CN105368826A - Primer pair for detecting CYP2C9 genetic typing through pyrosequencing method and kit - Google Patents
Primer pair for detecting CYP2C9 genetic typing through pyrosequencing method and kit Download PDFInfo
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- CN105368826A CN105368826A CN201510675176.1A CN201510675176A CN105368826A CN 105368826 A CN105368826 A CN 105368826A CN 201510675176 A CN201510675176 A CN 201510675176A CN 105368826 A CN105368826 A CN 105368826A
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Abstract
The invention relates to a primer pair for detecting CYP2C9 genetic typing through a pyrosequencing method and a kit, and belongs to the technical field of in vitro nucleic acid detection. The primer pair comprises a CYP2C9*2 forward amplification primer, a CYP2C9*2 reverse amplification primer, a CYP2C9*2 sequencing primer, a CYP2C9*3 forward amplification primer, a CYP2C9*3 reverse amplification primer and a CYP2C9*3 sequencing primer. Biotin labeling is conducted at the 5' end of the CYP2C9*2 forward amplification primer and the 5' end of the CYP2C9*3 reverse amplification primer. The kit comprises the amplification primers, a PCR reaction solution 1, a PCR reaction solution 2, the sequencing primers, uracil DNA glycosylase and Taq polymerase. The kit has the advantages of being accurate in detection result, high in specificity, short in detection period, easy to operate and capable of effectively meeting the clinical examination requirement.
Description
Technical field
The present invention relates to external nucleic acid detection technique field, particularly relate to primer pair and test kit that a kind of Manganic pyrophosphate complex initiation method detects CYP2C9 gene type.
Background technology
Cytochrome P450 2C9 (CytochromeP4502C9, CYP2C9) accounts for 20% of cytochromes P450 total amount, can catalyze metabolic many clinical commonly used drugs, and its importance in clinical medicine metabolism more and more comes into one's own.
CYP2C9 gene has high genetic polymorphism, its modal gene mutation site is CYP2C9*2 (rs1799853) and CYP209*3 (rs1057910), the enzymic activity of its coding reduces 30% and 80% than wild-type CYP2C9*1 respectively, affect the metabolism in vivo of its substrate drugs, cause the individual difference of curative effect of medication and toxicity, cause CYP2C9 transgenation individual lower to the dosage requirements of warfarin.Carry that to reach the time that Css needs after these two allelic individualities take warfarin longer, and have higher hemorrhage danger at the use initial stage, therefore dosage should be reduced when CYP2C9*2 or CYP2C9*3 genotype individuals takes Warfarin, but in the individual long therapeutic procedure of allelic mutation, the danger of excessively anti-freezing does not increase.Other mutant CYP2C9*4, the mutation frequency in crowd such as CYP2C9*5, CYP2C9*6 and CYP2C9*1l is very low, and it needs to study further on the impact of warfarin dosage requirements.CYP2C9*2 and CYP2C9*3 gene frequency has larger difference in not agnate, and wherein, in Caucasian, CYP2C9*2 and CYP2C9*3 gene frequency is 8%-20% and 6%-10% approximately respectively; In asian population, CYP2C9*2 allelotrope does not exist, and CYP2C9*3 gene frequency is approximately l%-4%; In the Black people of America, CYP2C9*2 gene frequency is approximately 2%-4%, CYP2C9*3 gene frequency is approximately l%-2%.
Warfarin is a kind of temparin derivative, is current one of most widely used oral anticoagulation thing clinically, for the thrombosis that prevention and therapy deep venous thrombosis, pulmonary infarction, cardiac valve replacement and atrial fibrillation cause.Warfarin treatment window is narrower, and very little dosage all may cause the generation of untoward reaction, and reaches identical action effect in Different Individual, can differ more than 10 times between high low dosage person.The raceme that warfarin is made up of S-warfarin and R-warfarin, S-warfarin reflects the anticoagulating active of 60%-70% in vivo, and R-warfarin accounts for 30%-40%, wherein S-warfarin is primarily of CYP2C9 metabolism, therefore CYP2C9 gene pleiomorphism has considerable influence to warfarin dose use.Clinical study results shows: when reaching identical anticoagulant effect, comparatively wild-type patient is low for the using dosage of CYP2C9*2 and CYP2C9*3 genotype patient warfarin; In addition, U.S. food Drug Administration (FDA) explicitly points out: when using warfarin, and suggestion detects CYP2C9 genotype.Therefore, in patient CYP2C9 genotype and clinical application there is significant dependency in the use that holds water of warfarin.
Manganic pyrophosphate complex initiation (Pyrosequencing) is a kind of based on being polymerized the DNA sequencing of principle (namely, determine the order of DNA nucleotide) method, belong to DNA sequence analysis technology of new generation, possess the ability of simultaneously a large amount of sample being carried out to sequencing analysis, and there is the advantage of high, quick, the directly perceived and low cost of high-throughput, specificity.Its ultimate principle is, by the enzyme cascade chemiluminescence reaction in 4 kinds of enzymatic same reaction systems, take turns in sequencing reaction at each, only add a kind of dNTP, if this dNTP and template are matched, polysaccharase just can be incorporated in primer strand and the tetra-sodium group (PPi) of mole number such as to be discharged, and PPi finally can be converted into visible light signal, and being converted into a peak value by PyrogramTM, the height of each peak value is directly proportional to the nucleotide number mixed in reaction; Then add lower a kind of dNTP, continue the synthesis of DNA chain; Finally by the situation analyzing peak value, reach the object measuring DNA sequence dna.But, pyrosequencing techniques is not also utilized to detect the product of CYP2C9 gene type in prior art.
At present, in the prior art, especially in hospital, the PCR-direct sequencing of the title that employing has " gold standard " detects CYP2C9 gene, but the susceptibility of the method is not high, can only detect that mutant cell ratio is at the tumor tissues of more than 10-20% and peripheral blood, mutant cell ratio is less than to tumor tissues and the peripheral blood of 10%, Standard PCR-direct sequencing is almost helpless; In addition, the method also have that cost is high, the shortcoming of the long and complex operation of sense cycle.
Summary of the invention
In order to solve aforesaid method detect in CYP2C9 genotyping process have that susceptibility is not high, sense cycle long, complex operation and the high technical problem of cost, the invention provides that a kind of susceptibility is high, high specificity, sense cycle are short, simple to operate and the Manganic pyrophosphate complex initiation method effectively meeting Clinical Laboratory requirement detects primer pair and the test kit of CYP2C9 gene type.
The invention provides the primer pair that a kind of Manganic pyrophosphate complex initiation method detects CYP2C9 gene type, the pleomorphism site of described CYP2C9 gene test is CYP2C9*2 and/or CYP2C9*3, and described primer pair comprises:
(1) for CYP2C9*2 allelotrope,
Amplimer is:
CYP2C9*2 forward amplimer: 5 '-GACGCTGCGGAATTTTGG-3 ' (SEQIDNO.1);
The reverse amplimer of CYP2C9*2:
5’-CAGTAAGGTCAGTGATATGGAGTAGGG-3’(SEQIDNO.2)；
CYP2C9*2 sequencing primer: 5 '-GGCTTCCTCTTGAACA-3 ' (SEQIDNO.3);
Wherein, 5 ' end of described CYP2C9*2 forward amplimer carries out biotin labeling;
(2) for CYP2C9*3 allelotrope,
Amplimer is:
CYP2C9*3 forward amplimer: 5 '-AGCCACATGCCCTACACAG-3 ' (SEQIDNO.4);
The reverse amplimer of CYP2C9*3: 5 '-CAGGCTGGTGGGGAGAAG-3 ' (SEQIDNO.5);
CYP2C9*3 sequencing primer: 5 '-GCACGAGGTCCAGAGA-3 ' (SEQIDNO.6);
Wherein, 5 ' end of the reverse amplimer of described CYP2C9*3 carries out biotin labeling.
Present invention also offers the test kit that a kind of Manganic pyrophosphate complex initiation method detects CYP2C9 gene type, the pleomorphism site of described CYP2C9 gene test is CYP2C9*2 and/or CYP2C9*3, and described test kit comprises:
(1) for CYP2C9*2 allelotrope,
CYP2C9*2 forward amplimer: 5 '-GACGCTGCGGAATTTTGG-3 ';
PCR reaction solution 1, described PCR reaction solution 1 is containing the reverse amplimer of CYP2C9*2: 5 '-CAGTAAGGTCAGTGATATGGAGTAGGG-3 ';
CYP2C9*2 sequencing primer: 5 '-GGCTTCCTCTTGAACA-3 ';
Wherein, 5 ' end of described CYP2C9*2 forward amplimer carries out biotin labeling;
(2) for CYP2C9*3 allelotrope,
The reverse amplimer of CYP2C9*3: 5 '-CAGGCTGGTGGGGAGAAG-3 ';
PCR reaction solution 2, described PCR reaction solution 2 is containing CYP2C9*3 forward amplimer: 5 '-AGCCACATGCCCTACACAG-3 ';
CYP2C9*3 sequencing primer: 5 '-GCACGAGGTCCAGAGA-3 ';
Wherein, 5 ' end of the reverse amplimer of described CYP2C9*3 carries out biotin labeling.
In a kind of preferred embodiment of described test kit provided by the invention, described test kit also comprises:
CYP2C9*2 positive reference substance 1, its wild homozygote plasmid of CYP2C9*2 for being inserted with nucleotide sequence shown in SEQIDNO.8;
CYP2C9*2 positive reference substance 2, its plasmid mixture formed with the CYP2C9*2 no mutant homozygote plasmid being inserted with nucleotide sequence shown in SEQIDNO.9 for the wild homozygote plasmid of described CYP2C9*2;
CYP2C*3 positive reference substance 1, its wild homozygote plasmid of CYP2C9*3 for being inserted with nucleotide sequence shown in SEQIDNO.10;
CYP2C*3 positive reference substance 2, its plasmid mixture formed with the CYP2C9*3 no mutant homozygote plasmid being inserted with nucleotide sequence shown in SEQIDNO.11 for the wild homozygote plasmid of described CYP2C9*3;
Wherein, plasmid vector is pMD18-T plasmid; The number ratio of CYP2C9*2 no mutant homozygote plasmid described in described CYP2C9*2 positive reference substance 2 and the wild homozygote plasmid of described CYP2C9*2 is 1:1; The number ratio of CYP2C9*3 no mutant homozygote plasmid described in described CYP2C9*3 positive reference substance 2 and the wild homozygote plasmid of described CYP2C9*3 is 1:1.
In a kind of preferred embodiment of described test kit provided by the invention, described test kit also comprises: quality control product (controloligo) and blank product, and the sequence of described quality control product is: TAYGGTTTGCA (SEQIDNO.7); Described blank product are water; The sequence of quality control product is by one of QIAGEN design and synthesis section of oligonucleotide chain, for detecting the property indices of PyroMarkQ24 sequenator.
In described PCR reaction solution 1 and PCR reaction solution 2, other components are conventional 10xPCRBuffer, dNTPS and H
2o, each component volume ratio configuration routinely (in 10xPCRBuffer, dNTPs, H2O and reaction solution, the volume ratio of primer is 5:3:37.5:1).
In described test kit, other reagent and solution are the conventional reagent of PCR and DNA Manganic pyrophosphate complex initiation, as the enzyme mixture be made up of archaeal dna polymerase, adenosine triphosphate sulfurylase, luciferase and bisphosphatase, the substrate mixture be made up of 5'-phosphosulfate and fluorescein, uracil dna glycosylase, Taq polysaccharase etc.
Present invention also offers the application of primer pair as above in the reagent for the preparation of detection CYP2C9 gene type.
Present invention also offers the application of test kit as above in the reagent for the preparation of detection CYP2C9 gene type.
Compared to prior art, primer pair and the test kit of Manganic pyrophosphate complex initiation method detection CYP2C9 gene type provided by the invention have following beneficial effect:
One, by utilizing Manganic pyrophosphate complex initiation law technology to devise highly sensitive and that specificity is good primer pair and test kit thereof, making described test kit when detecting CYP2C9 gene type, having accurately qualitative, the advantage of highly sensitive and high specificity; In addition, also have that sample preparation is simple, sequencing steps is simple, order-checking speed is fast, half hour completes and once go up the advantage that machine reacts, directly provides detection site frequency analysis and visual result;
Two, by utilizing Manganic pyrophosphate complex initiation law technology to devise highly sensitive and that specificity is good primer pair and test kit thereof, make described test kit when detecting CYP2C9 gene type, Real-Time Monitoring reaction process, reaction times short, PCR primer simple process can go up tetra-sodium sequencer, easy and simple to handle and high-throughput sample detection, and than gold standard method, namely the sensitivity of capillary electrophoresis sequencing is higher, is more suitable for the requirement for Clinical Laboratory;
Three, by being provided with blank product, positive reference substance and quality control product in described test kit, making described test kit when detecting CYP2C9 gene type, better can guarantee the accuracy of detected result.
Accompanying drawing explanation
Fig. 1 is warfarin mechanism of action and CYP2C9 effect schematic diagram;
Fig. 2 is the Manganic pyrophosphate complex initiation figure of clinical sample CYP2C9*2 wild-type;
Fig. 3 is the Manganic pyrophosphate complex initiation figure of clinical sample CYP2C9*2 sudden change heterozygous;
Fig. 4 is the Manganic pyrophosphate complex initiation figure of clinical sample CYP2C9*2 mutant homozygous type;
Fig. 5 is the Manganic pyrophosphate complex initiation figure of clinical sample CYP2C9*3 wild-type;
Fig. 6 is the Manganic pyrophosphate complex initiation figure of clinical sample CYP2C9*3 sudden change heterozygous;
Fig. 7 is the Manganic pyrophosphate complex initiation figure of clinical sample CYP2C9*3 mutant homozygous type;
Fig. 8 is the Manganic pyrophosphate complex initiation figure of clinical quality control product controloligo;
Fig. 9 is the Manganic pyrophosphate complex initiation figure of clinical CYP2C9*2 blank product;
Figure 10 is the Manganic pyrophosphate complex initiation figure of clinical CYP2C9*3 blank product;
Figure 11 to Figure 12 is the Manganic pyrophosphate complex initiation figure that CYP2C9*2 many groups designs primer; Wherein the sequencing result of Figure 11 is inaccurate, and the sequencing result of Figure 12 is true and reliable;
Figure 13 to Figure 14 is the Manganic pyrophosphate complex initiation figure that CYP2C9*3 many groups designs primer; Wherein the sequencing result of Figure 13 is inaccurate, and the sequencing result of Figure 14 is true and reliable.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is described in further detail.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Embodiment 1: the preparation of test kit
One, the Design and synthesis of primer and probe
For people CYP2C9 gene polynorphisms site CYP2C9*2 and CYP2C9*3 allelotrope, select special mutational site, use PyroMarkAssayDesign2.0 software, design primer; Wherein amplimer and sequencing primer are first through PAGE purifying, then through HPLC purifying, wherein 5 ' the end of SEQIDNO.1 and SEQIDNO.5 carries out biotin labeling.
Table 1. mutational site and type:
| Mutation | Base change |
| CYP2C9*2(rs1799853) | C>T |
| CYP2C9*3(rs1057910) | A>C |
Extension increasing sequence is as table 2:
Table 2. specificity amplification primer and primer sequence
Two, reference substance is selected
The one section of oligonucleotide chain TAYGGTTTGCAcontrololigo using synthetic is quality control product; DNase/RNase-Free water is blank product.
Three, PCR reaction solution composition
Table 3.PCR reaction solution 1 forms
| Material name | Volume (μ L) |
| 10x PCR Buffer | 5 |
| dNTP | 3 |
| The reverse amplimer of CYP2C9*2 | 1 |
| H 2O | 37.5 |
| Cumulative volume | 46.5μL |
Table 4.PCR reaction solution 2 forms
| Material name | Volume (μ L) |
| 10x PCR Buffer | 5 |
| dNTP | 3 |
| CYP2C9*3 forward amplimer | 1 |
| H 2O | 37.5 |
| Cumulative volume | 46.5μL |
Owing to increasing for two detection site, so there are 2 kinds of different PCR reaction solutions, respectively CYP2C9*2 and CYP2C9*3 site is detected.
Embodiment 2: the use of test kit
One, sample detection
Dissolve primer dry powder (it is 1 month that primer dissolves rear validity period).According to template number preparation system: get PCR reaction solution, add solvent primer, uracil dna glycosylase, Taq DNA polymerase, packing system, adding sample DNA, blank product or positive reference substance is template, composition PCR reaction system.Pcr amplification is carried out according to PCR response procedures.
The each main component of CYP2C9*2 and CYP2C9*3 system is as follows respectively:
The each main component of table 5.CYP2C9*2 system
The each main component of table 6.CYP2C9*3 system
This system response procedures is as follows:
Table 7.PCR response procedures
After having increased, sepharose inspection PCR result, to carry out next step program.
Two, Manganic pyrophosphate complex initiation
Carry out sequencing procedures according to Manganic pyrophosphate complex initiation standard operating procedure, key step is: the preparation of sample and purifying, then the sample after purifying is added upper machine order-checking in the MIX containing annealing liquid and sequencing primer.The corresponding dATP of working procedure, dTTP, dCTP, dGTP, enzyme mixture, substrate mixture is added in Manganic pyrophosphate complex initiation instrument agent bin.Quality control product controloligo carries sequencing primer, and ultimate density is 0.2 μM.
Three, result judges
Quality control product base recall rate is 100%; Blank product can't detect base frequency.
Four, quality control standard
All kinds of contrast quality control product judged result is as following table:
Table 8. quality control product standard testing result
| Reference substance | Standard test result |
| 1 | Quality control product | Peak height peak type is normal, sequence is accurate |
| 2 | Blank product | Base recall rate is 0 |
Five, report the test:
Result is as shown in Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6 and Fig. 7, and the judging criterion of sample result is as follows:
Sample detection result reported by table 9.
Fig. 1 is warfarin mechanism of action and CYP2C9 effect schematic diagram; The wild-type of what Fig. 2 and Fig. 5 showed respectively is CYP2C9*2 and CYP2C9*3 in clinical sample detected result, the sudden change heterozygous of what Fig. 3 and Fig. 6 showed respectively is CYP2C9*2 and CYP2C9*3 in clinical sample detected result, the mutant homozygous type of what Fig. 4 and Fig. 7 showed respectively is CYP2C9*2 and CYP2C9*3 in clinical detection result; The Manganic pyrophosphate complex initiation figure of quality control product controloligo, CYP2C9*2 and CYP2C9*3 blank product that what Fig. 8, Fig. 9 and Figure 10 showed respectively is in clinical detection result; Figure 11 to Figure 12 display be the Manganic pyrophosphate complex initiation design sketch of CYP2C9*2 many groups primer of design, wherein the primer of Figure 12 be best, is the CYP2C9*2 primer selected in our product; Figure 13 to Figure 14 display be the Manganic pyrophosphate complex initiation design sketch of CYP2C9*3 many groups primer of design, wherein the primer of Figure 14 be best, is the CYP2C9*3 primer selected in our product.
Primer pair and the test kit of Manganic pyrophosphate complex initiation method detection CYP2C9 gene type provided by the invention have following beneficial effect:
One, by utilizing Manganic pyrophosphate complex initiation law technology to devise highly sensitive and that specificity is good primer pair and test kit thereof, making described test kit when detecting CYP2C9*2 and CYP2C9*3 site, having accurately qualitative, the advantage of highly sensitive and high specificity; In addition, also have that sample preparation is simple, sequencing steps is simple, order-checking speed is fast, half hour completes and once go up the advantage that machine reacts, directly provides detection site frequency analysis and visual result;
Two, by utilizing Manganic pyrophosphate complex initiation law technology to devise highly sensitive and that specificity is good primer pair and test kit thereof, make described test kit when detecting CYP2C9*2 and CYP2C9*3 site, Real-Time Monitoring reaction process, reaction times short, PCR primer simple process can go up tetra-sodium sequencer, easy and simple to handle and high-throughput sample detection, and than gold standard method, namely the sensitivity of capillary electrophoresis sequencing is higher, is more suitable for the requirement for Clinical Laboratory;
Three, by being provided with blank product, positive reference substance and quality control product in described test kit, making described test kit when detecting CYP2C9*2 and CYP2C9*3 site, better can guarantee the accuracy of detected result.
The foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every equivalent flow process conversion utilizing description of the present invention to do, or be directly or indirectly used in other relevant technical field, be all in like manner included in scope of patent protection of the present invention.
SEQUENCELISTING
Help bio tech ltd in <110> Changsha three
<120> Manganic pyrophosphate complex initiation method detects primer pair and the test kit of CYP2C9 gene type
<130>2015
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<170>PatentInversion3.3
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Claims (8)
1. Manganic pyrophosphate complex initiation method detects a primer pair for CYP2C9 gene type, and it is characterized in that, the pleomorphism site of described CYP2C9 gene test is CYP2C9*2 and/or CYP2C9*3, and described primer pair comprises:
(1) for CYP2C9*2 allelotrope,
Amplimer is:
CYP2C9*2 forward amplimer: 5 '-GACGCTGCGGAATTTTGG-3 ';
The reverse amplimer of CYP2C9*2:
5’-CAGTAAGGTCAGTGATATGGAGTAGGG-3’；
CYP2C9*2 sequencing primer: 5 '-GGCTTCCTCTTGAACA-3 ';
Wherein, 5 ' end of described CYP2C9*2 forward amplimer carries out biotin labeling;
(2) for CYP2C9*3 allelotrope,
Amplimer is:
CYP2C9*3 forward amplimer: 5 '-AGCCACATGCCCTACACAG-3 ';
The reverse amplimer of CYP2C9*3: 5 '-CAGGCTGGTGGGGAGAAG-3 ';
CYP2C9*3 sequencing primer: 5 '-GCACGAGGTCCAGAGA-3 ';
Wherein, 5 ' end of the reverse amplimer of described CYP2C9*3 carries out biotin labeling.
2. Manganic pyrophosphate complex initiation method detects a test kit for CYP2C9 gene type, and it is characterized in that, the pleomorphism site of described CYP2C9 gene test is CYP2C9*2 and/or CYP2C9*3, and described test kit comprises:
(1) for CYP2C9*2 allelotrope,
CYP2C9*2 forward amplimer: 5 '-GACGCTGCGGAATTTTGG-3 ';
PCR reaction solution 1, described PCR reaction solution 1 is containing the reverse amplimer of CYP2C9*2: 5 '-CAGTAAGGTCAGTGATATGGAGTAGGG-3 ';
CYP2C9*2 sequencing primer: 5 '-GGCTTCCTCTTGAACA-3 ';
Wherein, 5 ' end of described CYP2C9*2 forward amplimer carries out biotin labeling;
(2) for CYP2C9*3 allelotrope,
The reverse amplimer of CYP2C9*3: 5 '-CAGGCTGGTGGGGAGAAG-3 ';
PCR reaction solution 2, described PCR reaction solution 2 is containing CYP2C9*3 forward amplimer: 5 '-AGCCACATGCCCTACACAG-3 ';
CYP2C9*3 sequencing primer: 5 '-GCACGAGGTCCAGAGA-3 ';
Wherein, 5 ' end of the reverse amplimer of described CYP2C9*3 carries out biotin labeling.
3. test kit according to claim 2, is characterized in that, described test kit also comprises:
CYP2C9*2 positive reference substance 1, its wild homozygote plasmid of CYP2C9*2 for being inserted with nucleotide sequence shown in SEQIDNO.8;
CYP2C9*2 positive reference substance 2, its plasmid mixture formed with the CYP2C9*2 no mutant homozygote plasmid being inserted with nucleotide sequence shown in SEQIDNO.9 for the wild homozygote plasmid of described CYP2C9*2;
CYP2C*3 positive reference substance 1, its wild homozygote plasmid of CYP2C9*3 for being inserted with nucleotide sequence shown in SEQIDNO.10;
CYP2C*3 positive reference substance 2, its plasmid mixture formed with the CYP2C9*3 no mutant homozygote plasmid being inserted with nucleotide sequence shown in SEQIDNO.11 for the wild homozygote plasmid of described CYP2C9*3;
Wherein, plasmid vector is pMD18-T plasmid.
4. test kit according to claim 3, is characterized in that, the number ratio of CYP2C9*2 no mutant homozygote plasmid described in described CYP2C9*2 positive reference substance 2 and the wild homozygote plasmid of described CYP2C9*2 is 1:1; The number ratio of CYP2C9*3 no mutant homozygote plasmid described in described CYP2C9*3 positive reference substance 2 and the wild homozygote plasmid of described CYP2C9*3 is 1:1.
5. test kit according to claim 2, is characterized in that, described test kit also comprises: quality control product and blank product, and the sequence of described quality control product is: TAYGGTTTGCA.
6. test kit according to claim 5, is characterized in that, described blank product are water.
7. the application of primer pair according to claim 1 in the reagent for the preparation of detection CYP2C9 gene type.
8. the application of test kit according to claim 2 in the reagent for the preparation of detection CYP2C9 gene type.
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| CN107201407A (en) * | 2017-06-27 | 2017-09-26 | 贵州省人民医院 | The primer and method of a kind of pyrosequencing method detection VKORC1 and CYP2C9 gene pleiomorphisms |
| CN108277269A (en) * | 2017-10-25 | 2018-07-13 | 长沙三济生物科技有限公司 | Primer pair and kit for detecting warfarin medication related gene polymorphism |
| CN108753947A (en) * | 2018-06-06 | 2018-11-06 | 无锡正则精准医学检验有限公司 | A kind of kit of detection androgens psilosis tumor susceptibility gene 20P11 gene pleiomorphisms |
| CN108796062A (en) * | 2018-06-06 | 2018-11-13 | 无锡正则精准医学检验有限公司 | A kind of kit of detection TGFBI gene pleiomorphisms |
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| CN107201407A (en) * | 2017-06-27 | 2017-09-26 | 贵州省人民医院 | The primer and method of a kind of pyrosequencing method detection VKORC1 and CYP2C9 gene pleiomorphisms |
| CN108277269A (en) * | 2017-10-25 | 2018-07-13 | 长沙三济生物科技有限公司 | Primer pair and kit for detecting warfarin medication related gene polymorphism |
| CN108753947A (en) * | 2018-06-06 | 2018-11-06 | 无锡正则精准医学检验有限公司 | A kind of kit of detection androgens psilosis tumor susceptibility gene 20P11 gene pleiomorphisms |
| CN108796062A (en) * | 2018-06-06 | 2018-11-13 | 无锡正则精准医学检验有限公司 | A kind of kit of detection TGFBI gene pleiomorphisms |
| CN113249463A (en) * | 2021-04-09 | 2021-08-13 | 湖南菲思特精准医疗科技有限公司 | Gene detection kit for angiotensin II receptor inhibitor medication and detection method and application thereof |
| CN113549685A (en) * | 2021-06-09 | 2021-10-26 | 湖南菲思特精准医疗科技有限公司 | Detection kit for celecoxib metabolic marker and detection method and application thereof |
| CN113584150A (en) * | 2021-06-17 | 2021-11-02 | 湖南菲思特精准医疗科技有限公司 | Detection kit for voriconazole metabolic marker, detection method and application thereof |
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