CN104561301A - Detection method for CYP2C9*3 gene polymorphism, as well as nucleic acid probe and kit for method - Google Patents
Detection method for CYP2C9*3 gene polymorphism, as well as nucleic acid probe and kit for method Download PDFInfo
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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Abstract
The invention discloses a detecting probe for CYP2C9*3 gene polymorphism. The nucleotide sequences of the detecting probe are selected from a sequence 1 and a sequence 2; a fluorescent group and a quenching group are respectively arranged at the ends (5'and 3'); the peak values of the wavelengths of the florescent light emitted from the fluorescent group are at different positions. Preferably, the quenching group is selected from MGB and BHQ2; preferably, the fluorescent group is selected from FAM, VIC, JOE, HEX, CY3, NED, TAMRA, ROX, TEXAS RED, CY5 and the like. The invention further discloses a primer pair used for gene amplification; the primer pair comprises a forward primer and a reverse primer; the forward primer is an oligonucleotide which consists of base sequences of a sequence 3; the reverse primer is an oligonucleotide which consists of base sequences of a sequence 4. The invention further discloses a kit which contains the primers and the probe, and a detecting method for the CYP2C9*3 gene polymorphism.
Description
Technical field
The present invention relates to a kind of detection probes and amplimer of CYP2C9*3 gene pleiomorphism, belong to biological technical field.
Background technology
Warfarin is widely used in the field oral anticoagulation thing such as antithrombotic treatments after the thrombus prevention of atrial fibrillation, palsy, venous thromboembolism, mechanical prosthetic valve implantation in recent decades.This medicine is racemization isomer, and wherein R type enantiomorph is different with S type enantiomorph action intensity, and the pharmacological action of S type is 3-6 times of R type.This medicine has very strong water-soluble, and absorb rapidly through gi tract, bioavailability is bordering on 100%.Within after oral administration 90 minutes, reach peak plasma concentrations, 36 ~ 42 hours transformation period.Be combined with plasma proteins in blood circulation (combination rate 98% ~ 99%).Store up two kinds of enantiomers in liver by different approaches metabolism, about 90%S type warfarin generation oxidative metabolism reaction, mainly by the CYP2C9 enzyme catalysis of liver cytochrome P 450 system, small part is by CYP3A4 catalysis.About 60%R type warfarin generation oxidative metabolism reaction, mainly by CYP1A2 and the CYP3A4 enzyme catalysis of liver cytochrome P 450 system, small part is by CYP2C19 catalysis.The dose-effect relationship of warfarin is by h and E factor (such as medicine, diet, various morbid state) impact, and these factors have an impact to its absorption, pharmacokinetics and pharmacodynamics.
Impact by inherited genetic factors: the dose response variability of warfarin medication is large.
Warfarin is different at the dosage of different race, age, or sex crowd.Vitamin K epoxide reductase mainly suppresses by S type warfarin, and the metabolism of S type warfarin is primarily of Cytochrome P450 (CYP2C9) catalysis.Large quantity research proves that Cytochrome P450 (CYP2C9) and vitamin K epoxide reductase complex body subunit 1 (VKORC1) are two and important determine the metabolism of warfarin and the gene of anticoagulant effect.Mainly contain the polymorphism in three sites in two genes, gene pleiomorphism affects initial dose and the maintenance dose of warfarin.Warfarin maintenance dose there are differences in different crowd, and Aisa people's dosage is lower.The gene pleiomorphism of CYP2C9*2 and CYP2C9*3 causes weakening the metabolism of S type warfarin, and then causes and decline to the removing of warfarin, Increased Plasma Half-life.The warfarin dose that above two kinds of sudden changes are lower compared with CYP2C9*1*1 needs of patients.CYP2C9*3 and VKORC1-1639A is dangerous allelotrope.Carry the warfarin dose that this allelic needs of patients is lower, simultaneously in the face of higher bleeding risk.Therefore gene pleiomorphism have impact on metabolite clearance and the maintenance dose of warfarin.Research display VKORC1-1639G>A and CYP2C9*2 and CYP2C9*3 genetic background can explain the variation of the warfarin dosage of about 50% in the past.The gene pleiomorphism of U.S. FDAs in 2007 to gene pleiomorphism increase information indicating: CYP2C9 and VKORC1 of warfarin have impact on drug dose during removing and the stable state of medicine, advises warfarin initial application comparatively low dosage for the patient that there is gene pleiomorphism.Current CYP2C9 and VKORC1 gene test commercialization, but the commercially produced product that can only use west detects in domestic medical institutions to warfarin sensitive gene.
China Medical Sciences Academy Fu Wai Hospital has abundant clinical data and clinical resources accumulation in the research of warfarin dosage detection technique, and this research simultaneously is also one of " 12 " great new drug initiative problem.Achievement in research is converted into the detection kit for clinical application and corresponding medicine guide, can facilitates and determine clinical medicine dose accurately and instruct doctor's medication, will great value be had to the clinical therapeutic efficacy improving cardiovascular disorder.
In prior art, TaqMan probe method is a kind of quantitative PCR technique of high special, and the core of its know-why is 5 '-3 ' exonuclease activity utilizing Taq enzyme, cuts off probe, produces fluorescent signal.Because probe and template are specific bindings, so the power of fluorescent signal just represents the quantity of template.
In the quantitative PCR reaction system of TaqMan probe method, comprise one couple of PCR primers and a probe.Probe only with template specificity combine, its binding site is between two primers.Probe 5 ' end is marked with reporter group (Reporter, R), as FAM, VIC etc., 3 ' end be marked with fluorescent quenching group (Quencher, Q), as BHQ etc.When probe is complete time, the fluorescent energy that reporter group is launched is quenched group absorptions, and instrument can't detect signal.Along with the carrying out of PCR, Taq enzyme runs into the probe be combined with template in chain extension process, and probe will cut off by its 5 '-3 ' 5 prime excision enzyme activity, and reporter group is away from quenching group, and its energy can not be absorbed, and namely produces fluorescent signal.So often through a PCR circulation, fluorescent signal is also the same with object fragment, has the process that a sync index increases.The intensity of signal just represents the copy number of the DNA of template.
The quenching group of MGB probe adopts non-fluorescence quenching group (NFQ), and itself does not produce fluorescence, greatly can reduce the intensity of background signal.Probe is also connected with MGB (Minor Groove Binder) modification group simultaneously, can by the Tm value raising about 10 DEG C of probe, because in order to obtain same Tm value, MGB probe can design shorter than common TaqMan probe, both reduce synthesis cost, and also make the success ratio of probe design greatly improve.Because when the DNA based composition of template is undesirable, short probe designs than the easier of length.
Disclosed in prior art, various fluorophor all can apply to the present invention, is exemplified below:
The peak value of the 1st group: fluorophor FAM its wavelength of fluorescence is at 520nm.
2nd group: fluorophor VIC, the peak value of its wavelength of fluorescence of JOE, HEX is at 550nm.
3rd group: fluorophor CY3, the peak value of its wavelength of fluorescence of NED, TAMRA is at 580nm.
The peak value of the 4th group: fluorophor ROX, TEXAS RED its wavelength of fluorescence is at 610nm.
The peak value of the 5th group: fluorophor CY5 its wavelength of fluorescence is at 670nm.
Fluorophor in preferred saltant type probe and wild-type probe is selected from FAM, VIC respectively.
Disclosed in prior art, various quenching group all can apply to the present invention.According to the detection probes of CYP2C9*3 gene pleiomorphism of the present invention, preferred quenching group is selected from MGB, BHQ2.
According to the detection probes of CYP2C9*3 gene pleiomorphism of the present invention, preferred probe is selected from following group:
CYP2C9*3 saltant type probe FAM-ccagagataccttgaccttctcc-MGB
CYP2C9*3 wild-type probe VIC-ccagagatacattgaccttctcc-MGB
CYP2C9*3 saltant type probe FAM-ccagagataccttgaccttctcc-BHQ2
CYP2C9*3 wild-type probe VIC-ccagagatacattgaccttctcc-BHQ2
CYP2C9*3 saltant type probe VIC-ccagagataccttgaccttctcc-MGB
CYP2C9*3 wild-type probe FAM-ccagagatacattgaccttctcc-MGB
CYP2C9*3 saltant type probe VIC-ccagagataccttgaccttctcc-BHQ2
CYP2C9*3 wild-type probe FAM-ccagagatacattgaccttctcc-BHQ2
CYP2C9*3 saltant type probe MGB-ccagagataccttgaccttctcc-FAM
CYP2C9*3 wild-type probe MGB-ccagagatacattgaccttctcc-VIC
CYP2C9*3 saltant type probe BHQ2-ccagagataccttgaccttctcc-FAM
CYP2C9*3 wild-type probe BHQ2-ccagagatacattgaccttctcc-VIC
CYP2C9*3 saltant type probe MGB-ccagagataccttgaccttctcc-VIC
CYP2C9*3 wild-type probe MGB-ccagagatacattgaccttctcc-FAM
CYP2C9*3 saltant type probe BHQ2-ccagagataccttgaccttctcc-VIC
CYP2C9*3 wild-type probe BHQ2-ccagagatacattgaccttctcc-FAM
Second aspect present invention provides a kind of reagent detecting CYP2C9*3 gene pleiomorphism, and described pack is containing above-mentioned every detection probes.
Third aspect present invention provides a kind of test kit detecting CYP2C9*3 gene pleiomorphism, it is characterized in that, described test kit comprises above-mentioned every detection probes.
In order to increase the melting temp (Tm value) of probe, probe of the present invention also can use lock nucleic acid, and capitalization can be used within the probe to be represent lock nucleic acid base.
The fourth aspect of technical scheme of the present invention also provides a kind of primer set for amplification pair, and described primer pair comprises forward primer and reverse primer;
The oligonucleotide that forward primer is made up of the base sequence of sequence 3;
The oligonucleotide be oppositely made up of to primer the base sequence of sequence 4.
Sequence 3E2-S-1:GTGGTGCACGAGGT
Sequence 4E2-A-1:TGTCACAGGTCACTGC
Described primer set for amplification is to for by gene amplification method amplification CYP2C9*3 gene.
Fifth aspect present invention additionally provides a kind of gene amplification reagent, and for the CYP2C9*3 gene that increases, described gene amplification reagent contains primer set for amplification pair of the present invention.
Sixth aspect present invention additionally provides a kind of gene amplification reagent box, it is characterized in that, comprise and template can be made to carry out the primer increased, described primer pair is selected from the primer set for amplification pair described in above-mentioned any one.
Seventh aspect present invention additionally provides a kind of method preparing amplified production, it is characterized in that, described method comprises the steps:
(I) with the nucleic acid in sample for template, use the primer set for amplification pair described in any one of preceding claim, in reaction solution, carry out the amplification step of CYP2C9*3 gene.
The method of amplified production provided by the invention, is characterized in that, in described step (I), adds the probe of hybridizing with the detected object site of CYP2C9*3 gene in described reaction solution further.
The method of amplified production provided by the invention, is characterized in that, described probe can be the probe of above-mentioned any one.
The method of amplified production provided by the invention, is characterized in that, the method also comprises following step (II) further
(II) step of the fluorescently-labeled fluorescence intensity in the described fluorescence labeling probe of described reaction solution is measured.
Eighth aspect present invention additionally provides a kind of reagent, it is characterized in that, described reagent contains the probe of above-mentioned any one and the primer set for amplification pair described in above-mentioned any one.
Ninth aspect present invention additionally provides a kind of test kit, it is characterized in that, described test kit contains the probe of above-mentioned any one and the primer set for amplification pair described in above-mentioned any one.
Preferred test kit of the present invention, containing, for example lower material:
Tris-HCl (pH 7.5-9.0) 10-100mmol/L
KCL 6-500mmol/L
MgCl
21.5-5.0mmol/L
dNTP 0.2mmol/L
Be selected from the primer 2 00-900nmol/L of the above-mentioned any one of the present invention
Be selected from the probe 100-900nmol/L of the above-mentioned any one of the present invention
Hot resistant DNA polymerase (Taq enzyme) 2.0-2.5U
The final concentration 200-700nM of preferred primer, the final concentration 200-500nM of preferred primer, the final concentration 300nM of most preferred primer.
The final concentration 200-700nM of preferred probe; The final concentration 300-500nM of preferred probe; The final concentration 400nM of most preferred probe.
Test kit of the present invention, preferably also containing genotype contrast outward with or internal reference.
Preferred genotype contrasts outward and is selected from mutant-type genotype contrast, wild type genotype contrast and heterozygous genotype contrast.
Tenth aspect present invention provides a kind of method detecting CYP2C9*3 gene pleiomorphism, it is characterized in that, uses the above-mentioned any one test kit of the present invention to detect CYP2C9*3 gene pleiomorphism.
The detection method of CYP2C9*3 gene pleiomorphism of the present invention, also comprises the steps:
(1) biological sample is carried out DNA extraction;
(2) DNA of extraction is added the present invention above-mentioned any one of test kit in reagent carry out pcr amplification;
(3) CYP2C9*3 gene pleiomorphism is determined by the variation of fluorescent signal.
The detection method of CYP2C9*3 gene pleiomorphism of the present invention, is characterized in that, preferred biological sample is whole blood.
The extraction of biological specimen DNA can use the technology of this area routine to carry out, such as, with reference to the method for RelaxGene poba gene group DNA extraction kit
1.EDTA anti-freezing venous blood 2mL-4mL, adds 5mL lysate CL cracking hemocyte, is transferred to by solution in totally aseptic 15mL centrifuge tube, puts upside down mixing; Centrifugal 5 minutes of 2,000 × g room temperatures, discard supernatant, retain precipitation;
2. add 5ml lysate CL in centrifuge tube, concuss until precipitation dissolve completely, centrifugal 5 minutes of 2,000 × g room temperatures;
3. abandon supernatant, add Proteinase K digestion liquid 2.5ml, vortex mixes; 56 DEG C of water-baths 10 minutes.
4. add 2.5ml Virahol, shake centrifuge tube gently, thread genomic dna is chosen.
5. in 70% ethanol, wash DNA, dry;
6. add 400ul damping fluid TB, dissolving DNA.
The present invention the 11 aspect provides a kind of method judging the usage quantity of warfarin, and it is characterized in that, the method comprises:
(1) step of CYP2C9*3 gene pleiomorphism is detected by the pleiomorphism detecting method described in any one provided by the invention;
(2) step of the dosage of warfarin is judged by the presence or absence of detected polymorphism.
Advantageous Effects:
Even the sample containing impurity.Such as whole blood or oral mucosa, can be rapider and easy carry out amplified reaction.In addition, if use primer pair of the present invention, amplified reaction can be carried out by amplification efficiency more excellent than ever, can also amplified reaction be shortened.Therefore according to primer of the present invention and containing its reagent, and their manufacture method of amplified production is used, analysis CYP2C9*3 gene polynorphisms that can be rapid and easy.
Primer set for amplification pair of the present invention, preferably uses when increasing the CYP2C9*3 gene in the biological samples such as whole blood sample.
By adding probe of the present invention in gene amplification system, after PCR reaction terminates, just allelic somatotype can be carried out only by the analysis of fluorescence curve.
And then owing to directly can check whole blood, oral mucosa suspension, therefore, it is possible to reduce the time and cost that spend.
Probe specificity of the present invention is strong, and detection sensitivity is high.
Square law device of the present invention is simple, easily is automated.
Using method of the present invention, when carrying out PCR, without the need to taking out amplified production, therefore there is not the danger of pollution.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of probe that effectively can detect CYP2C9*3 gene pleiomorphism, and provides and to detect CYP2C9*3 gene pleiomorphism, and for the test kit of the method.
For solving the problems of the technologies described above, the present invention adopts following technical scheme:
First aspect present invention provides the detection probes of CYP2C9*3 gene pleiomorphism, it is characterized in that, the nucleotide sequence of shown detection probes is selected from sequence 1 and sequence 2, uses the principle of TaqMan probe technology, has fluorophor and quenching group respectively at 5 ' and 3 ' end.In order to measure the fluorescent signal of saltant type probe and wild-type probe in PCR instrument simultaneously, the fluorophor that saltant type probe is connected with wild-type probe the peaks demand of the wavelength that fluoresces in different positions;
Sequence 1CYP2C9*3 saltant type probe 5 '-ccagagataccttgaccttctcc-3 '
Sequence 2CYP2C9*3 wild-type probe 5 '-ccagagatacattgaccttctcc-3 '.
As fluorescence dye, conventional dyestuff can be used.Fluorescence dye can use conventional method to the method that oligonucleotide combines.
Fluorochrome label can be used in probe two ends of the present invention.Preferably 5 ' end mark fluorescent group of probe, 3 ' end mark fluorescent quenching group.
Accompanying drawing explanation
Accompanying drawing is the amplification curve diagram of this site after quantitative fluorescent PCR.Two lines of different colours in figure, represent 2 kinds of allelotrope (A/C) respectively, and straight line representative does not have the allelotrope increased, the allelotrope of the curve representative amplification of rising.What light line detected is VIC fluorescence, and light line amplification represents wild-type (AA), and legend 2C9*3-A represents.What dark line detected is FAM fluorescence, and dark line amplification represents saltant type (CC), and legend 2C9*3-C represents.Article 2, the curve risen represents 2 kinds of allelotrope and all increases, and is heterozygous.
Fig. 1, CYP2C9*3 gene is the pcr amplification fluorescence curve of heterozygous.
Fig. 2, CYP2C9*3 gene is the pcr amplification fluorescence curve of saltant type.
Fig. 3, CYP2C9*3 gene is the pcr amplification fluorescence curve of wild-type.
Fig. 4, the pcr amplification fluorescence curve of sample 1, Fig. 4 with Fig. 3 is substantially identical, and experimenter's first is wild-type.
Fig. 5, the pcr amplification fluorescence curve of sample 2, Fig. 5 with Fig. 1 is substantially identical, and experimenter's second is heterozygous.
Fig. 6, the pcr amplification fluorescence curve of sample 3, Fig. 6 with Fig. 2 is substantially identical, and experimenter third is saltant type.
Fig. 7, the pcr amplification fluorescence curve of sample 4, Fig. 7 with Fig. 3 is substantially identical, and experimenter's first is wild-type.
Fig. 8, the pcr amplification fluorescence curve of sample 5, Fig. 8 with Fig. 1 is substantially identical, and experimenter's second is heterozygous.
Fig. 9, the pcr amplification fluorescence curve of sample 6, Fig. 9 with Fig. 2 is substantially identical, and experimenter third is saltant type.
Embodiment
Below in conjunction with embodiments of the invention, the present invention will be described, but the present invention is not by any restriction of following embodiment.
Embodiment 1
One, CYP2C9*3 gene probe and primer pair is prepared
Conventional technology is used to prepare CYP2C9*3 gene probe and CYP2C9*3 gene primer pair.
Concrete sequence is as follows:
CYP2C9*3 gene probe
* 3 saltant type probe FAM-ccagagataccttgaccttctcc-MGB
* 3 wild-type probe VIC-ccagagatacattgaccttctcc-MGB
CYP2C9*3 gene primer pair
Forward primer sequence E2-S-1:GTGGTGCACGAGGT
Reverse primer sequences E2-A-1:TGTCACAGGTCACTGC
Two, PCR reaction solution is configured,
Every part of consumption is as follows:
Commercially available 2 × PCR reaction solution (comprising taq enzyme, UNG enzyme, dNTP, dUTP) 10 μ L
Primer and probe mixture 1 μ L
Internal reference probe mixture 1 μ L
RNase-free water 7 μ L
Packing, divides by every pipe 19 μ l and is filled in 0.1ml PCR reaction tubes/plate, proceed to sample preparation district.
Three, template is added
Prepare the DNA sample that CYP2C9*3 shows as heterozygous, 1 μ l heterozygous DNA sample, negative controls 1 μ l are added into the above-mentioned composition of 19 μ l respectively and survey in PCR reaction solution, reaction system 20 μ l.Of short durationly centrifugally all reagent is focused on bottom reaction tubes (can not bubble be had), after determining to build pipe lid or sealer, carry out pcr amplification reaction immediately.
Four, pcr amplification
PCR instrument device program is run by following scheme:
(1) UNG reaction: 50 DEG C of 10min;
(2) denaturation: 95 DEG C of 60sec;
(3) PCR:(95 DEG C of 3sec → 60 DEG C 45sec) 45 circulations.
Measure the change increasing fluorescence intensity in time, the sense channel of 7500/7500fast PCR instrument is as follows respectively:
1st passage: 520nm detects saltant type.
2nd passage: 550nm detects wild-type.
Five, interpretation of result
The result that CYP2C9*3 shows as heterozygous indicates in FIG.These figure are the mutation analysis figure of the prolongation fluorescence intensity represented with the time, namely along with the mutation analysis figure of the increase fluorescence intensity of amplification cycles number of times.As shown in Figure 1, this fluorescence curve represents that in sample, CYP2C9*3 gene is heterozygous.
Embodiment 2
Step one is to four:
The method identical with embodiment 1 and step, replace with the DNA sample that CYP2C9*3 shows as saltant type by DNA sample, the DNA sample of 1 μ l saltant type be added in 19 μ lPCR reaction solutions.
Five, interpretation of result
The result that CYP2C9*3 shows as saltant type indicates in fig. 2.These figure are the mutation analysis figure of the prolongation fluorescence intensity represented with the time, namely along with the mutation analysis figure of the increase fluorescence intensity of amplification cycles number of times.As shown in Figure 2, this fluorescence curve represents that in sample, CYP2C9*3 gene is saltant type.
Embodiment 3
Step one is to four:
The method identical with embodiment 1 and step, replace with the DNA sample that CYP2C9*3 shows as wild-type by DNA sample, the DNA sample of 1 μ l wild-type be added in 19 μ lPCR reaction solutions.
Five, interpretation of result
The result that CYP2C9*3 shows as wild-type indicates in figure 3.These figure are the mutation analysis figure of the prolongation fluorescence intensity represented with the time, namely along with the mutation analysis figure of the increase fluorescence intensity of amplification cycles number of times.As shown in Figure 3, this fluorescence curve represents that in sample, CYP2C9*3 gene is wild-type.
Embodiment 4
One,
Carry out with it adopting venous blood 5mL from 3 experimenter's first, second and third, EDTA anti-freezing (obtaining sample 1,2 and 3).
Use the method for RelaxGene poba gene group DNA extraction kit
1., by EDTA anti-freezing venous blood 3mL, add 5mL lysate CL cracking hemocyte, solution is transferred in totally aseptic 15mL centrifuge tube, puts upside down mixing; Centrifugal 5 minutes of 2,000 × g room temperatures, discard supernatant, retain precipitation;
2. add 5ml lysate CL in centrifuge tube, concuss until precipitation dissolve completely, centrifugal 5 minutes of 2,000 × g room temperatures;
3. abandon supernatant, add Proteinase K digestion liquid 2.5ml, vortex mixes; 56 DEG C of water-baths 10 minutes.
4. add 2.5ml Virahol, shake centrifuge tube gently, thread genomic dna is chosen.
5. in 70% ethanol, wash DNA, dry;
6. add 400ul damping fluid TB, dissolving DNA obtains DNA sample.
Step 2 to four:
By three of gained parts of known type DNA samples, every part of 1 μ lDNA sample is added in the PCR reaction solution of the above-mentioned composition of 19 μ l, carrying out PCR and mensuration and measuring the change increasing fluorescence intensity in time similarly to Example 1.
The result of sample 1,2 and 3 at Fig. 4,5, indicate respectively in 6.These figure are the mutation analysis figure of the prolongation fluorescence intensity represented with the time, namely along with the mutation analysis of the increase fluorescence intensity of amplification cycles number of times is as Fig. 4, and 5, shown in 6.Fig. 4 with Fig. 3 is substantially identical, Fig. 5 with Fig. 1 is substantially identical, Fig. 6 with Fig. 2 is substantially identical.
Can determine CYP2C9*2 gene polynorphisms in sample according to fluorescence curve, analytical results is that experimenter's first is wild-type, experimenter's second is heterozygous, experimenter third is saltant type.Result is consistent with known results.
Embodiment 5
Gather Stomatocyte with swab stick from 3 experimenter's first, second and third, extract DNA, (sample 4,5 and 6) with methods such as boiling method or application mouth epithelial cells DNA extraction kit.1 μ lDNA sample being added into the following composition of 19 μ l surveys in PCR reaction solution, and identical with embodiment 4 carries out PCR and measure the change increasing fluorescence intensity in time.
The result of sample 4,5 and 6 at Fig. 7,8, indicate respectively in 9.As Fig. 7,8, shown in 9, can determine CYP2C9*3 gene polynorphisms in sample according to fluorescence curve, analytical results is that experimenter's first is wild-type, experimenter's second is heterozygous, experimenter third is saltant type.
Claims (24)
1. the detection probes of a CYP2C9*3 gene pleiomorphism, it is characterized in that, shown in the nucleotide sequence of detection probes be selected from sequence 1 and sequence 2, and 5 ' and 3 ' holds and has fluorophor and quenching group respectively, and fluorophor fluorescigenic wavelength peak in different positions;
Sequence 1CYP2C9*3 saltant type probe 5 '-ccagagataccttgaccttctcc-3 '
Sequence 2CYP2C9*3 wild-type probe 5 '-ccagagatacattgaccttctcc-3 '.
2. the detection probes of CYP2C9*3 gene pleiomorphism according to claim 1, is characterized in that, described quenching group is selected from MGB, BHQ2.
Fluorophor in described saltant type probe and wild-type probe is selected from different groups;
1st group: FAM,
2nd group: VIC, JOE, HEX
3rd group: CY3, NED, TAMRA
4th group: ROX, TEXAS RED
5th group: CY5.
3. the detection probes of CYP2C9*3 gene pleiomorphism according to claim 2, is characterized in that, the fluorophor in described saltant type probe and wild-type probe is selected from FAM, VIC respectively.
4. the detection probes of CYP2C9*3 gene pleiomorphism according to claim 2, is characterized in that, described quenching group is selected from MGB, BHQ2.
5. the detection probes of CYP2C9*3 gene pleiomorphism as claimed in one of claims 1-4, is characterized in that, described probe is selected from following group:
CYP2C9*3 saltant type probe FAM-ccagagataccttgaccttctcc-MGB
CYP2C9*3 wild-type probe VIC-ccagagatacattgaccttctcc-MGB
CYP2C9*3 saltant type probe VIC-ccagagataccttgaccttctcc-MGB
CYP2C9*3 wild-type probe FAM-ccagagatacattgaccttctcc-MGB
CYP2C9*3 saltant type probe FAM-ccagagataccttgaccttctcc-BHQ2
CYP2C9*3 wild-type probe VIC-ccagagatacattgaccttctcc-BHQ2
CYP2C9*3 saltant type probe VIC-ccagagataccttgaccttctcc-BHQ2
CYP2C9*3 wild-type probe FAM-ccagagatacattgaccttctcc-BHQ2
CYP2C9*3 saltant type probe MGB-ccagagataccttgaccttctcc-FAM
CYP2C9*3 wild-type probe MGB-ccagagatacattgaccttctcc-VIC
CYP2C9*3 saltant type probe MGB-ccagagataccttgaccttctcc-VIC
CYP2C9*3 wild-type probe MGB-ccagagatacattgaccttctcc-FAM
CYP2C9*3 saltant type probe BHQ2-ccagagataccttgaccttctcc-FAM
CYP2C9*3 wild-type probe BHQ2-ccagagatacattgaccttctcc-VIC
CYP2C9*3 saltant type probe BHQ2-ccagagataccttgaccttctcc-VIC
CYP2C9*3 wild-type probe BHQ2-ccagagatacattgaccttctcc-FAM.
6. detect a reagent for CYP2C9*3 gene pleiomorphism, it is characterized in that, described pack is containing the detection probes any one of claim 1-5.
7. detect a test kit for CYP2C9*3 gene pleiomorphism, it is characterized in that, described test kit comprises the detection probes any one of claim 1-5.
8. a primer set for amplification pair, described primer pair comprises forward primer and reverse primer;
The oligonucleotide that forward primer is made up of the base sequence of sequence 3;
The oligonucleotide be oppositely made up of to primer the base sequence of sequence 4.
Sequence 3CYP2C9*3 forward primer E2-S-1:GTGGTGCACGAGGT
Sequence 4CYP2C9*3 reverse primer E2-A-1:TGTCACAGGTCACTGC.
9. primer set for amplification pair according to claim 8, is characterized in that, described primer set for amplification is to the primer pair being CYP2C9*3 gene in amplification biological sample.
10. a gene amplification reagent, is characterized in that, described gene amplification reagent contains the primer set for amplification pair according to any one of claim 8-9.
11. 1 kinds of gene amplification reagent boxes, is characterized in that, comprise and template can be made to carry out the primer increased, described primer pair is selected from the primer set for amplification pair according to any one of claim 8-9.
12. 1 kinds of methods preparing amplified production, it is characterized in that, described method comprises the steps:
(I) with the nucleic acid in sample for template, use primer set for amplification pair according to any one of claim 8-9, in reaction solution, carry out the amplification step of CYP2C9*3 gene.
13., according to the method for the amplified production described in claim 12, is characterized in that, in described step (I), add the probe of hybridizing with the detected object site of CYP2C9*3 gene in described reaction solution further.
14. according to the method for the amplified production described in claim 12, and it is characterized in that, described probe is the probe be selected from any one of claim 1-5.
15. according to the method for the amplified production described in claim 14, and it is characterized in that, the method also comprises following step (II) further
(II) step of the fluorescently-labeled fluorescence intensity in the described fluorescence labeling probe of described reaction solution is measured.
16. 1 kinds of reagent, is characterized in that, described reagent contains and is selected from the probe any one of claim 1-5 and the primer set for amplification pair according to any one of claim 8-9.
17. 1 kinds of test kits, is characterized in that, described test kit contains and is selected from the probe any one of claim 1-5 and the primer set for amplification pair according to any one of claim 8-9.
18. test kits according to claim 17, is characterized in that, described test kit contains
Tris-HCl(pH 7.5-9.0) 10-100mmol/L
KCL 6-500mmol/L
MgCl
21.5-5.0mmol/L
dNTP 0.2mmol/L
Be selected from the primer 2 00-900nmol/L any one of claim 8-9
Be selected from the probe 100-900nmol/L any one of claim 1-5
Hot resistant DNA polymerase (Taq enzyme) 2.0-2.5U.
19. test kits according to claim 18, is characterized in that, described test kit contains
Tris-HCl(pH 7.5-9.0) 10-100mmol/L
KCL 6-500mmol/L
MgCl
21.5-5.0mmol/L
dNTP 0.2mmol/L
Be selected from the primer 2 00-900nmol/L any one of claim 8-9
Be selected from the probe 100-900nmol/L any one of claim 1-5
Hot resistant DNA polymerase (Taq enzyme) 2.0-2.5U
Genotype contrasts outward
Internal reference.
20. test kits according to claim 19, is characterized in that, described genotype contrasts outward and is selected from mutant-type genotype contrast, wild type genotype contrast and heterozygous genotype contrast.
21. 1 kinds of methods detecting CYP2C9*3 gene pleiomorphism, is characterized in that, use the test kit any one of claim 17-20 to detect CYP2C9*3 gene pleiomorphism.
The detection method of 22. CYP2C9*3 gene pleiomorphisms according to claim 21, it is characterized in that, the method comprises the steps:
(1) biological sample is carried out DNA extraction;
(2) reagent added by the DNA of extraction in the test kit any one of claim 17-20 carries out pcr amplification;
(3) CYP2C9*3 gene pleiomorphism is determined by the intensity of fluorescent signal.
The detection method of 23. CYP2C9*3 gene pleiomorphisms according to claim 22, it is characterized in that, described biological sample is whole blood.
24. 1 kinds of methods judging the usage quantity of warfarin, it is characterized in that, the method comprises:
(1) step of CYP2C9*3 gene pleiomorphism is detected by the pleiomorphism detecting method according to any one of claim 21-23;
(2) step of the dosage of warfarin is judged by detected gene pleiomorphism.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105274228A (en) * | 2015-10-27 | 2016-01-27 | 上海芯超生物科技有限公司 | Kit and method for warfarin medicine gene mutation site detection |
| CN105671153A (en) * | 2016-02-06 | 2016-06-15 | 厦门大学附属中山医院 | CYP2C9*3 detection parting kit based on probe AllGlo and parting method of CYP2C9*3 detection parting kit |
| CN107227371A (en) * | 2017-07-25 | 2017-10-03 | 重庆京因生物科技有限责任公司 | Primer, molecular beacon, kit and its detection method of CYP2C9*3 gene pleiomorphism quick detections |
| CN107653307A (en) * | 2016-07-26 | 2018-02-02 | 上海同科生物科技有限公司 | A kind of kit for quick detection gene associated with individualized medication of warfarin CYP2C9 and VKORC1 polymorphism |
| CN110819709A (en) * | 2019-12-16 | 2020-02-21 | 北京和合医学诊断技术股份有限公司 | Method for detecting CYP2C9 and VKORC1 gene polymorphism by fluorescent quantitative PCR (polymerase chain reaction) |
| CN110938688A (en) * | 2019-12-30 | 2020-03-31 | 武汉光谷联合医学检验所股份有限公司 | CYP2C9 gene polymorphism detection kit and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101760528A (en) * | 2008-12-26 | 2010-06-30 | 上海基康生物技术有限公司 | Medicine metabolic relevant loci detection method |
| CN103710432A (en) * | 2013-10-31 | 2014-04-09 | 上海百傲科技股份有限公司 | Specific primer pair and specific probe for CYP2C9 (cytochrome P450 2C9) and (vitamin K epoxide reductase complex subunit 1) VKORC1 gene chip detection |
-
2014
- 2014-12-31 CN CN201410852160.9A patent/CN104561301A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101760528A (en) * | 2008-12-26 | 2010-06-30 | 上海基康生物技术有限公司 | Medicine metabolic relevant loci detection method |
| CN103710432A (en) * | 2013-10-31 | 2014-04-09 | 上海百傲科技股份有限公司 | Specific primer pair and specific probe for CYP2C9 (cytochrome P450 2C9) and (vitamin K epoxide reductase complex subunit 1) VKORC1 gene chip detection |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105274228A (en) * | 2015-10-27 | 2016-01-27 | 上海芯超生物科技有限公司 | Kit and method for warfarin medicine gene mutation site detection |
| CN105671153A (en) * | 2016-02-06 | 2016-06-15 | 厦门大学附属中山医院 | CYP2C9*3 detection parting kit based on probe AllGlo and parting method of CYP2C9*3 detection parting kit |
| CN107653307A (en) * | 2016-07-26 | 2018-02-02 | 上海同科生物科技有限公司 | A kind of kit for quick detection gene associated with individualized medication of warfarin CYP2C9 and VKORC1 polymorphism |
| CN107227371A (en) * | 2017-07-25 | 2017-10-03 | 重庆京因生物科技有限责任公司 | Primer, molecular beacon, kit and its detection method of CYP2C9*3 gene pleiomorphism quick detections |
| CN110819709A (en) * | 2019-12-16 | 2020-02-21 | 北京和合医学诊断技术股份有限公司 | Method for detecting CYP2C9 and VKORC1 gene polymorphism by fluorescent quantitative PCR (polymerase chain reaction) |
| CN110938688A (en) * | 2019-12-30 | 2020-03-31 | 武汉光谷联合医学检验所股份有限公司 | CYP2C9 gene polymorphism detection kit and application thereof |
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