CN106319056A - Primer and probe set and kit for detecting VKORC1 (vitamin k epoxide reductase subunit 1) genetic typing - Google Patents
Primer and probe set and kit for detecting VKORC1 (vitamin k epoxide reductase subunit 1) genetic typing Download PDFInfo
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Abstract
The invention relates to a primer and probe set for detecting VKORC1 (vitamin k epoxide reductase subunit 1) genetic typing. The primer and probe set comprises a wild type and mutant type general downstream primer for VKORC1, a wild type and mutant type general downstream probe for VKORC1, a wild type upstream primer, a mutant type upstream primer, an internal control GAPDH upstream primer, an internal control GAPDH downstream primer and an internal control GAPDH probe. The invention further relates to a kit for detecting the VKORC1 genetic typing. The kit contains a PCR (polymerase chain reaction) liquid 1, a PCR liquid 2, a positive reference substance and a blank reference substance. The kit is high in sensitivity, the sensitivity can be up to one ten-thousandth, the lowest detectable limit is only 1-2 copies, and the kit is particularly applicable to detection of a low-mutation sample such as serum or plasma; compared with a sequencing method, a result can be observed in real time, a product is not required to be subjected to gel electrophoresis detection and is subjected to complete closed-tube operation, so that the risk of pollution of a PCR product is effectively reduced, the detection speed is fast, and the primer and probe set and the kit are applicable to high-flux sample detection.
Description
Technical field
The present invention relates to external nucleic acid detection technique field, particularly relate to a kind of primer detecting VKORC1 gene type and visit
Pin group and test kit.
Background technology
Warfarin is a kind of dicoumarol derivant, is one of the most most widely used current oral anticoagulation thing,
For preventing and treat the thrombosis that deep venous thrombosis, pulmonary infarction, cardiac valve replacement and atrial fibrillation cause.Warfarin treatment
Window is narrower, and the least dosage all may cause the generation of untoward reaction, and reaches identical action effect, just agent in Different Individual
More than 10 times can be differed between amount person.
Vitamin K epoxide reductase complex 1 (Vitamin kepoxide reductase subunit 1,
VKORC1) being the key enzyme in vitamin K circulation, warfarin blocks vitamin K and participates in cofactor form because suppressing this enzyme
The catalytic reaction of carboxylase, it is suppressed that factor Ⅱ, the functional activity of VII, Ⅸ, Ⅹ, thus produce anticoagulation.Both at home and abroad
Numerous studies find: VKORC1 gene mutation can increase this enzyme sensitivity to warfarin, thus strengthens anticoagulant effect, wherein than
Sudden change more typically has the G-1639A sudden change of promoter region and the C1173T sudden change of No. 1 intron.Research at present it turned out
Two sites of 1639G > A and 1173C > T are complete linkage.The China that VKORC1-1693AA (1173TT) genotype patient needs
Method woods dosage is substantially less than 1693GA or GG (1173TC or CC).U.S. food Drug Administration (FDA) explicitly points out:
When using warfarin, it is proposed that detection VKORC1 genotype.
Hospitals at Present is PCR-direct sequencing for " goldstandard " of VKORC1 gene test, but the behaviour of direct sequencing
Making program more complicated, sensitivity is the highest, is only capable of detecting the mutant etc. of more than 20-30%.Compared to direct sequencing,
The susceptiveness of ARMS-TaqMan method is higher, cost is lower.
ARMS is also referred to as ApoE gene (Allele Specific PCR, AS-PCR), and its principle is that PCR draws
When 3 ' end end bit bases of thing exist mispairing with its template DNA, typically amplification efficiency will be caused drastically to decline, design allele
When primer 3 ' base is matched with template, specific PCR amplimer, under strict conditions, only could occur that PCR expands
Signal, thus detect sudden change.
Real-time fluorescence quantitative PCR (Real-time Quantitative PCR, QPCR) technology in 1996 by the U.S.
Applied Biosystems company releases, owing to this technology not only achieves the PCR leap from qualitative to quantitative, and with often
Rule PCR compares, and it has specificity solution higher, effective PCR pollution problem, automaticity high, the most obtains
Extensively application.
QPCR technology is generally divided into sonde method and dye method, and this test kit uses TaqMan fluorescent probe: PCR amplification
Time add a specific fluorescent probe while pair of primers adding, this probe is an oligonucleotide, and two ends are marked respectively
Remember a reporter fluorescence group and a quenching fluorescence group.When probe is complete, the fluorescence signal that reporter group is launched is quenched
Group absorptions；During PCR amplification, probe enzyme action is degraded, is made reporter fluorescence group and cancellation by the 5'-3' 5 prime excision enzyme activity of Taq enzyme
Fluorophor separates, thus fluorescence monitoring system can receive fluorescence signal, and the most often one DNA of amplification, just has a fluorescence
Molecule is formed, it is achieved that the accumulation of fluorescence signal forms Complete Synchronization with PCR primer.
ARMS is not the most utilized to combine the product that QPCR technology carries out the genetic polymorphism detection of VKORC1.
Summary of the invention
In order to solve said method detection VKORC1 genotyping process in exist sensitivity the highest, detection cycle length, behaviour
Making loaded down with trivial details and that cost is high technical problem, the present invention provides a kind of sensitivity height, high specificity, detection cycle short, simple to operate
And effectively meet primed probe group and the test kit of the detection VKORC1 gene type of Clinical Laboratory requirement.
The invention provides a kind of primed probe group detecting VKORC1 gene type, described VKORC1 gene test is many
State property site is G1639A, and described primed probe group includes:
Amplification VKORC1 wild type and the general downstream primer of saltant type:
5’-GCCAGGCTTGTCTTAAACTCC-3’(SEQ ID NO.1)；
Amplification VKORC1 wild type and the general probe of saltant type:
5’FAM-ACCTCAAGTGATCCACCCACCTCGGC-TAMRA 3’(SEQ ID NO.2)；
The forward primer of amplification VKORC1 wild type:
5’-CCTGAAAAACAACCATTGGACG-3’(SEQ ID NO.3)；
The forward primer of amplification VKORC1 saltant type:
5’-CCTGAAAAACAACCATTGGACA-3’(SEQ ID NO.4)；And
The forward primer of amplification internal control GAPDH gene:
5’-CACATGGCCTCCAAGGAGTAA-3’(SEQ ID NO.5)；
The downstream primer of amplification internal control GAPDH gene:
5’-TGAGGGTCTCTCTCTTCCTCTTGT-3’(SEQ ID NO.6)；
The probe of amplification internal control GAPDH gene:
5’JOE-CTGGACCACCAGCCCCAGCAAG-TAMRA 3’(SEQ ID NO.7)。
Present invention also offers a kind of test kit detecting VKORC1 gene type, described VKORC1 gene test polymorphic
Property site is G1639A, and described test kit includes:
PCR reactant liquor 1, described PCR reactant liquor 1 is containing SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ
ID NO.5, SEQ ID NO.6 and the amplimer shown in SEQ ID NO.7 and probe；
PCR reactant liquor 2, described PCR reactant liquor 2 is containing SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.4, SEQ
ID NO.5, SEQ ID NO.6 and the amplimer shown in SEQ ID NO.7 and probe.
In a kind of preferred embodiment of the described test kit of present invention offer, described test kit also includes positive control
Product, it is the VKORC1 wild homozygote plasmid being inserted with SEQ ID NO.1 and SEQ ID NO.3 amplified production, is inserted with SEQ ID
The VKORC1 no mutant homozygote plasmid of NO.1 and SEQ ID NO.4 amplified production, is inserted with SEQ ID NO.5 and SEQ ID NO.6
The plasmid mixture of above-mentioned three kinds of plasmids composition of the internal control GAPDH plasmid of amplified production；
Wherein, plasmid vector is pMD18-T plasmid；VKORC1 no mutant homozygote plasmid in described plasmid mixture,
The quantity of VKORC1 wild homozygote plasmid and internal control GAPDH plasmid is than for 1:1:2.
In a kind of preferred embodiment of the described test kit of present invention offer, described test kit also includes blank
Product, described blank product are ultra-pure water.
In a kind of preferred embodiment of the described test kit of present invention offer, other components in described PCR reactant liquor 1-2
For 2 conventional x Mix Buffer and H2O, PCR response parameter and system be:
95℃ 30s；95 DEG C of 10s, 60 DEG C of 30s (collection fluorescence), carry out 50 circulations,
| Material name | Addition (μ L) |
| 2 x Mix Buffer (containing ROX) | 12.5 |
| Forward primer | 0.5 |
| Downstream primer | 0.5 |
| Probe | 0.5 |
| Internal control forward primer | 0.5 |
| Internal control downstream primer | 0.5 |
| Internal control probe | 0.5 |
| DNA profiling | 1 |
| Deionized water | 8.5 |
| Cumulative volume | 25 |
Present invention also offers primed probe group as above in preparation for the reagent detecting VKORC1 gene type
In application.
Compared to prior art, primed probe group and the test kit of the detection VKORC1 gene type that the present invention provides have
Following beneficial effect:
One, by utilize ARMS technology to combine QPCR Technology design is highly sensitive and specificity is good primed probe group and
Its test kit so that described test kit detect VKORC1 gene type time, have qualitative accurately, highly sensitive and high specificity
Advantage；Additionally, also have, sample treatment is simple, sequencing steps simple, order-checking speed is fast, it is anti-within one hour, to complete once to go up machine
The advantage that, detection site fluorescence curve and visual result should be directly given；
Two, by utilize ARMS technology to combine QPCR Technology design is highly sensitive and specificity is good primed probe group and
Its test kit so that described test kit when detecting VKORC1 gene type, can monitor in real time reaction process, response time short,
Easy and simple to handle and high flux sample detection, PCR reaction carries out fluorescent collecting, and ratio goldstandard method, i.e. capillary electrophoresis simultaneously
Sequencing sensitivity is higher, is particularly suited for mutation analysis and Clinical Laboratory；
Three, by being provided with blank product and positive reference substance in described test kit so that described test kit is in inspection
When surveying VKORC1 gene type, can preferably guarantee the accuracy of testing result.
Accompanying drawing explanation
Fig. 1 is warfarin mechanism of action and VKORC1 effect schematic diagram；
Fig. 2 is the amplified fluorescence curve chart of clinical sample VKORC1 wild type；
Fig. 3 is the amplified fluorescence curve chart of clinical sample VKORC1 sudden change heterozygous；
Fig. 4 is the amplified fluorescence curve chart of clinical sample VKORC1 mutant homozygous type；
Fig. 5 is the amplified fluorescence curve chart of clinical VKORC1 positive reference substance；
Fig. 6 is the amplified fluorescence curve chart of clinical VKORC1 blank product；
Fig. 7 to Fig. 8 is the amplified fluorescence curve chart of many groups of VKORC1 design primer；Wherein the sequencing result of Fig. 7 is inaccurate,
The sequencing result of Fig. 8 is true and reliable.
Detailed description of the invention
In order to make the purpose of the present invention, technical scheme and advantage clearer, below in conjunction with the accompanying drawings and embodiment, right
The present invention is described in further detail.Should be appreciated that specific embodiment described herein only in order to explain the present invention, and
It is not used in the restriction present invention.
Embodiment 1: the preparation of test kit
One, primer and the design of probe and synthesis
For people's VKORC1 gene polynorphisms site G1639A allele, select special mutational site, selection
Primer and probe design VKORC1 mutational site and near conserved region, it is to avoid have SNP in PBR territory and (pass through
The SNP of target-gene sequence is retrieved by online NCBI website), carry out Primer Blast by online NCBI website, confirm
The specific amplification of primer pair.Probe region between pair of primers, and avoid its calmodulin binding domain CaM to have SNP；Wherein
Amplimer and fluorescent probe first pass through PAGE purification, then through HPLC purification, wherein 5 ' the ends of purpose probe SEQ ID NO.2 are adopted
With fluorescent reporter group (FAM) labelling, the 5 ' ends of internal control probe SEQ ID NO.7 use fluorescent reporter group (JOE) labellings, and 3 '
End all uses fluorescent quenching group (TAMRA) labelling.
Table 1. mutational site and type
| Mutation | Base change |
| VKORC1(1639) | G>A |
Extension increasing sequence such as table 2:
Table 2. specificity amplification primer and probe sequence
Two, reference substance selects
Positive reference substance is made up of the mixture of 3 kinds of plasmids, VKORC1 no mutant homozygote plasmid in described plasmid mixture,
The quantity of VKORC1 wild homozygote plasmid and internal control GAPDH plasmid is than for 1:1:2；DNase/RNase-Free water is blank right
According to product.
Three, PCR reactant liquor composition
Table 3.PCR reactant liquor 1 forms
Table 4.PCR reactant liquor 2 forms
| Material name | Addition (μ L) |
| 2x Mix Buffer (containing ROX) | 12.5 |
| SEQ ID NO.4 | 0.5 |
| SEQ ID NO.1 | 0.5 |
| SEQ ID NO.2 | 0.5 |
| SEQ ID NO.5 | 0.5 |
| SEQ ID NO.6 | 0.5 |
| SEQ ID NO.7 | 0.5 |
| Deionized water | 8.5 |
| Cumulative volume | 24 |
By 2 kinds of different PCR reactant liquors, VKORC1 G1639A site is carried out typing detection.
Embodiment 2: the use of test kit
One, sample detection
Dissolve primed probe dry powder (after primer dissolves, effect duration is 1 month).System is prepared: take PCR anti-according to template number
Answering liquid, add solvent primer, probe, subpackage system, adding sample DNA, blank product or positive reference substance is template, group
Become PCR reaction system.PCR amplification is carried out according to PCR response procedures.
The each main component of VKORC1 system is as follows:
Table 5.PCR reactant liquor 1 forms
| Material name | Addition (μ L) |
| 2 x Mix Buffer (containing ROX) | 12.5 |
| SEQ ID NO.3 | 0.5 |
| SEQ ID NO.1 | 0.5 |
| SEQ ID NO.2 | 0.5 |
| SEQ ID NO.5 | 0.5 |
| SEQ ID NO.6 | 0.5 |
| SEQ ID NO.7 | 0.5 |
| DNA profiling | 1 |
| Deionized water | 8.5 |
| Cumulative volume | 25 |
Table 6.PCR reactant liquor 2 forms
| Material name | Addition (μ L) |
| 2 x Mix Buffer (containing ROX) | 12.5 |
| SEQ ID NO.4 | 0.5 |
| SEQ ID NO.1 | 0.5 |
| SEQ ID NO.2 | 0.5 |
| SEQ ID NO.5 | 0.5 |
| SEQ ID NO.6 | 0.5 |
| SEQ ID NO.7 | 0.5 |
| DNA profiling | 1 |
| Deionized water | 8.5 |
| Cumulative volume | 25 |
This system response procedures is as follows:
Table 7.PCR response procedures
Two, ABI7500 quantitative fluorescent PCR
Press right-hand member key, start ABI 7500.After start, machine left end " power " display lamp length is bright.Open door,
The reagent prepared is put into storehouse, the good position oneself put of note.
1) double-click " 7500Software v2.0.5 " icon and open software.In the window ejected, click on " OK " enter journey
Sequence.
2) click " New Experiment ", choose successively in " Experiment Properties " interface ejected
“7500(96wells)”、“Quantitaion-Standad Curve”、“Reagents " icon makes it brighten.
3) clicking on " Plate Setup ", the drop-down choosing in " Reporter " hurdle selects " FAM " in frame,
Drop-down choosing in " Quencher " hurdle selects " TAMRA " in frame, clicks on " Add New Target ", in " Reporter " hurdle
Drop-down choosing selects " JOE " in frame, and drop-down choosing the in " Quencher " hurdle selects " TAMRA " in frame；Repeatedly click on " Add New
Sample ", make " Sample Name " lower section eject sufficient amount of square frame, in these square frames, input unique volume of each sample
Number.
4) " Assign Targets and Samples " is clicked on, the drop-down choosing in " Passive Reference " hurdle
Select " ROX " in frame, choose the position, hole that reagent place puts, confirm corresponding with instrument, at " Assign sample (s) to the
Selected wells " beat " √ ", on " Assign target (s) to the selected wells " hurdle in square frame in hurdle
Beat " √ " in interior square frame, and the icon clicking on correspondence makes it brighten: " U " (sample to be tested), " S " (positive control), " N " (sky
White comparison).
5) reaction condition is arranged: clicks " Run Method " entrance reaction condition and arranges panel, the amplification journey needed for formulation
Sequence, arranges 95 DEG C 30 seconds in " Holding Stage " hurdle, cursor moves to " Cycling Stage " hurdle, at Section 1 " Step
1 " arrange 95 DEG C in 10 seconds, " Step 2 " arrange 60 DEG C 30 seconds, and click fluorescent collecting icon makes it brighten in this joint,
In " Number of Cycles " hurdle, input 50, and in " Reaction Volume Per Well " hurdle, input 25.
6) confirm errorless after, click on " START R ... " to file number and being saved in correspondence as time PCR, click on true
Fixed, bring into operation.One section of preheating is had, " IN USE " display lamp flicker when formally starting the cycle over after beginning.
7) after having run, open door, pour product into refuse receptacle, fill in instrument and use record.
Three, result judges
After reaction terminates, it is automatically adjusted baseline and threshold value.After setting, click on " Analyse " (analysis) button, i.e.
The Ct value of each sample can be obtained from " Ct " of " View Well Table " window.
Four, quality control standard
For VKORC1: the reaction tube Ct value being furnished with PCR reactant liquor 1 is set to Ct1, is furnished with the reaction tube Ct of PCR reactant liquor 2
Value is set to Ct2, △ Ct=Ct2-Ct1.
Ct≤32 of positive control ,-2≤△ Ct≤2；
Blank without S type amplification curve or shows without Ct value.
Conditions above all must meet in once experiment, and otherwise this experimental result is invalid.
Five, result report:
For sample to be checked:
1) if Ct 32, illustrate that DNA mass is the lowest, need again extract DNA or change sample；
2) if Ct-≤32, i.e. for Δ CT absolute value > sample of 12 is defined as corresponding homozygote ,-2≤Δ CT≤2
Sample is defined as the heterozygous of VKORC1.The test that then needs to reform of 2≤| Δ CT | the sample of≤12 is checked, result of reforming
Unanimously, then regard as sampling process or sample process process exists pollution, need to resample.
Fig. 1 is warfarin mechanism of action and VKORC1 effect schematic diagram；Fig. 2 is shown that in clinical sample testing result
The wild type of VKORC1；Fig. 3 is shown that the heterozygous of VKORC1 in clinical sample testing result；Fig. 4 is shown that clinical sample
The mutant homozygous type of VKORC1 in product testing result；Fig. 5 and Fig. 6 be shown that respectively the positives reference substance of Clinical detection result and
The amplified fluorescence curve chart of blank product.
Wherein, in Fig. 2 reactant liquor 1, FAM curve Ct1 value is 25.69, and in reactant liquor 2, FAM curve is without Ct1 value, △ Ct ＞ 12
It is judged to wild type.In Fig. 3 reactant liquor 1, FAM curve Ct1 value is 26.8, and in reactant liquor 2, FAM curve Ct2 value is 26.44, △ Ct
=26.8-26.44=0.36-2≤Δ CT≤2 are judged to heterozygous.In Fig. 4 reactant liquor 1, FAM curve is without Ct1 value, reactant liquor 2
Middle FAM curve Ct2 value is 25.22, and △ Ct ＞ 12 is judged to mutant homozygous type.The FAM curve of Fig. 5 reactant liquor 1 and 2 all has CT
Value, JOE curve the most all has CT value.The FAM curve of Fig. 6 reactant liquor 1 and 2 is all without CT value, and JOE curve is the most all without CT value.
Fig. 7 to Fig. 8 is shown that the amplified fluorescence curve chart of VKORC1 many groups primer of design, and wherein the primer of Fig. 8 is
Most preferably, it is primer selected in our product.
Primed probe group and the test kit of the detection VKORC1 gene type that the present invention provides have the advantages that
One, by utilize ARMS technology to combine QPCR Technology design is highly sensitive and specificity is good primed probe group and
Its test kit so that described test kit detect VKORC1 gene type time, have qualitative accurately, highly sensitive and high specificity
Advantage；Additionally, also have, sample treatment is simple, sequencing steps simple, order-checking speed is fast, it is anti-within one hour, to complete once to go up machine
The advantage that, detection site fluorescence curve and visual result should be directly given；
Two, by utilize ARMS technology to combine QPCR Technology design is highly sensitive and specificity is good primed probe group and
Its test kit so that described test kit when detecting VKORC1 gene type, can monitor in real time reaction process, response time short,
Easy and simple to handle and high flux sample detection, PCR reaction carries out fluorescent collecting, and ratio goldstandard method, i.e. capillary electrophoresis simultaneously
Sequencing sensitivity is higher, is particularly suited for mutation analysis and Clinical Laboratory；
Three, by being provided with blank product and positive reference substance in described test kit so that described test kit is in inspection
When surveying VKORC1 gene type, can preferably guarantee the accuracy of testing result.
The foregoing is only embodiments of the invention, not thereby limit the scope of the claims of the present invention, every utilize this
The equivalent flow process conversion that bright description is made, or directly or indirectly it is used in other relevant technical field, the most in like manner wrap
Include in the scope of patent protection of the present invention.
SEQUENCE LISTING
<110>help bio tech ltd in Changsha three
<120>primed probe group and the test kit of VKORC1 gene type are detected
<130> 2016
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213>artificial sequence
<400> 1
gccaggcttg tcttaaactc c 21
<210> 2
<211> 26
<212> DNA
<213>artificial sequence
<400> 2
acctcaagtg atccacccac ctcggc 26
<210> 3
<211> 22
<212> DNA
<213>artificial sequence
<400> 3
cctgaaaaac aaccattgga cg 22
<210> 4
<211> 22
<212> DNA
<213>artificial sequence
<400> 4
cctgaaaaac aaccattgga ca 22
<210> 5
<211> 21
<212> DNA
<213>artificial sequence
<400> 5
cacatggcct ccaaggagta a 21
<210> 6
<211> 24
<212> DNA
<213>artificial sequence
<400> 6
tgagggtctc tctcttcctc ttgt 24
<210> 7
<211> 22
<212> DNA
<213>artificial sequence
<400> 7
ctggaccacc agccccagca ag 22
Claims (6)
1. the primed probe group detecting VKORC1 gene type, it is characterised in that described VKORC1 gene test polymorphic
Property site is G1639A, and described primed probe group includes:
Amplification VKORC1 wild type and the general downstream primer of saltant type:
5’-GCCAGGCTTGTCTTAAACTCC-3’；
Amplification VKORC1 wild type and the general probe of saltant type:
5’FAM-ACCTCAAGTGATCCACCCACCTCGGC-TAMRA 3’；
The forward primer of amplification VKORC1 wild type:
5’-CCTGAAAAACAACCATTGGACG-3’；
The forward primer of amplification VKORC1 saltant type:
5’-CCTGAAAAACAACCATTGGACA-3’；And
The forward primer of amplification internal control GAPDH gene:
5’-CACATGGCCTCCAAGGAGTAA-3’；
The downstream primer of amplification internal control GAPDH gene:
5’-TGAGGGTCTCTCTCTTCCTCTTGT-3’；
The probe of amplification internal control GAPDH gene:
5’JOE-CTGGACCACCAGCCCCAGCAAG-TAMRA 3’。
2. the test kit detecting VKORC1 gene type, it is characterised in that the polymorphic position of described VKORC1 gene test
Point is G1639A, and described test kit includes:
PCR reactant liquor 1, described PCR reactant liquor 1 is containing SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID
Amplimer shown in NO.5, SEQ ID NO.6 and SEQ ID NO.7 and probe；
PCR reactant liquor 2, described PCR reactant liquor 2 is containing SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.4, SEQ ID
Amplimer shown in NO.5, SEQ ID NO.6 and SEQ ID NO.7 and probe.
Test kit the most according to claim 2, it is characterised in that described test kit also includes positive reference substance, it is
It is inserted with the VKORC1 wild homozygote plasmid of SEQ ID NO.1 and SEQ ID NO.3 amplified production,
It is inserted with SEQ ID NO.1 and the VKORC1 no mutant homozygote plasmid of SEQ ID NO.4 amplified production,
It is inserted with the matter of above-mentioned three kinds of plasmids composition of the internal control GAPDH plasmid of SEQ ID NO.5 and SEQ ID NO.6 amplified production
Grain mixture, wherein, plasmid vector is pMD18-T plasmid.
Test kit the most according to claim 3, it is characterised in that VKORC1 no mutant homozygote matter in described plasmid mixture
Grain, VKORC1 wild homozygote plasmid and internal control GAPDH plasmid quantity ratio for 1:1:2.
Test kit the most according to claim 2, it is characterised in that described test kit also includes blank product, described sky
White reference substance is ultra-pure water.
6. primed probe group as claimed in claim 1 is used for the application detecting in the reagent of VKORC1 gene type in preparation.
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