CN110295224A - Primer combination of probe and kit and application for instructing Rosiglitazone drug personalized medicine related gene to detect - Google Patents
Primer combination of probe and kit and application for instructing Rosiglitazone drug personalized medicine related gene to detect Download PDFInfo
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Abstract
The specific primer probe that the invention discloses a kind of for instructing Rosiglitazone drug personalized medicine related gene SNP combines, it is characterized in that, the primer combination of probe includes the specific primer probe combination for rs10192566, rs4149056, rs6467136 gene SNP.The invention also discloses the kit combined comprising the primer and its applications.The beneficial effects of the present invention are: a kind of parting kit and application for efficiently being used to instruct Rosiglitazone drug personalized medicine related gene SNP is provided, the testing result obtained using the present invention is accurately reliable, and cost is relatively low, has preferable practicability.
Description
Technical field
The present invention relates to vitro diagnostic techniques fields, and in particular to one kind is for instructing Rosiglitazone drug personalized medicine
The primer combination of probe and kit of related gene detection and application.
Background technique
Rosiglitazone (Rosiglitazone) belongs to Study of Thiazolidinedione derivatives as Insulin Sensitizer, mechanism of action and specificity
The γ receptor (PPAR γ) of peroxisome proliferator activator is related.By increasing skeletal muscle, liver, adipose tissue
To the sensibility of insulin, improves cell and the curative effect for reducing blood glucose is played to the utilization of glucose, can obviously reduce fasting blood
Sugar and insulin and C peptide level, also have reduction effect to postprandial blood sugar and insulin.But over administration can generate hypoglycemia, stomach
Enteron aisle reacts the adverse reactions such as (such as abdominal distension, diarrhea, nausea), dermoreaction, hematological system reaction (hemolytic anemia).
There are apparent individuation difference, the following toxic side effects also to seriously endanger sufferer for rosiglitazone in treating effect
Health.LPIN1, SLCO1B1, PAX4 genetic mutation are related with the curative effect of rosiglitazone in treating Type 2 Diabetes In China.Lipoprotein
It is the product of LPIN1 gene, is necessary to normal-fat tissue development and metabolism.The shortage of lipid 1 can lead to human adipose battalion
Support bad patient's fat cell developmental immaturity.LPIN1 genetic mutation may influence the anti-of rosiglitazone in treatment of type 2 diabetes mellitus
It answers, rs10192566 allele is patient's fasting blood-glucose of G allele, postprandial 2 hours blood glucose, glycosylated hemoglobins compared with nothing
The patient of allele is substantially reduced, and unrelated with age, gender and weight.TZDs is a kind of insulin sensitizer, be can be used as
The agonist of nuclear factor Peroxisome Proliferator-activated Receptors y, so as to cause the improvement of insulin sensitivity, especially in week
Enclose tissue.As common TZDs, Rosiglitazone has positive effect to the blood glucose for reducing type 2 diabetic patient for many years.It is clinical
Experiments have shown that Rosiglitazone can significantly improve insulin sensitivity, FPG, 2h blood glucose, glycosylated hemoglobin are reduced, so as to improve
Insulin sensitivity, PAX4rs6467136 variation are related with the curative effect of rosiglitazone in treating Type 2 Diabetes In China.Pass through detection
Rosiglitazone metabolism, transhipment or action target spot gene, can find early Rosiglitazone to the Different therapeutical effect of Different Individual, thus
It realizes " individuation " medication of Rosiglitazone, improves therapeutic effect, reduce the improper caused risk of dosage.
" drug metabolic enzyme and drug target technique of gene detection guide (tentative) " is pointed out: to drug metabolic enzyme and medicine
Object target gene is detected, and can be instructed clinical for the suitable drug of specific patient selection and dosage realization " individual
Change " medication, to improve the validity and safety of drug therapy.
The genetic polymorphism detection technology used currently on the market has PCR-RFLP, and cardinal principle is special by application
Property digestion with restriction enzyme pcr amplification product, and according to digestion site whether disappear judge its make a variation presence or absence, but should
Method is complicated for operation, and the cross contamination of PCR product is easily caused when sample size is more and is easy to appear that digestion is insufficient or digestion is excessive
And there are false negative or false positive results, reliability is low.Multiple PCR method is although specificity increases, this method
Principle be still based on the principle of regular-PCR, these factors such as primer specificity and Lo-Fi Taq enzyme can be caused to knot
The influence of fruit.Although DNA sequencing method is the current goldstandard for carrying out gene diagnosis, but complicated steps and complex processes, reagent price
Valuableness, the cross contamination being easy to appear between sample and cause sequencing fail；In addition the equipment of sequenator also has exceeded general clinic
Examine the tolerance range in laboratory.High-resolution melting curve method is a kind of quick, easy, economic, practical classifying method, but
Genotyping depends on the accuracy of instrument temperature control, and false positive is high.The method of Taqman probe is using specific fluorescence
The probe of label, high specificity, high sensitivity and easy to operate, quickly.
Summary of the invention
In order to clinical application easily occur not when solving the diagnosis of traditional clinical experience medical mode and Rosiglitazone drug medication
The technical issues of good reaction and no suitable reagent kit product.
The present invention is the detection kit combined using Taqman probe with fluorescent quantitative PCR technique, is suitable for clinic
Popularization and industrialization.
One of the objects of the present invention is to provide one kind for instructing Rosiglitazone drug personalized medicine related gene SNP
Primer combination of probe.
The second object of the present invention is to provide the kit of the primer combination of probe comprising the related gene SNP
The third object of the present invention is the application of the kit.
One of to achieve the purpose of the present invention, used technical solution is:
A kind of specific primer probe for instructing Rosiglitazone drug personalized medicine related gene SNP combines, institute
Stating primer combination of probe includes the specific primer probe group for rs10192566, rs4149056, rs6467136 gene SNP
It closes, specific as follows:
Rs10192566 primer probe sequence are as follows:
Rs10192566-F:5'-ACAGGAGACAAGCAACAAGGGT-3'；
Rs10192566-R:5'-ACTGGTGGCATTCTGGCATT-3'；
Rs10192566-P1:5'FAM-CTGGCAAAACAATGT-MGB 3'；
Rs10192566-P2:5'VIC-CTGGCAAAAGAATGT-MGB 3'；
Rs4149056 primer probe sequence are as follows:
Rs4149056-F:5'-GAAACACTCTCTTATCTACATAGGTTGT-3'；
Rs4149056-R:5'-CCTTCTTTAGCGAAATCATCAA-3'；
Rs4149056-P1:5'FAM-CCCATGAACACATATA-MGB 3'；
Rs4149056-P2:5'VIC-ACCCATGAACGCATATA-MGB 3'；
Rs6467136 primer probe sequence are as follows:
Rs6467136-F:5'-TGTGACTTTAGGTAATATTTTCTTGGAC-3'；
Rs6467136-R:5'-CGTGGGTTGTCTTTTCACTTTC-3'；
Rs6467136-P1:5'FAM-CTTTAAAAGTGCAAATGAC-MGB 3'；
Rs6467136-P2:5'VIC-CTTTAAAAGTGCAAGTGAC-MGB 3'.
In order to achieve the object of the present invention two, used technical solution is:
It is a kind of for instructing the PCR kit for fluorescence quantitative of Rosiglitazone drug personalized medicine related gene SNP, including
The PCR reaction solution of the described specific primer probe combination, the PCR reaction solution be respectively rs10192566, rs4149056,
The PCR reaction solution of rs6467136, the PCR reaction solution further include 2 × NuHi SNP Mix (Taq enzyme, dNTPs, MgCl2、PCR
Buffer, ROX reference fluorescent) and ultrapure water.
In a preferred embodiment of the invention, the PCR reaction solution ingredient is final concentration of: 1 × NuHi SNP
Mix, 0.5 μM of each specific primer and 0.2 μM of probe.
It in a preferred embodiment of the invention, further include positive reference substance I, positive reference substance II in the kit
And positive reference substance III；
The positive reference substance I be respectively rs10192566, rs4149056, rs6467136 gene loci CC type, TT type,
GG type plasmid；
The positive reference substance II is respectively rs10192566 gene loci CC type and GG type, rs4149056 gene loci
The plasmid of TT type and CC type, rs6467136 gene loci GG type and AA type by 1:1 quantity than heterozygosis；
The positive reference substance III is respectively rs10192566, rs4149056, rs6467136 gene loci GG type, CC
Type, AA type plasmid.
In order to achieve the object of the present invention three, used technical solution is:
A kind of application of the kit, the application are using the PCR kit for fluorescence quantitative, for people's gene
Group DNA carries out quantitative fluorescent PCR reaction, and the polymorphism of corresponding SNP site is distinguished according to the fluorescence signal that reaction process generates, and uses
To instruct Rosiglitazone drug personalized medicine.
The beneficial effects of the present invention are: it provides a kind of efficiently for instructing Rosiglitazone drug personalized medicine phase
The parting kit of correlation gene SNP and application, the testing result obtained using the present invention is accurately reliable, and cost is relatively low, has
Preferable practicability.
Detailed description of the invention
Fig. 1: the amplification curve diagram of the CC genotype of rs10192566 in the embodiment of the present invention (FAM is upper)；
Fig. 2: the amplification curve diagram of the CG genotype of rs10192566 in the embodiment of the present invention (FAM is upper)；
Fig. 3: the amplification curve diagram of the GG genotype of rs10192566 in the embodiment of the present invention (FAM is under)；
Fig. 4: the PCR fluorescence analysis figure of rs10192566 genotypic results in the embodiment of the present invention；
Fig. 5: the amplification curve diagram of the TT genotype of rs4149056 in the embodiment of the present invention (FAM is upper)；
Fig. 6: the amplification curve diagram of the TC genotype of rs4149056 in the embodiment of the present invention (FAM is under)；
Fig. 7: the amplification curve diagram of the CC genotype of rs4149056 in the embodiment of the present invention (FAM is under)；
Fig. 8: the PCR fluorescence analysis figure of rs4149056 genotypic results in the embodiment of the present invention；
Fig. 9: the amplification curve diagram of the GG genotype of rs6467136 in the embodiment of the present invention (FAM is upper)；
Figure 10: the amplification curve diagram of the AG genotype of rs6467136 in the embodiment of the present invention (FAM is upper)；
Figure 11: the amplification curve diagram of the AA genotype of rs6467136 in the embodiment of the present invention (FAM is under)；
Figure 12: the PCR fluorescence analysis figure of rs6467136 genotypic results in the embodiment of the present invention；
Figure 13: the amplification curve diagram of the CC genotype of primer combination of probe rs10192566-2 in comparative example (FAM is under)；
Figure 14: the amplification curve diagram of the CG genotype of primer combination of probe rs10192566-2 in comparative example (FAM is under)；
Figure 15: the amplification curve diagram of the GG genotype of primer combination of probe rs10192566-2 in comparative example (FAM is under)；
Figure 16: the PCR fluorescence analysis figure of primer combination of probe rs10192566-2 genotypic results in comparative example；
Figure 17: the amplification curve diagram of the TT genotype of primer combination of probe rs4149056-2 in comparative example (FAM is under)；
Figure 18: the amplification curve diagram of the TC genotype of primer combination of probe rs4149056-2 in comparative example (FAM is under)；
Figure 19: the amplification curve diagram of the CC genotype of primer combination of probe rs4149056-2 in comparative example (FAM is under)；
Figure 20: the PCR fluorescence analysis figure of primer combination of probe rs4149056-2 genotypic results in comparative example；
Figure 21: the amplification curve diagram of the GG genotype of primer combination of probe rs6467136-2 in comparative example (FAM is under)；
Figure 22: the amplification curve diagram of the AG genotype of primer combination of probe rs6467136-2 in comparative example (FAM is under)；
Figure 23: the amplification curve diagram of the AA genotype of primer combination of probe rs6467136-2 in comparative example (FAM is under)；
Figure 24: the PCR fluorescence analysis figure of primer combination of probe rs6467136-2 genotypic results in comparative example.
Specific embodiment
The principle of the present invention is mainly:
In the PCR reaction system of TaqMan probe method, including one couple of PCR primers and a pair of of probe.Probe and template
It specifically combines, binding site is between two primers.5 ' ends of probe are marked with reporter gene, and 3 ' ends are marked with fluorescence
Quenching group, when probe is complete, the fluorescent energy that reporter gene is emitted is quenched group absorptions, and instrument can't detect
Signal, with the progress of PCR, Taq enzyme encounters the probe in conjunction with template, 3 ' -5 ' exonucleases during chain extension
Activity will cut off probe, and reporter group cannot be absorbed far from quenching group, energy, i.e. generation fluorescence signal.With two
The different fluorescent dyes (such as FAM, HEX, VIC) of kind mark this pair of of probe respectively, for the different genotype of double equipotential SNP, just
It can complete to determine the genotype of single SNP site in a PCR reaction.
The present invention is further illustrated by the following examples, but these embodiments must not be used to explain to the present invention
Limitation.
Embodiment 1:
One, biological sample: biomaterial of the present invention is all from intra-company.For during the 9-12 month in 2018
The remaining crowd's anticoagulation of 100 detected of intra-company.
Two, the DNA for taking sample extracting to be detected, as pcr template: DNA extraction kit is Tiangeng biochemical technology (north
Capital) extracts kit " poba gene group DNA extraction kit " of Co., Ltd extracts (article No.: DP318).
1. taking whole blood 400ul, 800ul cell pyrolysis liquid CL is added to mix, 10000rpm/11500 × g, 1min are centrifuged, in abandoning
Clearly, it can be repeated once if cracking is not thorough.
2. 200ul buffer GS is added in precipitating, mixes, add 20ul Proteinase K, mixes.
3. 200ul buffer GB is added, mix, 56 DEG C, 10min, during which overturn for several times, until solution becomes clarification (if not
Clarification can extend the time to clarification).
4. adding 200ul dehydrated alcohol to mix, of short duration centrifugation.
5. shifting sample mixed liquor to be centrifuged to adsorption column CB3,13400 × g/12000rpm, 30s, filtrate is abandoned.
6. 500ul buffer GD is added to be centrifuged to adsorption column CB3,13400 × g/12000rpm, 30s, filtrate is abandoned.
7. 600ul buffer PW is added to be centrifuged to adsorption column CB3,13400 × g/12000rpm, 30s, filtrate is abandoned, is repeated
Once.
8. adsorption column CB3 is put into clean centrifuge tube, 13400 × g/12000rpm, filtrate is abandoned in 2min centrifugation, and room temperature is quiet
It sets 3 minutes, dries.
9. adding 100ulddH2O, is incubated at room temperature 2-5min, and 13400 × g/12000rpm, 2min centrifugation are collected elution and produced
Object, -20 DEG C of preservations.
Three, the design and synthesis of primer and probe: 1, for rs10192566, rs4149056 in human genome,
Rs6467136 gene loci (sequence is referring to mankind's whole genome sequence disclosed in ncbi database), uses Primer
3.0 software of Premier 5.0 and Primer Express, separately designs specific primer and probe.
Prepare specific primer and probe sequence, as shown in table 1 below:
Table 1
Note: Primer is with the corresponding rs number name of gene；F represents upstream primer, and R represents downstream primer, and P1 is represented
FAM probe, P2 represent VIC probe.
Four, prepare PCR reaction solution:
The primed probe that first group of addition gene loci is rs10192566, primer sequence are SEQ ID NO.1 and SEQ
ID NO.2, probe sequence are SEQ ID NO.3 and SEQ ID NO.4；
The primed probe that second group of addition gene loci is rs4149056, primer sequence are SEQ ID NO.5 and SEQ ID
NO.6, probe sequence are SEQ ID NO.7 and SEQ ID NO.8；
Third group adds the primed probe that gene loci is rs6467136, and primer sequence is SEQ ID NO.9 and SEQ ID
NO.10, probe sequence are SEQ ID NO.11 and SEQ ID NO.12；
Every group of PCR reaction solution further includes 2 × NuHi SNP Mix (Taq enzyme, dNTPs, MgCl2, PCR buffer, ROX ginseng
Than fluorescence) and ultrapure water；
The PCR reaction solution ingredient it is final concentration of: 1 × NuHi SNP Mix, 0.5 μM of each specific primer and 0.2 μ
M probe.
Five, PCR reaction system, as shown in table 2 below:
Table 2
PCR amplification program, as shown in table 3 below:
Table 3
Using ABI SteponePlus fluorescence quantitative PCR instrument (Applied Biosystems, U.S.'s Applied Biotechnology
Co., Ltd).
Six, experimental result:
PCR after reaction, using Stepone software V2.3, (give birth to by Applied Biosystems, U.S.'s application
Object Technology Co., Ltd.) it is analyzed, obtain 100 results are classified, probe according to the present invention, which is divided into, discerns 3
Totally 9 kinds of genotype of SNP site.
The amplification curve diagram of three kinds of genotype of rs10192566 is as shown in Figures 1 to 3, PCR fluorescence analysis figure such as Fig. 4 institute
Show；For the amplification curve diagram of three kinds of genotype of rs4149056 as shown in Fig. 5~7, PCR fluorescence analysis figure is as shown in Figure 8；
For the amplification curve diagram of three kinds of genotype of rs6467136 as shown in Fig. 9~11, PCR fluorescence analysis figure is as shown in figure 12.
Eight, kit test result of the invention is instructing the application in Rosiglitazone drug medication
Each site rs10192566, rs4149056, rs6467136 is obtained according to the result judgement in quantitative fluorescent PCR
Genotype.Shown in genotype the following table 4 corresponding with Rosiglitazone drug personalized medicine relationship:
Table 4
Comparative example 1: change the Genotyping of primer combination of probe sequence information detection people's anticoagulation tissue samples.
One, biological sample: biomaterial of the present invention is all from Pai Sennuo medical test institute.For 9-12 in 2018
In Pai Sennuo medical test 100 detected remaining crowd's anticoagulations during month.
Two, the DNA for taking sample extracting to be detected, as pcr template: DNA extraction kit is Tiangeng biochemical technology (north
Capital) extracts kit " poba gene group DNA extraction kit " of Co., Ltd extracts (article No.: DP318), and it is specific to extract
Step is the same as people's DNA extraction steps in embodiment 1.
Three, the design and synthesis of primer and probe:
1, for rs10192566, rs4149056, rs6467136 gene loci in human genome, (sequence is referring to NCBI number
According to mankind's whole genome sequence disclosed in library), using 3.0 software of Primer Premier 5.0 and Primer Express, divide
Two groups of primed probes are not designed.
Specific primer probe sequence and comparison primer probe sequence of the invention, as shown in table 5 below:
Table 5
Note: Primer is with the corresponding rs number name of gene；F represents upstream primer, and R represents downstream primer, and P1 is represented
FAM probe, P2 represent VIC probe.
Four, prepare PCR reaction solution:
First group is primer combination of probe rs10192566-1: addition primer sequence is SEQ ID NO.1 and SEQ ID
NO.2, probe sequence are SEQ ID NO.3 and SEQ ID NO.4；
Second group is primer combination of probe rs10192566-2: addition primer sequence is SEQ ID NO.13 and SEQ ID
NO.14, probe sequence are SEQ ID NO.15 and SEQ ID NO.16；
Third group is primer combination of probe rs4149056-1: addition primer sequence is SEQ ID NO.5 and SEQ ID
NO.6, probe sequence are SEQ ID NO.7 and SEQ ID NO.8；
4th group is primer combination of probe rs4149056-2: addition primer sequence is SEQ ID NO.17 and SEQ ID
NO.18, probe sequence are SEQ ID NO.19 and SEQ ID NO.20；
5th group is primer combination of probe rs6467136-1: addition primer sequence is SEQ ID NO.9 and SEQ ID
NO.10, probe sequence are SEQ ID NO.11 and SEQ ID NO.12；
6th group is primer combination of probe rs6467136-2: addition primer sequence is SEQ ID NO.21 and SEQ ID
NO.22, probe sequence are SEQ ID NO.23 and SEQ ID NO.24；
Every group of PCR reaction solution further includes 2 × NuHi SNP Mix (Taq enzyme, dNTPs, MgCl2, PCR buffer, ROX ginseng
Than fluorescence) and ultrapure water；
The PCR reaction solution ingredient it is final concentration of: 1 × NuHi SNP Mix, 0.5 μM of each specific primer and 0.2 μ
M probe.
Five, PCR reaction system, as shown in table 2 in embodiment 1.
Six, PCR amplification program, as shown in table 3 in embodiment 1.
Using ABI SteponePlus fluorescence quantitative PCR instrument (Applied Biosystems, U.S.'s Applied Biotechnology
Co., Ltd).
Seven, experimental result:
PCR after reaction, using Stepone software V2.3, (give birth to by Applied Biosystems, U.S.'s application
Object Technology Co., Ltd.) it is analyzed, analysis result is as follows:
The amplification curve diagram of three kinds of genotype of primer combination of probe rs10192566-1 is as shown in Figures 1 to 3, PCR fluorescence
Analysis chart is as shown in figure 4, amplification curve diagram and PCR fluorescence analysis figure are distinguished well；
For the amplification curve diagram of three kinds of genotype of primer combination of probe rs10192566-2 as shown in Figure 13~15, PCR is glimmering
Light analysis chart is as shown in figure 16, and amplification curve diagram and PCR fluorescence analysis figure are distinguished ineffective；
The amplification curve diagram of three kinds of genotype of primer combination of probe rs4149056-1 is as shown in Fig. 5~7, PCR fluorescence point
Analysis figure is as shown in figure 8, amplification curve diagram and PCR fluorescence analysis figure are distinguished well；
The amplification curve diagram of three kinds of genotype of primer combination of probe rs4149056-2 is as shown in Figure 17~19, PCR fluorescence
Analysis chart is as shown in figure 20, and amplification curve diagram and PCR fluorescence analysis figure are distinguished ineffective；
The amplification curve diagram of three kinds of genotype of primer combination of probe rs6467136-1 is as shown in Fig. 9~11, PCR fluorescence
Analysis chart is as shown in figure 12, and amplification curve diagram and PCR fluorescence analysis figure are distinguished well；
The amplification curve diagram of three kinds of genotype of primer combination of probe rs6467136-2 is as shown in Figure 21~23, PCR fluorescence
Analysis chart is as shown in figure 24, and amplification curve diagram and PCR fluorescence analysis figure are distinguished ineffective.
It can be seen that the differentiation that primer provided herein combines works well, and primer combination is also to pay greatly
It is obtained after amount creative work.
Sequence table
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aatgaaacac tctcttatct acataggttg tt 32
<210> 18
<211> 28
<212> DNA
<213> homo sapiens
<400> 18
ctttagcgaa atcatcaatg taagaaag 28
<210> 19
<211> 32
<212> DNA
<213> homo sapiens
<400> 19
cgaagcatat tacccatgaa cacatatatc ca 32
<210> 20
<211> 31
<212> DNA
<213> homo sapiens
<400> 20
aagcatatta cccatgaacg catatatcca c 31
<210> 21
<211> 26
<212> DNA
<213> homo sapiens
<400> 21
tgtgacttta ggtaatattt tcttgg 26
<210> 22
<211> 21
<212> DNA
<213> homo sapiens
<400> 22
tatttctccc attccgtggg t 21
<210> 23
<211> 20
<212> DNA
<213> homo sapiens
<400> 23
actttaaaag tgcaaatgac 20
<210> 24
<211> 19
<212> DNA
<213> homo sapiens
<400> 24
actttaaaag tgcaagtga 19
Claims (5)
1. a kind of specific primer probe for instructing Rosiglitazone drug personalized medicine related gene SNP combines, special
Sign is that the primer combination of probe includes the specificity for rs10192566, rs4149056, rs6467136 gene SNP
Primer combination of probe, specific as follows:
Rs10192566 primer probe sequence are as follows:
Rs10192566-F:5'-ACAGGAGACAAGCAACAAGGGT-3'；
Rs10192566-R:5'-ACTGGTGGCATTCTGGCATT-3'；
Rs10192566-P1:5'FAM-CTGGCAAAACAATGT-MGB3'；
Rs10192566-P2:5'VIC-CTGGCAAAAGAATGT-MGB3'；
Rs4149056 primer probe sequence are as follows:
Rs4149056-F:5'-GAAACACTCTCTTATCTACATAGGTTGT-3'；
Rs4149056-R:5'-CCTTCTTTAGCGAAATCATCAA-3'；
Rs4149056-P1:5'FAM-CCCATGAACACATATA-MGB3'；
Rs4149056-P2:5'VIC-ACCCATGAACGCATATA-MGB3'；
Rs6467136 primer probe sequence are as follows:
Rs6467136-F:5'-TGTGACTTTAGGTAATATTTTCTTGGAC-3'；
Rs6467136-R:5'-CGTGGGTTGTCTTTTCACTTTC-3'；
Rs6467136-P1:5'FAM-CTTTAAAAGTGCAAATGAC-MGB3'；
Rs6467136-P2:5'VIC-CTTTAAAAGTGCAAGTGAC-MGB3'.
2. a kind of fluorescence for instructing Rosiglitazone drug personalized medicine related gene SNP as described in claim 1 is fixed
Measure PCR kit, which is characterized in that including the PCR reaction solution that the specific primer probe combines, the PCR reaction solution
The respectively PCR reaction solution of rs10192566, rs4149056, rs6467136, the PCR reaction solution further include 2 × NuHi
SNP Mix (Taq enzyme, dNTPs, MgCl2, PCR buffer, ROX reference fluorescent) and ultrapure water.
3. a kind of fluorescence for instructing Rosiglitazone drug personalized medicine related gene SNP as claimed in claim 2 is fixed
Measure PCR kit, which is characterized in that the PCR reaction solution ingredient it is final concentration of: 1 × NuHi SNP Mix, 0.5 μM each
Specific primer and 0.2 μM of probe.
4. a kind of fluorescence for instructing Rosiglitazone drug personalized medicine related gene SNP as claimed in claim 2 is fixed
Measure PCR kit, which is characterized in that further include positive reference substance I, positive reference substance II and positive reference substance in the kit
Ⅲ；
The positive reference substance I is respectively rs10192566, rs4149056, rs6467136 gene loci CC type, TT type, GG type
Plasmid；
The positive reference substance II is respectively rs10192566 gene loci CC type and GG type, rs4149056 gene loci TT type
Plasmid with CC type, rs6467136 gene loci GG type and AA type by 1:1 quantity than heterozygosis；
The positive reference substance III is respectively rs10192566, rs4149056, rs6467136 gene loci GG type, CC type, AA
Type plasmid.
5. the application of kit as claimed in claim 2, which is characterized in that the application is to utilize the fluorescent quantitation
PCR kit carries out quantitative fluorescent PCR reaction for human gene group DNA, distinguishes phase according to the fluorescence signal that reaction process generates
The polymorphism of SNP site is answered, for instructing Rosiglitazone drug personalized medicine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110951858A (en) * | 2019-12-11 | 2020-04-03 | 上海派森诺生物科技股份有限公司 | Primer-probe combination for guiding detection of genes related to glibenclamide drug personalized administration, kit and application |
| CN111020025A (en) * | 2019-12-11 | 2020-04-17 | 上海派森诺生物科技股份有限公司 | Primer probe combination for guiding detection of related genes of metformin drug personalized medicine, kit and application |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014181107A1 (en) * | 2013-05-09 | 2014-11-13 | Medical Research Council | Genetic method of aiding the diagnosis and treatment of familial hypercholersterolaemia |
| CN106086224A (en) * | 2016-08-24 | 2016-11-09 | 上海派森诺生物科技股份有限公司 | A kind of detection multifarious primer sets of lactase gene and application thereof |
| KR101712454B1 (en) * | 2015-12-11 | 2017-03-07 | 대한민국 | Reagent for SNP-genotyping of a gene related with anticancer drug-metabolizing enzyme and transporter, the kit comprising the same, and the method for the SNP-genotyping |
| CN108998517A (en) * | 2018-09-06 | 2018-12-14 | 武汉康录生物技术股份有限公司 | A kind of mankind SLCO1B1 and ApoE genetic polymorphism detection kit and its preparation method and application |
| CN110205371A (en) * | 2019-05-14 | 2019-09-06 | 南京派森诺基因科技有限公司 | Primer combination of probe and kit and application for instructing Fluvastatin drug personalized medicine related gene to detect |
| CN110205370A (en) * | 2019-05-14 | 2019-09-06 | 南京派森诺基因科技有限公司 | Primer combination, kit and method for instructing Pravastatin drug personalized medicine related gene to detect |
| CN110241197A (en) * | 2019-05-28 | 2019-09-17 | 南京派森诺基因科技有限公司 | Primer combination of probe and kit and application for instructing Atorvastatin drug personalized medicine related gene to detect |
| CN110295225A (en) * | 2019-05-28 | 2019-10-01 | 南京派森诺基因科技有限公司 | Primer combination and kit and method for instructing Lovastatin drug personalized medicine related gene to detect |
| CN110387412A (en) * | 2019-07-08 | 2019-10-29 | 上海派森诺生物科技股份有限公司 | Primer combination and kit and method for instructing Rosuvastatin drug personalized medicine related gene to detect |
| CN110951858A (en) * | 2019-12-11 | 2020-04-03 | 上海派森诺生物科技股份有限公司 | Primer-probe combination for guiding detection of genes related to glibenclamide drug personalized administration, kit and application |
-
2019
- 2019-05-28 CN CN201910451777.2A patent/CN110295224A/en not_active Withdrawn
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014181107A1 (en) * | 2013-05-09 | 2014-11-13 | Medical Research Council | Genetic method of aiding the diagnosis and treatment of familial hypercholersterolaemia |
| KR101712454B1 (en) * | 2015-12-11 | 2017-03-07 | 대한민국 | Reagent for SNP-genotyping of a gene related with anticancer drug-metabolizing enzyme and transporter, the kit comprising the same, and the method for the SNP-genotyping |
| CN106086224A (en) * | 2016-08-24 | 2016-11-09 | 上海派森诺生物科技股份有限公司 | A kind of detection multifarious primer sets of lactase gene and application thereof |
| CN108998517A (en) * | 2018-09-06 | 2018-12-14 | 武汉康录生物技术股份有限公司 | A kind of mankind SLCO1B1 and ApoE genetic polymorphism detection kit and its preparation method and application |
| CN110205371A (en) * | 2019-05-14 | 2019-09-06 | 南京派森诺基因科技有限公司 | Primer combination of probe and kit and application for instructing Fluvastatin drug personalized medicine related gene to detect |
| CN110205370A (en) * | 2019-05-14 | 2019-09-06 | 南京派森诺基因科技有限公司 | Primer combination, kit and method for instructing Pravastatin drug personalized medicine related gene to detect |
| CN110241197A (en) * | 2019-05-28 | 2019-09-17 | 南京派森诺基因科技有限公司 | Primer combination of probe and kit and application for instructing Atorvastatin drug personalized medicine related gene to detect |
| CN110295225A (en) * | 2019-05-28 | 2019-10-01 | 南京派森诺基因科技有限公司 | Primer combination and kit and method for instructing Lovastatin drug personalized medicine related gene to detect |
| CN110387412A (en) * | 2019-07-08 | 2019-10-29 | 上海派森诺生物科技股份有限公司 | Primer combination and kit and method for instructing Rosuvastatin drug personalized medicine related gene to detect |
| CN110951858A (en) * | 2019-12-11 | 2020-04-03 | 上海派森诺生物科技股份有限公司 | Primer-probe combination for guiding detection of genes related to glibenclamide drug personalized administration, kit and application |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110951858A (en) * | 2019-12-11 | 2020-04-03 | 上海派森诺生物科技股份有限公司 | Primer-probe combination for guiding detection of genes related to glibenclamide drug personalized administration, kit and application |
| CN111020025A (en) * | 2019-12-11 | 2020-04-17 | 上海派森诺生物科技股份有限公司 | Primer probe combination for guiding detection of related genes of metformin drug personalized medicine, kit and application |
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