CN106350595A - Primer, kit and method for detecting human CYP2C9 genetic typing - Google Patents
Primer, kit and method for detecting human CYP2C9 genetic typing Download PDFInfo
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Abstract
The invention belongs to the technical field of genetic typing, and particularly relates to a primer, a probe, a kit and a method for detecting human CYP2C9 genetic typing. The primer and the probe comprise the following nucleotide sequences: an upstream primer SEQ ID NO.1: 5'-CAGCTAAAGTCCAGGAAGAGAT-3', a downstream primer SEQ ID NO.2: 5'-TTCTGAATTTAATGTCACAGGT-3', and a probe SEQ ID NO.3: 5'-CAGAGATACATTGACCTTCTCCCCACC-3'. The kit comprises the primer and the probe, dNTPs (Deoxyribonucleoside Triphosphate), MgCl2, mixed enzyme prepared from UNG (Uracil-N-Glycosylase) and DNA (Deoxyribose Nucleic Acid) polymerase having the activity of flap endonuclease, and positive and negative reference substances. The method for detecting the human CYP2C9 genetic typing by using the kit is capable of detecting different types on a target nucleic acid SNP (Single Nucleotide Polymorphism) site, so that the operation process is simplified, the cost is reduced, the resolution ratio of a detection result is high, and quickness and accuracy are realized.
Description
Technical field
The invention belongs to genotyping technique field and in particular to a kind of primer of detection mankind's cyp2c9 gene type and
Probe, test kit, method.
Background technology
In cytochrome p450 superfamily, the metabolic enzyme related to drug metabolism is mainly cyp1, cyp2, cyp3 family.
Cyp2c9 is the important member of cyp2c subfamily, and the medicine of metabolism clinical about 10%, including warfarin, phenytoin, toluene sulphur fourth
Urea, fluoxetine etc..This enzyme has genetic polymorphism, there is individuality, racial diversity, leads to Different Individual, the medicine generation of race
Ability of thanking exists different.The modal allele of Chinese han population is cyp2c9*3, occurs 1075 nucleotide a's > c
Mutation, 359 of aminoacid sequence are changed into leu from ile, lead to a β-pleated sheet of substrate identification to be destroyed, and then reduce enzyme
Catalytic capability, its enzyme activity is only equivalent to the 4～6% of wild type.The different genotype of cyp2c9 directly affect curative effect of medication and
The power of toxic and side effects, the therefore genotype according to cyp2c9 come instruct accurately, safe medication there is important clinical meaning.
Method for detecting gene pleiomorphism mainly has sequencing, pcr-rflp (polymerase chain reaction-restriction fragment
Length polymorphism), gene chips, fluorescent quantitation pcr method.Accurately, but high cost, time-consuming for sequencing result.pcr-rflp
Gene pleiomorphism, low cost, visual result, but operating process vulnerable to pollution are detected by the method for restriction enzyme digestion and electrophoresis, leads to mistake
Result.Gene chips detect genotype by nucleic acid hybridization, and advantage is that flux is high, and shortcoming is relatively costly, complex operation, knot
The difficult interpretation of fruit.Conventional fluorescent quantitation pcr method can be divided into fluorescence pcr sonde method and fluorescence pcr melting curve according to its principle difference
Method.The former is directed to target snp site and designs two kinds of probes, mates with saltant type with wild type respectively, using the ct value difference of amplification
Different come interpretation genotype.The latter is directed to target snp site and designs a kind of probe, the standard based on probe and the intermolecular hybrid of target nucleic acid
Really property (i.e. on snp site, different bases cause the difference of melting temperature) carrys out interpretation genotype.Traditional fluorescent quantitation pcr method inspection
The accuracy surveyed depends on the difference of the affine degree between probe and corresponding allele, the i.e. difference of its solution temperature (tm)
Not, when this difference is not very big, often lead to final testing result and there is deviation, accuracy is not high.With technology
Constantly bring forth new ideas, a kind of be developed based on the new snp detection technique of flap Cobra venom endonuclease and double fluorescence labeling probe.
The method is using the dna polymerase with flap endonuclease activity, and is being excised in valvular structure using this polymerase
5 ' ends single-stranded when cutting position be located between first base of double stranded section of valvular structure and second base this
Characteristic, in conjunction with the dna probe being modified with double fluorescent labelling and quenching group it is achieved that detecting target core with a fluorescent probe
Typing on sour snp site.
Detection method currently for cyp2c9 gene type limits to said gene chip method, conventional fluorescent quantitation pcr spy
The skill of handling needles, its shortcoming is increasingly difficult to meet the requirement of Clinical Laboratory.In the new snp detection technique using double fluorescence labeling probe
The research of detection cyp2c9 gene type, also nobody's report.
Content of the invention
Present invention aim to overcome that the deficiencies in the prior art, by autonomous Design primer and probe, and combine based on lobe
Shape Cobra venom endonuclease and the new snp detection technique of double fluorescence labeling probe, provide a kind of detection mankind's cyp2c9 gene type
Primer and probe, test kit, method, with solve currently for cyp2c9 gene type detection confinement problems.
In order to realize foregoing invention purpose, the technical solution used in the present invention is as follows:
For detecting primer and the probe of mankind's cyp2c9 gene type, comprise following nucleotide sequence:
Forward primer seq id no:1:5 '-cagctaaagtccaggaagagat-3 '；
Downstream primer seq id no:2:5 '-ttctgaatttaatgtcacaggt-3 '；
Probe seq id no:3:5 '-cagagatacattgaccttctccccacc-3 '.
The primer that the present invention provides and probe can accurately detect cyp2c9 gene mutation site region.
The present invention also proposes a kind of test kit of detection mankind's cyp2c9 gene type, and described test kit comprises pcr reaction
Liquid, described pcr reactant liquor includes above-mentioned primer and probe.
The test kit low cost that the present invention provides, using simple, can accurately detect cyp2c9 gene type.
The present invention also provides a kind of method that utilization mentioned reagent box detects mankind's cyp2c9 gene type, and it includes as follows
Step:
Extract human genome dna；
Described dna is made into reaction system with described test kit, carries out fluorescent quantitation pcr amplification；
Described cyp2c9 gene type is analyzed according to described amplification.
The method of the detection cyp2c9 gene type that the present invention provides, can detect target nucleic acid with a fluorescent probe
Multiple typings on snp site, simplify operating process, have saved cost, testing result high resolution, accurate rapidly.
In addition, the present invention also provides the purposes using mentioned reagent box in detection cyp2c9 gene type.This purposes can
Abolish the limitation of existing cyp2c9 gene type detection, hinge structure, be more easy to meet the requirement of Clinical Laboratory.
Brief description
Fig. 1 is cyp2c9 gene 1075 site wild type testing result figure；
Fig. 2 is cyp2c9 gene 1075 site homozygous mutant testing result figure；
Fig. 3 is cyp2c9 gene 1075 site heterozygous testing result figure；
Fig. 4 is cyp2c9 gene 1075 site homozygous mutant contrast test testing result figure.
Specific embodiment
In order that the technical problem to be solved in the present invention, technical scheme and beneficial effect become more apparent, below in conjunction with
Drawings and Examples, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only used
To explain the present invention, it is not intended to limit the present invention.
The embodiment of the present invention provides a kind of primer for detecting mankind's cyp2c9 gene type and probe, comprises following core
Nucleotide sequence:
Forward primer seq id no:1:5 '-cagctaaagtccaggaagagat-3 '；
Downstream primer seq id no:2:5 '-ttctgaatttaatgtcacaggt-3 '；
Probe seq id no:3:5 '-cagagatacattgaccttctccccacc-3’.
The 10th nucleotide of this probe is complementary with corresponding sequence on target nucleic acid to the sequence between 3 ' ends, this cyp2c9 base
The sporting c.1075a of cause > c.The primer of the present embodiment and probe can accurately detect cyp2c9 gene mutation site region.
Specifically, hold respectively with two kinds of different fluorescent reporter group labellings it is preferable that fluorescence report at probe 5 ' end and 3 '
Accuse group to be selected from: fam, hex, tet, vic, rox, cy5, cy3, joe, alex, cal；The 11st nucleotide site in probe
Use fluorescent quenching group labelling.In the present embodiment, 5 ' ends of dna probe and 3 ' ends have one to send green fluorescence respectively
(fam) and red fluorescence (vic) group double labeling, and the next base in snp site (holds the 3 ' directions held along 5 '
Ground 11 bases) on be modified with a quenching group (zen for idt companytmInternal quencher), this quenching group energy
Enough green fluorescence group and the fluorescence of red fluorescence group are quenched simultaneously.By snp site to it on double fluorescent labelling dna probe
Sequence between 3 ' ends is complementary with corresponding sequence on target nucleic acid, and by the sequence snp site to its 5 ' end and target core
In acid, corresponding sequence is not complementary, therefore forms valvular structure.
The embodiment of the present invention also provides a kind of test kit of detection mankind's cyp2c9 gene type, and this test kit comprises by upper
State primer and the pcr reactant liquor of probe composition, wherein: forward primer concentration is: 0.1-0.5 μm；Downstream primer concentration is: 0.1-
0.5μm；Concentration and probe concentration is: 0.1-0.5 μm.Specifically in the present embodiment, 0.5 μm of forward primer；0.5 μm of downstream primer；Probe
0.5μm.This test kit low cost, using simple, can accurately detect cyp2c9 gene type.
Specifically, this test kit also contains dntps, mgcl2.Wherein dntps concentration is: 0.2-0.5mm；mgcl2Concentration
For: 0.5-2.5mm；In the present embodiment, preferably dntps 0.5mm；mgcl22.5mm.Specifically, this test kit also comprises ung
Enzyme and dna polymerase, this dna polymerase has flap endonuclease activity.
Because dna polymerase has flap endonuclease activity, when forward primer is extended at fluorescent probe, glimmering
On light probe, the sequence complementary with target nucleic acid can not excised by polymerase.Cutting position valvular structure double stranded section
Between one base and second base.When the base in snp site and the corresponding base complementrity on fluorescent probe on target nucleic acid
When, then excise between snp site and next base.Therefore, green fluorescence group is separated with quenching group, thus producing green
Color fluorescence.Continue complementary with target nucleic acid sequence on cutting fluorescent probe with polymerase, quenching group also with the other end
Red fluorescence group separates, thus producing red fluorescence.Therefore, in the luminous efficiency of green fluorescence group and red fluorescence group
In the case of being substantially the same, if the base in snp site on target nucleic acid and the corresponding base complementrity on fluorescent probe, record
The intensity of green fluorescence generally equal with the intensity of red fluorescence.When the base on the snp site of target nucleic acid and fluorescence
The base of the relevant position not mutual added time on probe, the position that polymerase is cut on fluorescent probe is being modified with quenching group
Base after.Therefore, after on fluorescent probe, the sequence complementary with target nucleic acid is not removed, green fluorescence group
Remain in this with quenching group to be removed in the sequence got off, the fluorescence of therefore green fluorescence group is still quenched group and quenches
Go out, thus not producing green fluorescence, but quenching group is separated with red fluorescence group, thus producing red fluorescence, now records
Green fluorescence intensity negligible compared with the intensity of red fluorescence.As the snp containing half simultaneously in sample of nucleic acid
The base in the target nucleic acid of the base in site and the corresponding base complementrity on fluorescent probe and second half snp site not with fluorescence
The target nucleic acid of the corresponding base complementrity on probe, in this case, the base in snp site and the corresponding alkali on fluorescent probe
The complementary target nucleic acid of base produces green fluorescence and red fluorescence simultaneously, and the base in snp site not with fluorescent probe on right
The target nucleic acid answering base complementrity only produces red fluorescence.Because the content of two kinds of target nucleic acids is equal, in green fluorescence group
In the case of being substantially the same with the luminous efficiency of red fluorescence group, the intensity of the described green fluorescence recording is about red glimmering
The half of the intensity of light.
The test kit of the present embodiment detects that the specificity of snp derives from the dna polymerization with flap endonuclease activity
Enzyme action removes the accuracy of cutting position during valvular structure, rather than the standard based on the hybridization between dna probe and target nucleic acid
Really property (i.e. on snp site, different bases cause the difference of melting temperature), therefore, this test kit detect snp accuracy and can
It is significantly higher than prior art by property.
The test kit reaction system of the embodiment of the present invention adopts ung-dutp decontamination system, and wherein, affiliated ung enzyme is permissible
The uracil glycosidic bond that selective hydrolysis rupture in the double-strand containing du or single-stranded dna, the dna chain having disappearance base of formation,
Under alkaline medium and high temperature, the further hydrolytic cleavage of meeting, therefore adds appropriate dutp in reaction system, in pcr amplification
Before, can effectively prevent from testing the pollution that indoor amplified production leads to using ung enzyme effect, thus including sample to whole detection system
The concentration of product, interference factor carry out quality control.And ung enzyme can be inactivated when 95 DEG C, do not affect follow-up pcr expanding effect.
More specifically, embodiment of the present invention test kit is provided with positive reference substance and negative controls, positive reference substance bag
Containing two kinds of plasmids, negative controls are te buffer.This two kinds of plasmids comprise the 1075a wild-type nucleic acid of cyp2c9 gene respectively
Sequence and 1075c mutant nucleic acid sequence.The ratio composition of two kinds of plasmid your concentration ratios 1:1 of massage.By positive, negative right
According to making the present embodiment technical method more accurately true.
The embodiment of the present invention also provides a kind of method of detection mankind's cyp2c9 gene type, and it comprises the steps:
Extract human genome dna；
Dna is made into reaction system with mentioned reagent box, carries out fluorescent quantitation pcr amplification；
Cyp2c9 gene type is analyzed according to amplification.
Specifically, this reaction system is 40ul.Wherein, dna mass: 10-500ng, preferably 100ng；Pcr reactant liquor
34.5ul, mixed enzyme 0.5ul.The response procedures of fluorescent quantitation pcr amplification are: 50 DEG C of 2min；95 DEG C of denaturations 2min；95 DEG C of changes
Property 15s；62 DEG C of extension 45s；35 circulations.
This method carries out fluorescence pcr reaction using flap Cobra venom endonuclease with reference to double fluorescent labelling dna probe and achieves
Detect the multiple typings on target nucleic acid snp site with a fluorescent probe, simplify operation, saved cost.Based on the party
Method, accurately detection cyp2c9 gene type.
Finally, according to embodiments of the invention, the present inventor can naturally expect mentioned reagent box in detection cyp2c9
Purposes in gene type.This purposes can abolish the limitation of existing cyp2c9 gene type detection, hinge structure, is more easy to
Meet the requirement of Clinical Laboratory.
Priority of the present invention carried out test of many times, now lift A partial experiment result as reference pair invention carry out detailed further
Thin description, is described in detail with reference to specific embodiment.
Embodiment 1 detects the test kit of mankind's cyp2c9 gene type
Primer and probe design
Inventor can detect primer and the spy of 1075 single nucleotide polymorphisms according to ncbi people's cyp2c9 gene order, design
Pin nucleotide sequence is as follows:
Forward primer seq id no:1:5 '-cagctaaagtccaggaagagat-3 '；
Downstream primer seq id no:2:5 '-ttctgaatttaatgtcacaggt-3 '；
Probe seq id no:3:5 '-cagagatacattgaccttctccccacc-3’.
Wherein, the 10th nucleotide of probe is complementary with corresponding sequence on target nucleic acid to the sequence between 3 ' ends, and probe is used
Double fluorescent reporter group labellings, its 5 ' end fam labelling, vic labelling is used at 3 ' ends, the 11st t labelling quenching group zen of probetm
internal quencher.
The preparation of cyp2c9 fluorescent quantitation pcr reactant liquor
The configuration of the present embodiment pcr reaction solution concentration is as follows: buffer, dntps (containing dutp) 0.5mm；mgcl22.5mm；
0.5 μm of forward primer；0.5 μm of downstream primer；0.5 μm of probe.
Mixed enzyme
In test kit, mixed enzyme used is mixed by uracil glycosylase enzyme (ung enzyme) and dna polymerase, wherein,
Dna polymerase is the dna polymerase with flap endonuclease activity, selected from the taq of new england biolabs company
Dna polymerase.The mixing concentration of enzymatic activity of this composition is 2-4u.
Reference substance
Design positive reference substance sequence according to ncbi data base's cyp2c9 gene 1075 single nucleotide polymorphisms, synthesize sequence
Row, build dna recombiant plasmid.Cyp2c9 positive reference substance includes 2 kinds of plasmids and is respectively 1075a wild type, 1075c saltant type.
Specifically mixed by molar concentration rate 1:1 by 1075a wild type, 1075c saltant type recombiant plasmid.And negative controls are
Te buffer.
According to above-mentioned pcr reactant liquor, mixed enzyme, reference substance organize cost implementation offer for detecting the mankind
The test kit of cyp2c9 gene type.
It should be understood that the content of each component is not limited to above-mentioned concentration in pcr reactant liquor, as long as meeting all available as follows
In follow-up test it may be assumed that dntps concentration 0.2-0.5mm；mgcl2Concentration 0.5-2.5mm；Forward primer concentration 0.1-0.5 μm；Under
0.1-0.5 μm of primer concentration of trip；Concentration and probe concentration 0.1-0.5 μm.
The fluorescent quantitation pcr detection of embodiment 2cyp2c9 gene type and analysis
(1) extraction of human blood sample genome dna
The present embodiment utilizes the poba gene group that Bao'an branch company of Shenzhen HYK Gene Technology Co., Ltd. produces to extract
Purification kit extracts sample dna, and concrete operations are as follows:
(1) from extracts kit take out 1.5ml centrifuge tube several, each pipe add anticoagulation 200 μ l.
(2) add 500 μ l buffer clg solution in anticoagulation, cover tightly centrifugation lid, whirlpool shakes more than 20s, fully
Reverse mixing is it is necessary to abundant mixing or whirlpool concussion are to guarantee to discharge genome dna.
(3) 100 μ l buffer pp solution, whirlpool concussion or back and forth reverse 10s are added.
(4) 12,000g centrifugation 5min.
(5) take out dna purification column from extracts kit, the supernatant in step (4) is transferred to purification column, 8000g
Centrifugation 1min.For improving dna extracted amount, after centrifugation, filtrate can rewind be centrifuged once again.
(6) abandon filtrate after post, dna purification column was put in same collecting pipe, add 750 μ l bufferings toward in purification column
Liquid w, after room temperature places 2min, 8000g centrifugation 1min (confirms that buffer w has added dehydrated alcohol).
(7) abandoned filtrate after post, added 750 μ l buffer buffer w toward in purification column, 8000g was centrifuged 1min
(confirming that buffer w has added dehydrated alcohol).
(8) abandoned filtrate after post, by purification column 12,000g is centrifuged 2min.
(9) purification column is transferred in new 1.5ml centrifuge tube, room temperature is placed 5min and fully volatilized ethanol.Plus 70～100
μ l collection liquid eb (collection liquid eb is preheating to 65 DEG C and is conducive to improving the elution efficiency of dna), room temperature places 1min, 12,000 centrifugations
2min eluting genome dna.Can be then added to crossing the eluent sucking-off after post on purification column film, room temperature placement 1min, 12,000g
Centrifugation 1min can improve the elution amount of dna.
Finally, the concentration of genome dna and quality are passed through micro ultraviolet spectrophotometer and are measured od260And od280Light absorption value
(the nanophotometertmp-clas micro-spectrophotometer of German implen, Beijing triumphant AudioCodes skill Development Co., Ltd
K2800 nucleic acids instrument), dna concentration is in 2ng/ μ l～100ng/ μ l；od260/od280Value, between 1.5～2.0, meets follow-up
Test requirements document.
The dna sample of purification preserves at 4 DEG C and is less than 7 days；The dna sample failing in time detection preserves in -20 DEG C, preserves
Time is less than 3 months；Purification dna sample multigelation is less than 5 times.
(2) reagent prepares
Take out pcr reactant liquor from test kit, thaw on ice, calculate the pcr reaction tube required for each reaction system
Number, pipe number is n (n=n sample number+1 negative control+1 positive control, n >=3).According to the form below 1 takes the pcr of amount of calculation to react respectively
Liquid and mixed enzyme, in suitable centrifuge tube, overturn and mix 10 times, and 2000g is centrifuged 10 seconds, the different reaction system of labelling.
Table 1
(3) it is loaded
Positive control and negative control take out, and thaw on ice, mix in turbula shaker, and 2000g is centrifuged 10 seconds.To institute
It is separately added into sample, positive control, each 5ul of negative control in the pcr reaction tube setting, cover tightly lid, and make a record.Mix
Pcr reaction system in each pcr reaction tube and added sample.Pcr reaction tube 2000g is centrifuged 10 seconds.It is transferred to detection zone,
It is placed on pcr instrument, order put by record sample.
It should be noted that above-mentioned sample, positive control dna mass concentration are between 2ng/ μ l～100ng/ μ l, that is, often
Individual reaction system can meet test requirements document for 10-500ng.
(4) pcr amplification
Abi7500 fluorescent quantitation pcr instrument, selects fam (reporter:fam；Quencher:none) sense channel；vic
(reporter:vic；Quencher:none) sense channel.Reference fluorescent (passive reference) is set to none, threshold
Value line is set to 50000.
Pcr amplification program: 50 DEG C of 2min；95 DEG C of denaturations 2min；95 DEG C of degeneration 15s；62 DEG C of prolongation 45s；35 circulations,
Collect fluorescence in 62 DEG C of 45s, that is, phosphor collection is arranged on " step 3:62 DEG C, 45s ".
(5) Analysis of test results
As reaction system testing result is more than 37.5 in vic sense channel no ct or ct, then the genome dna of judgment sample
Concentration is less than test limit.In the case that reaction system testing result is less than or equal to 37.5 in vic sense channel ct, by following
Table 2 judges (i.e. the fluorescence intensity of comparative sample fam passage and vic passage), and cyp2c9 gene type is as shown in table 3 below.
Table 2
Table 3
In the present embodiment testing result, the testing result in cyp2c9 gene 1075 site has 3 kinds.As shown in Figure 1: fam
Fluorescence intensity and vic channel fluorescence intensity are close, and that is, 1075 sites are aa wild type；As shown in Figure 2: fam channel fluorescence intensity
Compare vic channel fluorescence intensity negligible, that is, 1075 sites are cc homozygous mutant；As shown in Figure 3: fam fluorescence intensity
For vic channel fluorescence intensity half, that is, 1075 sites are ac heterozygous.
The present embodiment devises common taqman probe and carries out contrast test, for wild (fam labelling) and mutational site
(vic labelling) separately designs common taqman probe.The result of detection homozygous mutant sample is as shown in figure 4, the open country of fam labelling
Raw type coupling probe has obvious amplified signal (now should not having amplified signal or negligible in theory), and its result is relatively
Dual labelled probe accuracy designed by the present invention substantially reduces.
It follows that this cyp2c9 gene type detection method, there is simple to operate, rapid, high resolution, result more accurate
True feature.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all essences in the present invention
Any modification, equivalent and improvement made within god and principle etc., should be included within the scope of the present invention.
Claims (10)
1. it is used for detecting primer and the probe of mankind's cyp2c9 gene type, comprise following nucleotide sequence:
Forward primer seq id no:1:5 '-cagctaaagtccaggaagagat-3 '；
Downstream primer seq id no:2:5 '-ttctgaatttaatgtcacaggt-3 '；
Probe seq id no:3:5 '-cagagatacattgaccttctccccacc-3 '.
2. primer as claimed in claim 1 and probe it is characterised in that described probe 5 ' end and 3 ' hold respectively with two kinds different
Fluorescent reporter group labelling, described fluorescent reporter group is selected from: fam, hex, tet, vic, rox, cy5, cy3, joe, alex,
cal；
11st nucleotide site fluorescent quenching group labelling of described probe.
3. c.1075a primer as claimed in claim 1 or 2 and probe it is characterised in that the sporting of described cyp2c9 gene
>c.
4. a kind of detection mankind's cyp2c9 gene type test kit it is characterised in that described test kit comprises pcr reactant liquor,
Described pcr reactant liquor includes described primer as arbitrary in claim 1-3 and probe.
5. test kit as claimed in claim 4 is it is characterised in that in described pcr reactant liquor, forward primer concentration is: 0.1-
0.5μm；Downstream primer concentration is: 0.1-0.5 μm；Concentration and probe concentration is: 0.1-0.5 μm.
6. test kit as claimed in claim 4 is it is characterised in that described test kit is also included by ung enzyme and dna polymerase group
The mixed enzyme becoming, described dna polymerase has flap endonuclease activity；And/or
Described test kit also includes positive reference substance and negative controls；Described positive reference substance includes two kinds of plasmids, and described two
Kind of plasmid comprises 1075a wild type nucleic acid sequence and the 1075c mutant nucleic acid sequence of described cyp2c9 gene respectively, and described two
Plant your concentration ratio 1:1 of plasmid massage to mix；Described negative controls are te buffer；And/or
Described test kit also includes dntps, mgcl2, described dntps concentration is: 0.2-0.5mm；mgcl2Concentration is: 0.5-
2.5mm.
7. a kind of method of detection mankind's cyp2c9 gene type, it comprises the steps:
Extract human genome dna；
Described dna is made into reaction system with the arbitrary described test kit of claim 4-6, carries out fluorescent quantitation pcr amplification；
Described cyp2c9 gene type is analyzed according to described amplification.
8. method as claimed in claim 7 is it is characterised in that described reaction system is 40ul, wherein:
Pcr reactant liquor 34.5ul, mixed enzyme 0.5ul, dna mass 10-500ng.
9. method as claimed in claim 7 or 8 is it is characterised in that the response procedures of described fluorescent quantitation pcr amplification are: 50
℃2min；95 DEG C of denaturations 2min；95 DEG C of degeneration 15s；62 DEG C of extension 45s；35 circulations.
10. purposes in detection cyp2c9 gene type for the test kit of claim 4-6.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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) |
| CN112301096A (en) * | 2019-07-26 | 2021-02-02 | 杭州丹威生物科技有限公司 | Novel nucleic acid probe labeling method |
| CN114369688A (en) * | 2022-03-22 | 2022-04-19 | 深圳联合医学科技有限公司 | Compositions, kits, methods and uses for detecting a variant of SARS-CoV-2 Oncuronte |
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| CN104164506A (en) * | 2014-08-13 | 2014-11-26 | 深圳联合医学科技有限公司 | Method for real-time PCR (polymerase chain reaction) detection of single nucleotide polymorphism and application of method |
| CN105177116A (en) * | 2015-06-02 | 2015-12-23 | 武汉友芝友医疗科技有限公司 | Human CYP2C9 and VKORC1 genetic polymorphism detection kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104164506A (en) * | 2014-08-13 | 2014-11-26 | 深圳联合医学科技有限公司 | Method for real-time PCR (polymerase chain reaction) detection of single nucleotide polymorphism and application of method |
| CN105177116A (en) * | 2015-06-02 | 2015-12-23 | 武汉友芝友医疗科技有限公司 | Human CYP2C9 and VKORC1 genetic polymorphism detection kit |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112301096A (en) * | 2019-07-26 | 2021-02-02 | 杭州丹威生物科技有限公司 | Novel nucleic acid probe labeling method |
| 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) |
| CN114369688A (en) * | 2022-03-22 | 2022-04-19 | 深圳联合医学科技有限公司 | Compositions, kits, methods and uses for detecting a variant of SARS-CoV-2 Oncuronte |
| CN114369688B (en) * | 2022-03-22 | 2022-06-03 | 深圳联合医学科技有限公司 | Compositions, kits, methods and uses for detecting a variant of SARS-CoV-2 Oncuronte |
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