CN103710423A - Susceptible noninvasive detection kit for obesity (genes like LEPR) - Google Patents
Susceptible noninvasive detection kit for obesity (genes like LEPR) Download PDFInfo
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- CN103710423A CN103710423A CN201210373799.XA CN201210373799A CN103710423A CN 103710423 A CN103710423 A CN 103710423A CN 201210373799 A CN201210373799 A CN 201210373799A CN 103710423 A CN103710423 A CN 103710423A
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Abstract
The invention provides a method for detecting obese gene mutation site, a detection probe and a detection kit. The kit comprises a specific primer and a detection probe of the pleomorphic locus gene type of three mononucleotides which comprise C825T on a GNB3 gene, Gln223Arg on a LEPR gene and -866G/A on a UCP2 gene, a PCR reaction assembly, a PCR product purifying assembly and a DNA sequencing reaction assembly. The kit assesses a risk level of obesity at the genetic level by detecting the pleomorphic locus gene type of three mononucleotides which are closely related to obesity, and prevents generation of obesity in advance. A sampling method comprises sampling by oral mucosa cells without pain and wound, and avoids cross infection. The gene noninvasive detection kit has advantages of accurate and reliable sequencing detection result, no need of expensive special Import Instrument, and easy popularization.
Description
Technical field
The invention belongs to biology field, relate to medical science and biotechnology, be specifically related to a kind of gene detecting kit of obesity, the risk class occurring from gene aspect assess disease according to detected result, and instruct fat high risk population prevention ahead of time and treatment.
Background technology
Obesity refers to that the too much and (or) local body fat content of body fat total content is too much, is by the chronic metabolic disease due to the acting in conjunction of h and E factor.Along with improving constantly of standard of living, the change of mode of life, China resident Overweight and obesity rate is the trend of obvious rising.The sickness rate of Single Obesity increases year by year, and international paediatrics circle of Yi Bei is classified one of important topic of 21 century children's health as.Obesity can be divided into two classes according to fat accumulation position: a class increases with overall fat, and fat generation is located as feature in buttocks, thigh etc., is called subcutaneous lipids type fat, that is adiposis universalis; Another kind ofly with fat, mainly accumulate in belly, especially intraperitoneal is feature, is called visceral adiposity, that is centric obesity.The Chronic Non-Communicable Diseasess such as Overweight and obesity and hypertension, diabetes B are closely related, wherein, centric obesity and cardiovascular disorder and insuline resistance syndrome there is higher dependency.
The fat cause of disease is quite complicated, and inherited genetic factors occupies critical role in fat mechanism.The gene that relates to energy expenditure or energy intake is relevant with fat generation.The mankind's obesity patient shows as high leptin level mostly, has leptin resistance state, so leptin receptor (Leptin receptor, LEPR) regulates body to ingest as leptin and the signal transmission intermediary of energy metabolism is paid much attention to.And the relation of research leptin receptor polymorphism and each index of energy metabolism has very important meaning for the common genetic background that discloses obesity and relative disease thereof.Because the 668th Nucleotide A of LEPR gene extron → G replaces, L-glutamic acid is replaced (Gln223Arg) by arginine.The charging property in this site, by neutral steer positive charge, can affect the function of LEPR and change its signal transmission capacity.Infer that Gln223Arg gene pleiomorphism also may produce impact to LEPR signal transduction pathway, thereby participated in the pathogenesis of leptin resistance obesity.
Uncoupling protein (UCP) is mitochondrial inner membrane transport vehicle, and the gene pleiomorphism of UCP can affect transcribing of mRNA and protein expression, and then affects basal metabolic rate(BMR), thereby changes people's obesity-prone.(uncoupling proteins 2, UCP2) assignment of genes gene mapping, in 11q13 karyomit(e), is one of UCP family member to uncoupling protein 2.Quantity research shows greatly, and UCP2 gene is one of obesity-related gene.In the research discovery male sex of Zou Hengyun etc.-866G/A polymorphism is relevant with obesity, in the male sex, GG genotype individual relative AA genotype has higher obesity rates, this is consistent to 698 Austrian results of study with Harald etc., relative G allelotrope, the allelic carrier of A has lower obesity rates.A series of research also shows that the diseases such as UCP2 gene pleiomorphism and coronary heart disease, 2 type diabetes also have dependency.
GNB3 gene in G protein ' beta ' 3 subunit exists polymorphic C825T site.Polymorphism on 825 of GNB3 genes refers to 825 changes that cytidylic acid(CMP) (C) and thymic acid (T) have occurred of the 10th exon in GNB3 gene.Chinese scholars shows a large amount of crowds' C825T alleles analysis research, and polymorphism and the obesity in this site exist dependency.Also find that GNB3 gene 825 loci polymorphisms are relevant with the recovery of pregnant woman's normal type in postpartum.GNB3 genotype is relevant with body fat content, and genotype 825T/T lipid content is significantly higher than genotype 825T/C and 825C/C.
Therefore, set up simply, obesity-prone gene tester fast, the high risk population that examination in time goes out to be liable to obesity, carries out health guidance and prevention targetedly, for ensureing that human security health has very important significance.
Summary of the invention
Based on Gln223Arg, UCP2 gene on C825T, LEPR gene on GNB3 gene-3 SNPs loci gene types of 866G/A can be used to assess obesity onset risk rank, the invention provides a kind of obesity gene detecting kit.
This test kit comprises:
The Auele Specific Primer of 3 SNPs loci gene types of detect on GNB3 gene on C825T, LEPR gene on Gln223Arg, UCP2 gene-866G/A to and DNA sequencing primer.
PCR reaction component (comprising Taq enzyme, dNTP mixed solution, reaction buffer, ddH2O etc.).
PCR product purification assembly (comprising SAP enzyme, ExoI enzyme, ddH2O etc.).
DNA sequencing reaction assembly (comprising BigDye mix, EDTA solution, 100% ethanolic soln, 70% ethanolic soln, HIDI solution, ddH2O etc.).
Component and the content of test kit of the present invention comprise:
PCR reaction system: 10 * PCR reaction buffer 2.5ul; 25mM dNTP mixed solution 0.2ul; 5U/ul Taq archaeal dna polymerase 0.125ul; 20uM Auele Specific Primer is to (every each 0.25ul of primer); DdH2O 19.175ul.
PCR product purification system: 1U/ul SAP enzyme 0.75ul; 10U/ul ExoI enzyme 0.375ul; DdH2O 3.875ul.
Sequencing reaction system: 25%BigDye mix 1ul; 3.2uM DNA sequencing primer 1ul; 125mM EDTA solution 1ul; 100% ethanolic soln 15ul; 70% ethanolic soln 30ul; HIDI solution 8ul; DdH2O 2ul.
This test kit detects for a person-portion, and the storage temperature of test kit is-20 ℃.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Unless otherwise defined, the same meaning that all specialties of using in literary composition and scientific words and one skilled in the art are familiar.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.
The use of embodiment 1. detection kit.
1, extracting DNA profiling
Scraping person under inspection's oral mucosa epithelial cell, with phenol chloroform method extracting genomic dna.
2, pcr amplification reaction
Use the PCR reaction component in detection kit, wherein, contain following primer pair:
(1) GNB3(C825T) forward primer: 5 '-TTCCTGCCGCTTGTTTGA-3 '
GNB3(C825T) reverse primer: 5 '-AAATGCCAGGAACCCAGAA-3 '
(2) LEPR(Gln223Arg) forward primer: 5 '-AAACTCAACGACACTCTCCTT-3 '
LEPR(Gln223Arg) reverse primer: 5 '-ACTGACATTAGAGGTGAC-3 '
(3) UCP2(-866G/A) forward primer: 5 '-CACGCTGCTTCTGCCAGGAC-3 '
UCP2(-866G/A) reverse primer: 5 '-AGGCGTCAGGAGATGGACCG-3 '.
The reaction system of pcr amplification is: 10 * PCR reaction buffer 2.5ml; The dNTP mixed solution 0.2ml of 25mM, 5U/ul Taq enzyme 0.125ml, DNA profiling 1ml (12-15ng left and right), each 0.25ml of 20uM forward primer reverse primer, ddH2O 19.175ml.
Reaction conditions is: 94 ℃ of sex change in 4 minutes and enzyme activate, 94 ℃ of sex change in 30 seconds, and 55 ℃ of annealing in 30 seconds, 72 ℃ extend for 1 minute, circulates 28 times, and last 72 ℃ of prolongations are more than 5 minutes.
3, pcr amplification product purifying
Use the PCR product purification assembly in detection kit, reaction system is cumulative volume 25ul, comprises PCR product 20ul, 1U/ul SAP enzyme 0.75ul, 10U/ul ExoI enzyme 0.375ul, deionized water 3.875ul.
On ABI2720 type pcr amplification instrument, react, reaction conditions is 37 ℃, 15min, 72 ℃, 20min.
4, DNA sequencing reaction
Use the sequencing reaction assembly in detection kit, wherein, contain following DNA sequencing primer:
(1) GNB3(C825T) sequencing primer: 5 '-TTCCTGCCGCTTGTTTGA-3 '
(2) LEPR(Gln223Arg) sequencing primer: 5 '-AAACTCAACGACACTCTCCTT-3 '
(3) UCP2(-866G/A) sequencing primer: 5 '-CACGCTGCTTCTGCCAGGAC-3 '.
The system of reaction is cumulative volume 5ul, comprises PCR purified product 1ul, 25%Bigdye mix1ul, 3.2uM DNA sequencing primer 1ul, deionized water 2ul.
On ABI2720 type pcr amplification instrument, react, reaction conditions is 98 ℃ of 2min, carries out 96 ℃ of 30s of 25 circulations, 55 ℃ of 30s, 60 ℃ of 4min.
After reaction finishes, add 125mM EDTA solution 1ul and 100% ethanolic soln 15ul, under room temperature, precipitate 15min; At 4 ℃, the centrifugal 30min of the rotating speed of 3600rpm/min, removes supernatant liquor gently; Add 70% ethanolic soln 30ul, the centrifugal 15min of 3600rpm/min, removes supernatant liquor gently; Room temperature adds HIDI solution 8ul after placing 20min, puts into sequenator.
5, gene type assay
The those skilled in the art that are familiar with DNA sequencing technology can determine by identification DNA sequencing collection of illustrative plates the genotype in the SNP site of detecting.
Embodiment 2. carries out the service of obesity gene test for crowd.
1. sample and extracting DNA
By hospital laboratory doctor, instruct person under inspection to use oral cavity sampling wiper to carry out mouth epithelial cells sampling, adopt phenol chloroform method to carry out the DNA extracting of mouth epithelial cells.
2. genotype detection
Use test kit provided by the invention, on Gln223Arg, UCP2 gene on C825T, LEPR gene on the GNB3 gene of person under inspection's genomic dna-3 SNPs sites of 866G/A are carried out respectively DNA sequencing, are determined the genotype in these 3 SNPs sites.
3. obesity gene high risk population risk-assessment
By to the genotypic analysis of person under inspection SNPs, provide obesity risk-assessment report.3 SNP locus genes of describe in detail in report on person under inspection GNB3 gene on C825T, LEPR gene on Gln223Arg, UCP2 gene-866G/A detect information and genetic risk assessment result.In addition, according to person under inspection's risk class, and to person under inspection, describe and understand obesity gene test report by doctor in detail.
Claims (7)
1. examination obesity high risk population's a gene noninvasive detection kit, is characterized in that: 3 genotypic Auele Specific Primers of mononucleotide polymorphism site of detect on GNB3 gene on C825T, LEPR gene on Gln223Arg, UCP2 gene-866G/A and DNA detection probe, Taq enzyme, dNTP mixed solution, reaction buffer, SAP enzyme, ExoI enzyme, BigDye mix, EDTA solution, 70% ethanolic soln, 100% ethanolic soln, HIDI solution, ddH
2o etc.
2. test kit according to claim 1, it is characterized in that: described Auele Specific Primer to refer to on Gln223Arg, UCP2 gene on C825T, LEPR gene on GNB3 gene-3 single nucleotide polymorphism (SNPs) site of 866G/A, can specific amplification go out to comprise the primer pair of the DNA fragmentation in these 3 SNPs sites.
3. test kit according to claim 1, it is characterized in that: described DNA sequencing primer be on Gln223Arg, UCP2 gene on C825T, LEPR gene on GNB3 gene-3 SNPs sites of 866G/A and designing, can go out by DNA sequencing technology specific detection the DNA sequencing primer of above-mentioned 3 SNPs loci gene types.
4. according to the test kit shown in claim 1, it is characterized in that, 3 pairs of contained specific primer sequences are as follows:
(1) GNB3(C825T) forward primer: 5 '-TTCCTGCCGCTTGTTTGA-3 '
GNB3(C825T) reverse primer: 5 '-AAATGCCAGGAACCCAGAA-3 '
(2) LEPR(Gln223Arg) forward primer: 5 '-AAACTCAACGACACTCTCCTT-3 '
LEPR(Gln223Arg) reverse primer: 5 '-ACTGACATTAGAGGTGAC-3 '
(3) UCP2(-866G/A) forward primer: 5 '-CACGCTGCTTCTGCCAGGAC-3 '
UCP2(-866G/A) reverse primer: 5 '-AGGCGTCAGGAGATGGACCG-3 '.
5. test kit according to claim 1, is characterized in that, 3 contained DNA sequencing primer sequences are as follows:
(1) GNB3(C825T) sequencing primer: 5 '-TTCCTGCCGCTTGTTTGA-3 '
(2) LEPR(Gln223Arg) sequencing primer: 5 '-AAACTCAACGACACTCTCCTT-3 '
(3) UCP2(-866G/A) sequencing primer: 5 '-CACGCTGCTTCTGCCAGGAC-3 '.
6. test kit according to claim 1, is characterized in that the component of test kit and content comprise:
(1) PCR reaction system: 10 * PCR reaction buffer 2.5ul, 25mM dNTP mixed solution 0.2ul, 5U/ul Taq archaeal dna polymerase 0.125ul, 20uM Auele Specific Primer pair, every each 0.25ul of primer, ddH
2o 19.175ul;
(2) PCR product purification system: 1 U/ul SAP enzyme 0.75ul, 10 U/ul ExoI enzyme 0.375ul, ddH
2o 3.875ul;
(3) sequencing reaction system: 25% BigDye mix1ul, 3.2uM DNA sequencing primer 1ul, 125mM EDTA solution 1ul, 100% ethanolic soln 15ul, 70% ethanolic soln 30ul, HIDI solution 8ul, ddH
2o 2ul.
7. this test kit detects application for a person-portion, and the storage temperature of test kit is-20 ℃.
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| CN201210373799.XA CN103710423A (en) | 2012-10-02 | 2012-10-02 | Susceptible noninvasive detection kit for obesity (genes like LEPR) |
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| CN201210373799.XA CN103710423A (en) | 2012-10-02 | 2012-10-02 | Susceptible noninvasive detection kit for obesity (genes like LEPR) |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834459A (en) * | 2017-01-21 | 2017-06-13 | 张垒 | It is a kind of by detecting rs6046310 come the kit of specific detection obesity |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834459A (en) * | 2017-01-21 | 2017-06-13 | 张垒 | It is a kind of by detecting rs6046310 come the kit of specific detection obesity |
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Application publication date: 20140409 |