CN107190051A - A kind of kit for detecting melbine personalized medicine associated SNP positions - Google Patents
A kind of kit for detecting melbine personalized medicine associated SNP positions Download PDFInfo
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- CN107190051A CN107190051A CN201610143499.0A CN201610143499A CN107190051A CN 107190051 A CN107190051 A CN 107190051A CN 201610143499 A CN201610143499 A CN 201610143499A CN 107190051 A CN107190051 A CN 107190051A
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Abstract
Patent of the present invention discloses the kit machine detection method that a kind of utilization high-resolution melt curve analysis analysis method detects melbine personalized medicine associated SNP positions.The kit carries out parting, including SLC2A2 gene rs5398 SNP sites and the SNP of SCL47A1 genes rs 228966 to 2 SNP sites of melbine medication.Kit includes 2 × HRM buffer solutions, 25mM MgCl2,1uM forward primer, 1uM reverse primers and 1uM probes.Above-mentioned 2 SNP site partings can be completed by analyzing the melting temperature of different genotype, Molecular diagnosis foundation is provided to melbine personalized medicine so as to reach.
Description
Technical field
Patent of the present invention is related to a kind of detection kit PCR amplification method for detecting SNP site, and the kit and its core PCR amplification method of melbine personalized medicine associated SNP positions are detected especially with high-resolution melt curve analysis analytical technology.
Background technology
Diabetes are one of universally acknowledged diseases of dangerous human health, and China is that diabetes prevalence is high, and number of patients occupies first place in the world close to 100,000,000, its to patient health harm and heavy burden is caused to social economy.Therefore, diabetes are to defend one of disease that planning commission pays close attention to, and are defended the entry of planning commission's landing classification diagnosis and treatment.In addition, diabetes are also one of two representative diseases in U.S.'s " accurate medical science plan ".Data above illustrates the urgency and importance for preventing and treating diabetes.Melbine is domestic and international guide（ADA, EASD, IDF and CDS etc.）In by the line oral drugs as diabetes B clinical treatment, with stronger blood sugar reducing function, cardiovascular protective effect, 2 diabetes mellitus types can be reduced suffer from the risk of malignant tumour, reduce Impaired Glucose Tolerance Treated（IGT）Person develops into the probability of diabetes, and critical role is occupied in diabetes control.Inquired into for such a most popular Remedies for diabetes and determine its individual Different therapeutical effect and cause the gene polymorphism sites of side effect, it is the first step for realizing the accurate medical science of diabetes, pass through Genotyping examination, instruct personalized medicine to treat, be favorably improved treatment level and glycemic control of China diabetic using melbine.
The content of the invention
SLC2A2 and SCLl47A1 genetic polymorphism directly or indirectly influences melbine transhipment in vivo and metabolic process.The present invention provides the kit and its core PCR amplification method of a kind of high-resolution melt curve analysis analysis detection melbine personalized medicine associated SNP positions.
The technical solution adopted by the present invention：A kind of kit for detecting melbine personalized medicine associated SNP positions, described kit can carry out amplification parting detection to SLC2A2 gene rs5398 SNP sites and the SNP sites of SCL47A1 genes rs 228966 simultaneously.Detection SNP amplification condition be：2 × HRM buffer solutions, 25mM MgCl2, 1uM
Forward primer, 1uM reverse primers and 1uM probes, the saltant type for carrying correspondence SNP site by expanding post analysis amplified production melt curve analysis to judge whether is showed, wherein melting temperature has difference between homozygous mutant melting curve, homozygous wildtype melting curve, the combination of wild type and saltant type then occur it is bimodal, as shown in figures 1-6.Therefore, it can be good at distinguishing genotype using this method, result judgement is accurate, easy, quick, it is easy to accomplish computer automation detection and data report.
The pair of primers and probe of the detection SLC2A2 gene rs5398 SNP sites are as follows：
Forward primer：5’-TGTGTTTGCTTTCTATCCAGGAC-3’
Reverse primer：5’-GGGCTGAGCCACTCTTCTTT-3’
Probe：5’-GGCCTTTACCCTGTTCACATTTTTTAAAGTT/3AmM/-3’
The pair of primers and probe of the SNP sites of SCL47A1 genes rs 228966 are as follows：
Forward primer：5’-TGCTAAGCATCGTAACCTGGG-3’
Reverse primer：5’-CTGGTGGGAAAACTTGGTCCT-3’
Probe：5’-TCCACAGTAGCGTGGAAGTTCCCGGCTAGAC/3AmM/-3’
The present invention is analyzed using high-resolution melt curve analysis analytical technology, PCR reaction conditions：95 DEG C 15 minutes, then 95 DEG C 15 seconds, 60 DEG C 20 seconds and 72 DEG C 30 seconds, 65 circulations, the temperature of time per unit is risen to 4.8 DEG C/s to 95 DEG C, with 2.5 DEG C/s to 60 DEG C, with 4.8 DEG C/s to 72 DEG C, then 72 DEG C 1 minute.HRM analysis conditions：With 4.8 DEG C/s to 95 DEG C of denaturation PCR primer, 55 DEG C of degree hybridization are then reduced to 2.5 DEG C/s.95 DEG C of periods, 25 fluorescence signals of every degree Celsius of acquisition are increased at 55 DEG C with 4.8 DEG C/s clumps.
Brief description of the drawings
Accompanying drawing 1：SLC2A2 gene rs5398 SNP C/C type HRM results.
Accompanying drawing 2：SLC2A2 gene rs5398 SNP T/T type HRM results.
Accompanying drawing 3：SLC2A2 gene rs5398 SNP C/T type HRM results.
Accompanying drawing 4：The G/G type HRM results of SCL47A1 genes rs 228966.
Accompanying drawing 5：The A/A type HRM results of SCL47A1 genes rs 228966.
Accompanying drawing 6：The G/A type HRM results of SCL47A1 genes rs 228966.
Accompanying drawing 7：SLC2A2 gene rs5398 SNP clinical sample testing results.
Accompanying drawing 8：The SNP clinical sample testing results of SCL47A1 genes rs 228966.
Embodiment
Embodiment one.
Step 1：Configure PCR reaction systems:Include 2 × HRM master mix, 25mM MgCl2,1uM forward primer and 1uM reverse primers, and 1uM probes.
Step 2：PCR is expanded：95 DEG C 15 minutes, then 95 DEG C 15 seconds, 60 DEG C 20 seconds and 72 DEG C 30 seconds, 65 circulations, the temperature of time per unit is risen to 4.8 DEG C/s to 95 DEG C, with 2.5 DEG C/s to 60 DEG C, with 4.8 DEG C/s to 72 DEG C, then 72 DEG C 1 minute.
Step 3：HRM is analyzed：With 4.8 DEG C/s to 95 DEG C of denaturation PCR primer, 55 DEG C of degree hybridization are then reduced to 2.5 DEG C/s.95 DEG C of periods, 25 fluorescence signals of every degree Celsius of acquisition are increased at 55 DEG C with 4.8 DEG C/s clumps.
Step 4：Gene sequencing carries out testing result confirmation
97 parts of clinical samples are analyzed using the above method, SLC2A2 gene rs5398 SNP sites and the genetic mutation in SCL47A1 gene rs 228966SNP sites are detected using the HRM methods of foundation, genotype meets with gene sequencing result 100%, sees accompanying drawing 7-8.
Claims (5)
1. a kind of kit for detecting melbine personalized medicine associated SNP positions, its feature is including primer, the probe of detection SLC2A2 gene rs5398 SNP sites, primer, the probe of the SNP sites of detection SCL47A1 genes rs 228966 with, the kit.
2. the kit of detection melbine personalized medicine associated SNP positions according to claim 1, it is characterized in that, the kit includes a forward primer, reverse primer and the probe of detection gene SLC2A2 gene rs5398 SNP sites, a forward primer, reverse primer and the probe of the SNP sites of detection gene SCL47A1 genes rs 228966.
3. the kit of detection melbine personalized medicine associated SNP positions according to claim 2, it is characterised in that the primer probe sequence of the detection gene SLC2A2 gene rs5398 SNP sites is as follows：
Forward primer：5’-TGTGTTTGCTTTCTATCCAGGAC-3’
Reverse primer：5’-GGGCTGAGCCACTCTTCTTT-3’
Probe：5’-GGCCTTTACCCTGTTCACATTTTTTAAAGTT/3AmM/-3’.
4. the kit of detection melbine personalized medicine associated SNP positions according to claim 2, it is characterised in that the primer probe sequence of the SNP sites of detection gene SCL47A1 genes rs 228966 is as follows：
Forward primer：5’-TGCTAAGCATCGTAACCTGGG-3’
Reverse primer：5’-CTGGTGGGAAAACTTGGTCCT-3’
Probe：5’-TCCACAGTAGCGTGGAAGTTCCCGGCTAGAC/3AmM/-3’.
5. the detection method that the kit of detection melbine personalized medicine associated SNP positions according to claim 1 is used, it is characterised in that the method used is high-resolution melt curve analysis analysis method（HRM）, method is as follows：
Reaction condition：95 DEG C 15 minutes, then 95 DEG C 15 seconds, 60 DEG C 20 seconds and 72 DEG C 30 seconds, 65 circulations, the temperature of time per unit is risen to 4.8 DEG C/s to 95 DEG C, with 2.5 DEG C/s to 60 DEG C, with 4.8 DEG C/s to 72 DEG C, then 72 DEG C 1 minute；HRM analysis conditions：With 4.8 DEG C/s to 95 DEG C of denaturation PCR primer, 55 DEG C of degree hybridization are then reduced to 2.5 DEG C/s；95 DEG C of periods, 25 fluorescence signals of every degree Celsius of acquisition are increased at 55 DEG C with 4.8 DEG C/s clumps.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110699440A (en) * | 2018-07-09 | 2020-01-17 | 西安医臻生物医药科技有限公司 | Primer and method for detecting SNP (single nucleotide polymorphism) locus of gene related to metformin personalized medicine |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101008032A (en) * | 2006-01-26 | 2007-08-01 | 北京华安佛医药研究中心有限公司 | Uses and method for polymorphic point genetype for predicting sulfaurea drugs effect |
| WO2007128884A1 (en) * | 2006-05-09 | 2007-11-15 | Oy Jurilab Ltd | Novel genes and markers in type 2 diabetes and obesity |
| CN101619351A (en) * | 2009-01-23 | 2010-01-06 | 中南大学 | Method for detecting related gene mutations of diabetes drug treatment as well as special chip and kit thereof |
| CN103014138A (en) * | 2011-09-23 | 2013-04-03 | 深圳北京大学香港科技大学医学中心 | Method for determining correlation between single nucleotide polymorphism rs6887695 of IL-12b gene of Chinese Han people and systemic lupus erythematosus |
| CN104059991A (en) * | 2014-07-10 | 2014-09-24 | 光瀚健康咨询管理(上海)有限公司 | Method and kit for detecting susceptible loci rs2241766 of type 2 diabetes mellitus |
| CN104131090A (en) * | 2014-07-18 | 2014-11-05 | 北京大学人民医院 | Kit for prediction of sulfonylurea drug effects |
| CN104846069A (en) * | 2015-03-25 | 2015-08-19 | 刘坤 | Amplimer and probe for detecting AQP7 gene SNP multiple samples multiple sites by Q-PCR method and application thereof |
| CN105112502A (en) * | 2015-07-07 | 2015-12-02 | 博奥颐和健康科学技术（北京）有限公司 | Complete primers for detecting SNP site combination associated with diabetes, and applications thereof |
| CN105189778A (en) * | 2012-10-30 | 2015-12-23 | 香港中文大学 | Novel biomarkers for diabetic kidney diseases and use thereof |
-
2016
- 2016-03-14 CN CN201610143499.0A patent/CN107190051A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101008032A (en) * | 2006-01-26 | 2007-08-01 | 北京华安佛医药研究中心有限公司 | Uses and method for polymorphic point genetype for predicting sulfaurea drugs effect |
| WO2007128884A1 (en) * | 2006-05-09 | 2007-11-15 | Oy Jurilab Ltd | Novel genes and markers in type 2 diabetes and obesity |
| CN101619351A (en) * | 2009-01-23 | 2010-01-06 | 中南大学 | Method for detecting related gene mutations of diabetes drug treatment as well as special chip and kit thereof |
| CN103014138A (en) * | 2011-09-23 | 2013-04-03 | 深圳北京大学香港科技大学医学中心 | Method for determining correlation between single nucleotide polymorphism rs6887695 of IL-12b gene of Chinese Han people and systemic lupus erythematosus |
| CN105189778A (en) * | 2012-10-30 | 2015-12-23 | 香港中文大学 | Novel biomarkers for diabetic kidney diseases and use thereof |
| CN104059991A (en) * | 2014-07-10 | 2014-09-24 | 光瀚健康咨询管理(上海)有限公司 | Method and kit for detecting susceptible loci rs2241766 of type 2 diabetes mellitus |
| CN104131090A (en) * | 2014-07-18 | 2014-11-05 | 北京大学人民医院 | Kit for prediction of sulfonylurea drug effects |
| CN104846069A (en) * | 2015-03-25 | 2015-08-19 | 刘坤 | Amplimer and probe for detecting AQP7 gene SNP multiple samples multiple sites by Q-PCR method and application thereof |
| CN105112502A (en) * | 2015-07-07 | 2015-12-02 | 博奥颐和健康科学技术（北京）有限公司 | Complete primers for detecting SNP site combination associated with diabetes, and applications thereof |
Non-Patent Citations (5)
| Title |
|---|
| NABODITA KAUL等: "Genes, Genetics, and Environment in Type 2 Diabetes:Implication in Personalized Medicine", 《DNA AND CELL BIOLOGY》 * |
| 吕申等: "《病理检验技术》", 31 August 2014, 人民军医出版社 * |
| 周国华: "《SNP检测技术与个体化药物治疗》", 28 February 2015, 苏州大学出版社 * |
| 李建娥: "PRKAA2基因多态性与二甲双胍疗效的相关性研究", 《中国药理学通报》 * |
| 陈培贤: "SLC22A1，SLC22A2，SLC47A1，SLC47A2，ATM基因多态性对二甲双胍药物疗效及不良反应的影响", 《万方学位论文数据库》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110699440A (en) * | 2018-07-09 | 2020-01-17 | 西安医臻生物医药科技有限公司 | Primer and method for detecting SNP (single nucleotide polymorphism) locus of gene related to metformin personalized medicine |
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Effective date of registration: 20190423 Address after: 310018 69 No. 12 Street, Hangzhou Economic and Technological Development Zone, Zhejiang Province Applicant after: Kangyong Biotechnology Co., Ltd. Address before: 310018 69 Jingkai Street, Hangzhou City, Zhejiang Province Applicant before: CHINESE PEPTIDE COMPANY |
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Application publication date: 20170922 |