CN105132416A

CN105132416A - Detection primer group and kit for CYP2C9*3 gene amplification

Info

Publication number: CN105132416A
Application number: CN201510515084.7A
Authority: CN
Inventors: 刘沛; 丁朋举; 李江浩; 王林海
Original assignee: BEIJING SINO-MDGENE TECHNOLOGY Co Ltd
Current assignee: BEIJING SINO-MDGENE TECHNOLOGY Co Ltd
Priority date: 2015-08-20
Filing date: 2015-08-20
Publication date: 2015-12-09

Abstract

The invention relates to a kit for SNP, in particular to a detection primer group and kit for CYP2C9*3 gene amplification. The CYP2C9*3 primer group comprises upstream and downstream primers and a fluorescent primer to a target gene; a 5' terminal of the fluorescent primer is labeled with a fluorescent group. The kit comprises the primer group, a negative quality control and a positive quality control. By using the primer group and the kit, genotype of CYP2C9*3 can be determined quickly and simply, detection sensitivity is high, detection time is short, less pollution is caused, operating is simple and convenient, the cost is low, and the primer group and the kit are suitable for large-scale clinical development; therefore, CYP2C9*3 gene amplification can be detected quickly, effectively and accurately, and timely disease treatment and therapeutic effect monitoring can be guaranteed.

Description

A kind of detection primer sets of CYP2C9*3 gene amplification and test kit

Technical field

The present invention relates to the test kit of a kind of SNP, specifically, relate to a kind of detection primer sets and test kit of CYP2C9*3 gene amplification.

Background technology

Warfarin is a kind of coumarins oral anticoagulation of clinical conventional treatment thrombotic diseases, has the dual function of anti-freezing and thrombolysis.The anticoagulant therapy index range of warfarin is narrow, if underdosage, does not reach result for the treatment of, and dosage then can cause bleeding little over amount, and severe one jeopardizes patients ' lives.But warfarin plasma drug level and curative effect exist obvious individual difference and racial difference, there are some researches show, different patient uses the dosage of anticoagulation medicine warfarin can differ 20 times, and Asian maintenance dose is lower than white people by 30% ~ 40%.Therefore, how clinically safety, reasonable employment warfarin are the Focal point and difficult point that many investigators pay close attention to for a long time.

In recent years, along with the development of pharmacogenomics and illustrating of warfarin pharmacological action molecular mechanism, the effect of mononucleotide gene pleiomorphism in warfarin consumption individual difference is more and more subject to people's attention.At present, the known gene relevant with pharmacokinetics to the pharmacodynamics of warfarin reaches more than 30 and plants, wherein the gene pleiomorphism of cytochrome P450 2C9 gene (CytochromeP4502C9, CYP2C9) affects one of topmost inherited genetic factors of warfarin consumption individual difference.Warfarin metabolism can be directly the product of non-activity by cytochrome pathways, and warfarin is the antagonist (VKA) of vitamin K, produces anticoagulation by suppressing liver epoxidoreductase.Therefore, CYP2C9 gene determines the anticoagulant effect of warfarin.

Namely CYP2C9 is the topmost metabolic enzyme of warfarin.Up to the present, the allelotrope of the CYP2C9 found has 30 kinds, wherein common with * l (wild-type), * 3 (Ile359Leu, SNPrs1057910).Carry the allelic patient of variability, the activity of its warfarin metabolic enzyme is starkly lower than wild-type, and its hemorrhage danger increases by 2 ~ 3 times.

Warfarin has R and S two hypotypes, and wherein the effect of S type antivitamin K is the most obvious, and R type is substantially inoperative in vivo.The metabolism of S type warfarin can be non-activity or SA product by cytochrome pathways CYP2C9 gene, S type warfarin directly acts on vitamin K epoxide reductase, the oxidized form of vitamin K and reduced form can not be transformed mutually, thus suppress the function of vitamin K, also suppress the activation of some thrombin simultaneously.Large quantity research proves, CYP2C9 gene pleiomorphism can affect the initial using dosage of warfarin, and CYPC9*3AA type dosage is the highest, and AC heterozygous is taken second place, and CC type is minimum.The dosage of warfarin in different race, age, or sex crowd varies, and very large reason is CYP2C9 gene polynorphisms.And in CYP2C9 gene, use the relevant rule of pharmacogenetics substantially can determine the initial using dosage of warfarin by the polymorphism of detection site and then in conjunction with patient's relevant clinical information.

U.S. FDA approval gene diagnosis and test item have about 2000, wherein 1290 multinomial completely approval be used for clinical, about 1000 just in approval process.Facts have proved, genetic polymorphism detection is effective.China should use warfarin and untapped crowd reaches about 90%, and patients with atrial fibrillation only has 6.6% proper use of warfarin, and its reason is the correct dose that can not determine that warfarin uses, can not specification monitoring INR.Like this, atrial fibrillation causes apoplexy patient obviously to increase, and the patients with cerebral apoplexy of clinical statistics display nearly 1/3 is caused by atrial fibrillation.If clinician determines the initial using dosage of warfarin by the means of gene test, safe handling warfarin, will reduce disability rate and the lethality rate of atrial fibrillation.

It is below the classifying method now to CYP2C9*3.

Taqman method: for the specific probe of SNP site variation design, probe mark cost is higher; Adopt fluorescent quenching and two end-labelling, have the defect that cancellation is difficult to thoroughly, background is higher; Employing enzyme is exo-acting, by the impact of enzyme performance time quantitative.

DHPLC technology and mass spectroscopy: need respectively to use high performance liquid chromatograph and mass spectrograph, apparatus expensive, be difficult to promote clinical examining in survey.

DNA chip: because the advantages such as high-throughput are widely applied in SNP detects, but it relies on the difference of wild-type and mutated genes hybridization kinetics to detect mutational site, because the hybridization kinetics difference between different loci is different, when carrying out multidigit point and detect simultaneously, condition is difficult to control, repeatability is poor, easily occurs false positive false negative result.

Micrometering sequence: as a kind of mutation analysis based on array, is also faced with the key issues such as the variation how reducing false negative rate and target nucleotide sequences composition at present.

High temperature conjunction enzyme process: high temperature conjunction enzyme detection reaction (ligasedetectionreaction, LDR) is a kind of novel gene pleiomorphism detecting method come into the picture gradually in recent years, this technological operation is simple, stable, reliable results.Be commonly called as little order-checking.

Restriction Enzyme blanking method (RFLP): accuracy is better, and expense is also lower. but somatotype can only be carried out to the polymorphic site that a part contains ready-made restriction enzyme site.

SNaPshot method: based on the typing method of fluorescent mark single-basic extension principle, is generally used for 10-30 SNP site analysis.

DNA sequencing method: relatively accurate, price is more expensive.The secondary structure of DNA chain easily causes artifacts, makes sequencing result occur deviation.

High resolving power melting curve method (HRM): be the SNP research tool the method for rising in recent years, without the need to designing probe, cost is low, but result precision is lower.

Amplification refractory mutation system (Amplificationrefractorymutationsystem, ARMS)): this technology is by relating to specific primer, selectively increase wild or sudden change masterplate, determine whether as wild-type or saltant type by the amount of amplified production, utilize 3 ' of the PCR primer principle of holding last bit base could effectively must increase with its template DNA complementation, design ApoE gene amplimer, under strict conditions, only could there is pcr amplification band when primer 3 ' base and template are matched, thus detect sudden change.Different primers adding, 3 T distinguish the fragment amplified.

Capillary electrophoresis (capillaryelectrophoresis, CE) also known as HPCE (highperformancecapillaryelectrophoresis, HPCE), be a class be split tunnel with kapillary, take high-voltage dc as the Novel liquid-phase isolation technique of motivating force.In fact capillary electrophoresis comprises electrophoresis, chromatogram and intersection content thereof, and it makes analytical chemistry be able to enter from microlitre level receive premium on currency and put down, and make single cell analysis, and even single molecule analysis becomes possibility.

The domestic research to CYP2C9*3 at present still belongs to blank, does not more have ripe detection method or reagent.

In view of this, special proposition the present invention.

Summary of the invention

The invention provides a kind of detection primer sets of CYP2C9*3 gene amplification;

The invention provides a kind of detection kit of CYP2C9*3 gene amplification.

For solving the problems of the technologies described above, the present invention adopts the basic conception of technical scheme to be: the detection primer sets of CYP2C9*3 gene amplification, and described CYP2C9*3 detects primer sets and comprises:

CYP2C9*3-PD-F1: the nucleotide sequence as shown in SEQIDNO:1, or be substituted by the nucleotide sequence shown in SEQIDNO:1, lack, add the nucleotide sequence formed;

CYP2C9*3-PD-F2: the nucleotide sequence as shown in SEQIDNO:2, or be substituted by the nucleotide sequence shown in SEQIDNO:2, lack, add the nucleotide sequence formed;

CYP2C9*3-PD-R: the nucleotide sequence as shown in SEQIDNO:3, or be substituted by the nucleotide sequence shown in SEQIDNO:3, lack, add the nucleotide sequence formed;

Described CYP2C9*3-PD-R is fluorescent primer, and its 5 ' end is marked with fluorophor;

As the preferred technical solution of the present invention, described fluorophor is selected from by 6-Fluoresceincarboxylic acid, chlordene-6-methyl fluorescein, VC fluorescence dye, four chloro-6-Fluoresceincarboxylic acids, carboxy-X-rhodamine, 6-carboxyl tetramethylrhodamine, Sulforhodamine, 6-carboxyl-4 ', 5 '-two chloro-2 ', at least one in 7 '-dimethoxyfluorescein succinimide ester, Hua Jing 3, Hua Jing 5 or flower cyanines 5.5.

As the preferred technical solution of the present invention, the fluorophor of described CYP2C9*3-PD-R is 6-Fluoresceincarboxylic acid.

CYP2C9*3 gene amplification detection kit, comprises the detection primer sets of above-described CYP2C9*3 gene amplification.

As the preferred technical solution of the present invention, also comprise containing Mg ²⁺, Taq enzyme and dNTPs PCR reaction solution.

As the preferred technical solution of the present invention, also comprise negative quality control product, positive quality control product;

Described negative quality control product is purified water; Described positive quality control product is deactivation whole blood.

As the preferred technical solution of the present invention, described test kit is divided into negative quality control product, positive quality control product, CYP2C9*3 reaction system;

Described CYP2C9*3 reaction system comprises CYP2C9*3PCR reaction solution 12.5 μ L, CYP2C9*3 primer mixed solution 6.5 μ L, and sample to be tested DNA2 μ L, purified water 4 μ L, is made into 25 μ L reaction systems altogether;

Consisting of of wherein said CYP2C9*3 primer mixed solution: the CYP2C9*3-PD-F1 of 1.5 μ L, the CYP2C9*3-PD-F2 of 1.5 μ L, the CYP2C9*3-PD-R of 1.5 μ L.

The construction process of CYP2C9*3 positive quality control product comprises the following steps:

(1) use asepsis injector at person under inspection's arm bending place or the back of the hand place venous blood samples 3 ~ 5mL, be placed in EDTA anticoagulant tube (purple lid), put upside down mixing gently;

(2) genome extraction is carried out with sky root whole blood test kit;

(3) gene of purifying utilizes uv analysis method to measure absorbancy, and calculating concentration, optimum concn is 50ng/ml.

The using method of CYP2C9*3 gene amplification detection kit, detailed process is as follows:

(1) sample process and nucleic acid extraction;

(2) PCR reaction is carried out;

(3) detected by Genetic Analyser, obtain fragment analysis result.

The treatment process of described sample to be tested comprises: magnetic bead extraction method, post formulation, boiling lysis and cetyl trimethylammonium bromide method, preferred post formulation.

As the preferred technical solution of the present invention, the sample described in step (1) is human genome DNA, and OD260/OD280 is in 1.8-2.0, DNA total amount at 3-15 μ g, and the final concentration of dilution DNA is to 50-125ng/ μ L.

As the preferred technical solution of the present invention, the final concentration of dilution DNA is to 50ng/ μ L.

As the preferred technical solution of the present invention, in step (2), the reaction conditions of PCR reaction is: 37 DEG C of insulations are after 2 minutes; 95 DEG C of denaturations 3 minutes, 92 ~ 95 DEG C, 10 ~ 15 seconds, 55 ~ 65 DEG C, 10 ~ 35 seconds, totally 35 ~ 45 circulations; Be preferably: 37 DEG C of insulations are after 2 minutes; 95 DEG C of denaturations 3 minutes; 94 DEG C, 15 seconds; 60 DEG C, 35 seconds; 72 DEG C, 20 seconds; Totally 35 circulations.

The technology of the present invention principle is, chooses the region of the SNP of CYP2C9, and design specificity ARMS primer, then detects the SNP type that whole blood DNA carries out CYP2C9 with capillary electrophoresis.

After adopting technique scheme, the present invention compared with prior art has following beneficial effect:

Utilize detection primer sets of the present invention and test kit can determine CYP2C9*3 gene type fast, easily, have that detection sensitivity is high, the time is short, decreasing pollution, simple to operation, and cost is low, is suitable for larger scale clinical and carries out.Thus realize CYP2C9*3 gene amplification fast, effectively and detect accurately, thus can ensure case diagnosis and treatment timely and result for the treatment of monitoring.

Detection primer of the present invention, by fluorescent quantitative PCR, during gene-detecting apparatus detection reaction result, when corresponding peaks appears in sense channel, can accurately judge corresponding type.

Accompanying drawing explanation

Fig. 1-1,1-2,1-3,1-4,1-5,1-6,1-7 are the sequencing result figure of embodiment 7 samples;

Fig. 2 is the sequencing result figure of positive quality control product;

Fig. 3 is the sequencing result figure of negative quality control product.

Embodiment

Be further explained explanation below in conjunction with specific embodiments and the drawings to the present invention, agents useful for same of the present invention is commercially available.

1. sample process

1.1 sample process and nucleic acid extraction

The extraction of sample, positive quality control product and negative quality control product uses TIANampBloodDNAKit (CatNO.DP318) test kit, and concrete operations see this test kit specification sheets, but will note following item:

1.1.1 when sample, positive quality control product are extracted, by 100 μ L buffer solution elution.

1.1.2, when negative quality control product extracts, add 4 μ LCYP2C9*3 in the negative quality control product of 200 μ L and participate in extracting, by 100 μ L buffer solution elution.

1.1.3 extract DNA to need to measure concentration with ultraviolet spectrophotometer, sample pollutes without albumen or RNA, OD260/OD280=1.8 ~ 2.0, OD260/OD230 >=2.0; DNA content requires: recommend DNA applied sample amount 50 ~ 100ng; The DNA extracted detects immediately, and in-20 ± 5 DEG C of preservations.

1.2 prepare

Take out each component of test kit before experiment starts, fully thaw and sustained oscillation mixing in 15 seconds, centrifugal 15 seconds of 2000rpm.

Prepare the PCR instrument example reaction pipe of respective numbers, except sample, also should comprise negative quality control product and positive quality control product reaction tubes.

2. reagent configuration

2.1 determine stoichiometric number 10,10=sample number to be checked (7)+quality control product number (2)+1.Calculate the amount of each reagent be added in reaction mixture, be calculated as follows:

Table 2 system is prepared

Reagent	PCR reaction solution (μ L)	Primer mixed solution (μ L)	Purified water (μ L)
				CYP2C9*3	12.5×10	6.5×10	4.0×10

2.2 get 1.5mL centrifuge tube (sterilizing) configures reaction system, and reagent all adds rear vortex and mixes 10 seconds, centrifugal 10 seconds of 2000rpm.

Then above-mentioned mixed solution 23 μ L/ pipe point is filled in aseptic PCR reaction tubes by 2.3.

3. application of sample

By extracting sample DNA, CYP2C9*3 (A/C) positive quality control product, the negative quality control product of CYP2C9*3 add in PCR reaction tubes by table 3 specified amount respectively, cover tightly pipe lid, liquid on tube wall all gets rid of and (avoids bubble to produce) at the bottom of pipe by centrifugal 10 seconds of 2000rpm, then carries out pcr amplification reaction immediately.Sample putting position is as table 4:

Table 3 application of sample

Title

Composition

Application of sample amount

Sample

Sample DNA

2μL

Table 4 sample putting position

Title	1	2	3	4	5	6	7	8	9
										CYP2C9*3	①	②	③	④	⑤	⑥	⑦	STD1	NTC1

Note: 1.-7. representative sample; STD1 representative CYP2C9*3 (A/C) positive quality control product; NTC1 represents negative quality control product.

4.PCR amplification program is arranged

Table 5PCR amplification instrument 960 type PCR reaction conditions

5. sample preparation

For CYP2C9*3 site, get 1 μ LPCR product stoste, mark and 8 μ LHi-DiFormamide are placed in 0.2mL single tube mesoscale eddies and vibrate 10 seconds in 1 μ LLIZ500 molecular weight, centrifugal 15 seconds of 2000rpm.

6. instrument preparation, capillary electrophoresis and detection

Operate the computer with reference to ABI3500Dx Genetic Analyser user manual.The product handled well is transferred on 96 hole automatic sampling pallets, automatic sampling pallet is placed in instrument, shuts instrument door.Start capillary electrophoresis.

7. interpretation of result

CYP2C9*3 loci gene type decision table

Note: "+" indicates peak figure, "-" indicates without peak figure.

The sequencing result of 7 samples to be checked is as shown in Fig. 1-1 to 1-7, and as can be seen from Fig. 1-1, sample 1 to be checked occurs unimodal near 123.1bp, illustrates that this sample is AA homozygosity; As can be seen from Fig. 1-2, sample 2 to be checked occurs unimodal near 126.6bp, illustrates that this sample is CC homozygosity; As can be seen from Fig. 1-3, sample 3 to be checked occurs bimodal near 123.1bp and 126.6bp, illustrates that this sample is AC heterozygous; As can be seen from Fig. 1-4, sample 4 to be checked occurs unimodal near 126.6bp, illustrates that this sample is CC homozygosity; As can be seen from Fig. 1-5, sample 5 to be checked occurs unimodal near 123.1bp, illustrates that this sample is AA homozygosity; As can be seen from Fig. 1-6, sample 6 to be checked occurs bimodal near 123.1bp and 126.5bp, illustrates that this sample is AC heterozygous; As can be seen from Fig. 1-7, sample 7 to be checked occurs bimodal near 123.1bp and 126.6bp, illustrates that this sample is AC heterozygous.

Fig. 2 is the sequencing result figure of positive quality control product, upper as can be seen from figure, occurs bimodal, illustrate that this sample is AC heterozygous near 123.1bp and 126.4bp, consistent with the sample type added;

Fig. 3 is the sequencing result figure of negative quality control product, and upper as can be seen from figure, near 123 ± 1bp and 126 ± 1bp, all do not occur peak, illustrate that this sample does not belong to any type, the negative quality control product in fact in the present invention is purified water, also there will not be any peak value.

The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims

The detection primer sets of 1.CYP2C9*3 gene amplification, is characterized in that, described CYP2C9*3 detects primer sets and comprises:

CYP2C9*3-PD-F1: the nucleotide sequence as shown in SEQIDNO:1, or be substituted by the nucleotide sequence shown in SEQIDNO:1, lack, add the nucleotide sequence formed;

CYP2C9*3-PD-F2: the nucleotide sequence as shown in SEQIDNO:2, or be substituted by the nucleotide sequence shown in SEQIDNO:2, lack, add the nucleotide sequence formed;

CYP2C9*3-PD-R: the nucleotide sequence as shown in SEQIDNO:3, or be substituted by the nucleotide sequence shown in SEQIDNO:3, lack, add the nucleotide sequence formed.

Wherein, described CYP2C9*3-PD-R is fluorescent primer, and its 5 ' end is marked with fluorophor.
2. the detection primer sets of CYP2C9*3 gene amplification as claimed in claim 1, it is characterized in that, described fluorophor is selected from by 6-Fluoresceincarboxylic acid, chlordene-6-methyl fluorescein, VC fluorescence dye, four chloro-6-Fluoresceincarboxylic acids, carboxy-X-rhodamine, 6-carboxyl tetramethylrhodamine, Sulforhodamine, 6-carboxyl-4 ', 5 '-two chloro-2 ', at least one in 7 '-dimethoxyfluorescein succinimide ester, Hua Jing 3, Hua Jing 5 and Hua Jing 5.5.
3. the detection primer sets of CYP2C9*3 gene amplification as claimed in claim 2, it is characterized in that, the fluorophor of described CYP2C9*3-PD-R is 6-Fluoresceincarboxylic acid, and the fluorophor of HER2-PD-R is HEX.
4.CYP2C9*3 gene amplification detection kit, is characterized in that, comprises the detection primer sets of the CYP2C9*3 gene amplification described in any one of claim 1-3.
5. CYP2C9*3 gene amplification detection kit as claimed in claim 4, is characterized in that, also comprise containing Mg ²⁺, Taq enzyme and dNTPs PCR reaction solution.
6. CYP2C9*3 gene amplification detection kit as claimed in claim 4, is characterized in that, also comprise negative quality control product, positive quality control product;

Described negative quality control product is purified water; Described positive quality control product is deactivation whole blood.
7. CYP2C9*3 gene amplification detection kit as claimed in claim 4, it is characterized in that, described test kit is divided into negative quality control product, positive quality control product, CYP2C9*3 reaction system;

Described CYP2C9*3 reaction system comprises CYP2C9*3PCR reaction solution 12.5 μ L, CYP2C9*3 primer mixed solution 6.5 μ L, and sample to be tested DNA2 μ L, purified water 4 μ L, is made into 25 μ L reaction systems altogether;

Consisting of of wherein said CYP2C9*3 primer mixed solution: the CYP2C9*3-PD-F1 of 1.5 μ L, the CYP2C9*3-PD-F2 of 1.5 μ L, the CYP2C9*3-PD-R of 1.5 μ L.
The using method of 8.CYP2C9*3 gene amplification detection kit, it is characterized in that, detailed process is as follows:

(1) sample process and nucleic acid extraction;

(2) PCR reaction is carried out;

(3) carry out order-checking by Genetic Analyser to detect, obtain fragment analysis result.
9. using method as claimed in claim 8, it is characterized in that, the sample described in step (1) is human genome DNA, and OD260/OD280 is in 1.8-2.0, DNA total amount at 3-15 μ g, and the final concentration of dilution DNA is to 50-125ng/ μ L.
10. using method as claimed in claim 9, it is characterized in that, the final concentration of dilution DNA is to 50ng/ μ L.