:
Be configured to the sepharose solution of 2.5 % by weight with 1 * TAE damping fluid, microwave-oven-heating dissolves agarose fully, the DyeGeneGreen nucleic acid dye (TIANGEN Biotech (Beijing) Co., Ltd.) that adds 1/20000 volume, falling to get after glue PCR product (10 μ L) and 6 * Loading Buffer(can be available from Beijing promise rich Rider Science and Technology Ltd.) loading analysis after mixing.Under 90V voltage after electrophoresis 30min, observations and Taking Pictures recording under the ultraviolet gel imaging system.389bp and 237bp band to occur be AA type (* 1/*1), 389bp occurs and the 201bp band is GG type (* 3/*3), 389bp, 237bp occurs and the 201bp band is AG type (* 1/*3).The results are shown in Figure 3.14 and 15 is different human blood genomic dna CYP3A5SNP (rs776746) somatotype detected result, and the result of the positive control in Fig. 2 compares, and analyzes the AG type (* 1/*3) that is.
Reappear this experiment 10 times, find that result is consistent, circulation ratio is very good.
Comparative Examples
389bp and 237bp band are that AA type (* 1/*1), 389bp and 201bp band are that GG type (* 3/*3), 389bp, 237bp and 201bp band are AG type (* 1/*3), the corresponding the swimming whether above-mentioned position of the electrophoresis of investigation Comparative Examples product product occurs brings the evaluation circulation ratio, thereby investigates the impact of test conditions.
Comparative Examples 1
Carry out the PCR reaction according to the mode identical with embodiment 2, difference is, and is as follows under the PCR reaction conditions: 94 ℃ of denaturation 10min; 94 ℃ of sex change 30sec, 60 ℃ of renaturation 30sec, 70 ℃ are extended 40sec, 40 circulations; 70 ℃ are extended 5min.Re-start 10 PCR reactions, and amplified production is carried out electrophoresis, find that result is without any reproduction.
Comparative Examples 2
Carry out the PCR reaction according to the mode identical with embodiment 2, difference is, with HotStarTaq Plus Master Mix(QIAGEN) replace to respectively the ExTaq of Hotstar, Takara company of Hotstar, the Promega company of Takara company and the rTaq of Takara company, and carry out respectively 10 PCR reactions, and amplified production is carried out electrophoresis.Find, when the enzyme reaction mixture is the rTaq of the ExTaq of Hotstar, Takara company of Hotstar, the Promega company of Takara company and Takara company, find that result is without any circulation ratio.
Comparative Examples 3
Carry out PCR reaction according to the mode identical with embodiment 2, difference is, in design PCR reaction system, the concentration ratio of peripheral primer and described inner primer is that the concentration of the peripheral primer of 1:1(is 0.714 μ M, and the concentration of inner primer is 0.714 μ M).Re-start 10 PCR reactions, and amplified production is carried out electrophoresis, find that result is without any reproduction.
Comparative Examples 4
Carry out PCR reaction according to the mode identical with embodiment 2, difference is, in design PCR reaction system, the concentration ratio of peripheral primer and described inner primer is that the concentration of the peripheral primer of 2:1(is 1.428 μ M, and the concentration of inner primer is 0.714 μ M).Re-start 10 PCR reactions, and amplified production is carried out electrophoresis, find that result is without any reproduction.
Claims (8)
1. test kit that is used for CYP3A5SNP (rs776746) somatotype rapid detection, it is characterized in that, comprising: lid, box body, can embed described box body the container that the enzyme reaction mixture is housed, can embed described box body the container that primer mixture is housed, can embed the container that positive control is housed of described box body and can embed the container that water is housed of described box body.
2. the test kit for CYP3A5SNP (rs776746) somatotype rapid detection according to claim 1, it is characterized in that, in the described container that primer mixture is housed, peripheral primer 1, peripheral primer 2, inner primer 1 and inner primer 2 are housed, the nucleotide sequence of described peripheral primer 1 is as shown in SEQ IDNO:l, the nucleotide sequence of described peripheral primer 2 is as shown in SEQ ID NO:2, the nucleotide sequence of described inner primer 1 is as shown in SEQ ID NO:3, and the nucleotide sequence of described inner primer 2 is as shown in SEQ ID NO:4.
3. the test kit for CYP3A5SNP (rs776746) somatotype rapid detection according to claim 2, is characterized in that, the concentration ratio of described peripheral primer and described inner primer is 1:5 ~ 1:7.
4. the test kit for CYP3A5SNP (rs776746) somatotype rapid detection according to claim 3, it is characterized in that, the concentration of described peripheral primer 1 and described peripheral primer 2 is 0.714 μ M, and the concentration of described inner primer 1 and described inner primer 2 is 4.286 μ M.
5. the test kit for CYP3A5SNP (rs776746) somatotype rapid detection according to claim 1, is characterized in that, described positive control is the masterplate DNA of GG type (* 3/*3), AA type (* 1/*1), AG type (* 1/*3).
6. the test kit for CYP3A5SNP (rs776746) somatotype rapid detection according to claim 1, is characterized in that, described enzyme reaction mixture comprises archaeal dna polymerase, dNTP and MgCl
2
7. detection method that is used for CYP3A5SNP (rs776746) somatotype rapid detection, the method comprises the following steps:
1) in right to use requirement 1 to 6, the described test kit of any one prepares the PCR reaction system;
2) carry out the PCR reaction according to following condition, to obtain amplified production:
94 ℃ of-98 ℃ of denaturation 10-20min;
94 ℃ of-98 ℃ of sex change 20-40sec, 54 ℃ of-56 ℃ of renaturation 20-40sec, 68 ℃-72 ℃ are extended 20-40sec, 25-45 circulation;
68 ℃-72 ℃ are extended 5-10min;
3) amplified production being carried out agarose gel electrophoresis detects;
4) after electrophoresis detection, 389bp and 237bp band occurring is AA type (* 1/*1), and 389bp and 201bp band occurring is GG type (* 3/*3), and 389bp, 237bp and 201bp band occurring is AG type (* 1/*3).
8. detection method according to claim 7, is characterized in that, in described step 2) in carry out PCR reaction according to following condition:
95 ℃ of denaturation 15min;
95 ℃ of sex change 30sec, 55 ℃ of renaturation 30sec, 72 ℃ are extended 30sec, 42 circulations;
72 ℃ are extended 7min.
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Cited By (3)
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| CN103361433A (en) * | 2013-07-26 | 2013-10-23 | 天津市秀鹏生物技术开发有限公司 | Primer group and kit for detecting genetic typing of human cytochrome P450 enzyme system 3A5(CYP3A5) |
| CN105602947A (en) * | 2015-11-16 | 2016-05-25 | 北京晋祺生物科技有限公司 | CYP3A5 gene detection primer group, reaction system composed thereof and application |
| CN105861689A (en) * | 2016-05-06 | 2016-08-17 | 北京晋祺生物科技有限公司 | Primer combination, reagent kit and method for detecting CYP3A5*3 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103361433A (en) * | 2013-07-26 | 2013-10-23 | 天津市秀鹏生物技术开发有限公司 | Primer group and kit for detecting genetic typing of human cytochrome P450 enzyme system 3A5(CYP3A5) |
| CN105602947A (en) * | 2015-11-16 | 2016-05-25 | 北京晋祺生物科技有限公司 | CYP3A5 gene detection primer group, reaction system composed thereof and application |
| CN105602947B (en) * | 2015-11-16 | 2019-01-11 | 北京晋祺生物科技有限公司 | The detection primer group of CYP3A5 gene, its reaction system and application for constituting |
| CN105861689A (en) * | 2016-05-06 | 2016-08-17 | 北京晋祺生物科技有限公司 | Primer combination, reagent kit and method for detecting CYP3A5*3 |
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Effective date of registration: 20150203 Address after: Branch of Beijing economic and Technological Development Zone of Beijing City fourteen street 101111 No. 99 No. eighteen building two unit 401 room Applicant after: iPhase Biosciences (Beijing) Co., Ltd. Address before: 101111 Beijing City, Daxing District economic and Technological Development Zone Branch 14 Street No. 99, building 18, unit 2, room 1832 Applicant before: iPhase Pharmaceutical Services Co., Ltd. |
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Effective date of registration: 20210803 Address after: Room 1832, building 18, 99 Kechuang 14th Street, Beijing Economic and Technological Development Zone, Daxing District, Beijing Patentee after: IPHASE PHARMACEUTICAL SERVICES Address before: 101111 Room 401, unit 2, building 18, No. 99, Kechuang 14th Street, Beijing Economic and Technological Development Zone, Beijing Patentee before: IPHASE BIOSCIENCES (BEIJING) Co.,Ltd. |
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