CN101240327A - Kit for detecting child fatness inheritance susceptible risk - Google Patents

Abstract

The invention discloses an agent box for detecting baby fatness impressionable risks. The agent box comprises specificity primer pair and DNA sequencing primer for detecting number rs429358 SNP site and rs7412 SNP site on apolipoprotein E gene (APOE), specificity primer pair and specificity fluorescent detecting probe pair for detecting number rs320 SNP site on lipoproteins lipase gene (LPL), number rs13306517 SNP site on thin element gene (LEP), number rs1801282 SNP site on peroxisome hyperplasy activate receptor Gamma (PPAR Gamma) gene, fluorescent definite quantity PCR general component, PCR reaction component, PCR outcome yield cleanse component ANDDNA sequencing reaction component etc.. The agent box of the invention assesses baby fatness impressionable risks by detecting synchronously mononucleotide polymorphism site gene type on APOE, LPL, LEP and PPAR Gamma genes correlative closely to baby fatness impressionable risks.

Description

A kind of test kit that detects child fatness inheritance susceptible risk

Technical field

The present invention relates to molecular biology and medical field, more specifically, the present invention relates to a kind of test kit that detects child fatness inheritance susceptible risk, assess child fatness inheritance susceptible risk by the mononucleotide polymorphism site genotype that detects simultaneously on apolipoprotein E gene (APOE), lipoprotein lipase gene (LPL), leptin (LEP) encoding gene and the peroxysome hyperplasia activated receptor γ gene (PPAR γ).

Background technology

Obesity is a kind of heat metabolic disturbance, takes in heat and surpasses the heat that consumes, cause that body fat gathers too much due to.It is generally acknowledged to surpass and be obesity by the accurate body weight of height (length) institute's mark 20%.At present, China's obesity of childhood sickness rate has ascendant trend year by year, and from bringing up to below 5% more than 10%, the obeisity sickness rate reaches 5%-8%.Puppy fat and many Adulthood chronic diseases such as hypertension, hyperlipidemia, diabetes, atherosclerotic cardiovascular and cerebrovascular diseases have very substantial connection, and obesity causes the morbidity of these diseases and mortality ratio sharply to rise, and the population health level is descended.Therefore, correctly be familiar with puppy fat disease, from the juvenile set about the phase prevention and treatment of obesity very important.

The puppy fat cause of disease mainly contains: it is too much to take in heat; The very few heat exhaustion of moving is few; Hereditation; Nervous center is regulated; The endocrine metabolism imbalance; Psychological factor.A lot of to the gene research of obesity in recent years, human obesity belongs to polygenic inheritance, and heredity plays an easily outbreak usefulness in the obesity morbidity, interact with other pathogenic factorss.Known, four kinds of genes and the puppy fat of coding apo E (APOE), lipoprotein lipase (LPL), leptin (LEP) and peroxysome hyperplasia activated receptor γ (PPAR γ) have substantial connection.

APOE is the apoprotein of a main chylomicron, can with the receptors bind on liver cell or the peripheral cell, be very important for the proteic normal katabolism of triglyceride level.The modal three kinds of hypotypes of APOE albumen are APOE2 ,-E3 and-E4, constitute by three kinds of haplotype ε 2, ε 3, ε 4 respectively.The difference of these three kinds of hypotypes on amino acid coding is mainly in two sites, and site A is No. 130 amino-acid residue, and site B is No. 176 amino-acid residue.Wherein the E3 hypotype is the most common hypotype, is defined as the wild-type of APOE gene.The SNP of Cys130Arg correspondence is rs429358, and base is changed to T471C; The SNP of Cys176Arg correspondence is rs7412, and base is changed to T609C.Sanghera DK etc. are by discovering that to one group of 9-10 year young girl's sample the APOE genetic polymorphism has a significant effect to the relation of blood lipid level and obese degree and blood lipid level.Xiang Wei etc. adopt the fat and healthy juvenile APOE genotype of the polymerase chain reaction 2 restriction fragment length polymorphism methods analysts of improvement, find that also the fat juvenile of simple form has the variation of APOE gene pleiomorphism.Zhang Xiao etc. have detected 324 8 ~ 12 years old simple fat juveniles' blood fat, measure body weight and height, calculate BMI, use polymerase chain reaction,PCR to detect their APOE genotype simultaneously, find that the APOE gene pleiomorphism may have certain influence to the relation of simple fat juvenile BMI and blood fat.

Lpl gene encoding apolipoprotein lipase, this enzyme is expressed in heart, muscle and fatty tissue, with the proteic form functionating of homodimer, its molecular biological effect comprises two portions, a part is the lytic enzyme effect of triglyceride, and another part then is the bridge of receptor-mediated lipoprotein endocytosis.Site mutation on the LPL albumen is not only closely related with lipoprotein metabolism, blood lipid level, but also relevant with the fat susceptibility of individuality.Lipoprotein lipase LPL mainly participates in the lipid metabolism path in vivo, as the hydrolysis of katalaze enzyme catalysis vldl or low-density lipoprotein.The rs320 site is be positioned at No. 8 intron of lpl gene polymorphic, to the change of G original HindIII restriction enzyme site is disappeared by T.Jemaa etc. are to 236 routine Paris, FRA obese patients (the 162 routine women and the 74 routine male sex, actual measurement body weight＞120% ideal body weight) analysis of lpl gene polymorphism shows, HindIII polymorphism relevant with BMI (P＜0.05), and the variation of this gene increases individual fat susceptibility.Li Rong etc. in 2003 by discovering, the measurement index of reflection central obesities such as fat group H+H+ genotype person waistline, VA is significantly higher than non-H+H+ genotype person, illustrates that the centrality accumulation of lpl gene Hind III polymorphism and obese people fat has relation.

Leptin (Leptin) claim the fat suppression element again, is the coded protein of ob gene (as ob).Leptin is a kind of by fatty tissue excretory polypeptide, participates in a plurality of energy metabolisms in the body.Its acceptor is positioned on the film of target cells such as hypothalamus and adipocyte; It is to be a huge receptor family that the latter has corresponding signal transduction body.Leptin is brought into play following extensively physiological function by leptin receptor: depress appetite, and minimizing is ingested; Change the autonomic nerve activity, increase metabolic rate, impel body temperature rise, reduce fat stores; Reduce plasma glucose content, insulin secretion is descended, body weight and energy i (in vivo) store and descend, and avoid obesity, insulin resistant, diabetes and sterile; Combine with the specificity transport protein,, transfer a series of nerve-body fluid factors, regulate energy metabolism, body fat and body weight are controlled on the relative constant level to hypothalamus carrier fat information.Rs13306517 is that to be positioned at a G132A of ob gene polymorphic, causes that the protein 25 position Gln synonym of ob genes encoding is replaced, and important dependency may be arranged with the activity of leptin, thereby relevant with the obese degree of individuality.Discovering Japanese population such as Ohshiro Y in 2000, in 53 routine obese individuals, the allelic frequency of occurrences of leptin 25CAG is apparently higher than 132 routine non-obese individuals, therefore think that leptin gene 25CAG allelotrope may be relevant with the generation of obesity, infer that perhaps it can become a new genetic marker of Japanese population even world crowd obesity susceptibility.Wu Hua etc. in the research of the sudden change of leptin gene and puppy fat disease to the 197 routine juveniles of Han nationality, regular 68 examples of recombinating wherein, slight fat group 81 examples, in/the fat group of severe 48 examples, analyze.Result of study be presented at regular reorganization with in/severe is fat organize between the distributional difference of the genotype frequency in Codn25 site and gene frequency all exist significance (P=0.000, P=0.001); Among leptin gene Codn25 (CAA/CAG) sudden change and the juvenile of Han nationality/and there is dependency in the severe obesity, and may there be the difference of obese degree in this genovariation to the influence of Han nationality's puppy fat susceptibility.

PPAR γ full name peroxysome hyperplasia activated receptor γ, encoded protein belongs to nuclear receptor peroxysome hyperplasia activated receptor subfamily.The PPAR family member has mainly found three kinds of hypotype: α, δ, three kinds of hypotypes of γ at present.PPAR γ encoded protein is the γ hypotype, mainly the metabolic differentiation of responsible lipid and sugar, lipid metabolism etc.Rs1801282 is that to be positioned on No. three karyomit(e) a C/G on the PPAR γ gene polymorphic, causes that the 12nd amino acid of albumen of PPAR γ genes encoding becomes Ala (A) by Pro (P).A plurality of abroad colonies, find in the research of fat case-control above 2000 pairs, polymorphic and the fat relevant a plurality of indexes of A12P on PPAR γ 2 genes as: BMI, waist-to-hipratio etc. has relevant, and pointing out this polymorphic site is important obesity generation related locus.Find in the investigation of Beamer to the Caucasia obese people that the A12 haplotype has higher weight index, and think that this site may be relevant with the multifactor fat syndromes that causes of the mankind.People such as Zhang Defeng discover in the obese individuals that to extracting 104 routine non-obese individuals and 104 routine obese individuals among the Shijiazhuang area health check-up crowd it is to cause that the Serum Leptin Levels secretion is increased, obesity has one of inherited genetic factors of substantial connection that PPAR γ 2 genes have the Ala12 sudden change.Lu Jin etc. measure peroxisome proliferator activated receptor γ 22 (the PPAR γ 22) genotype of 232 routine Chinese populations with PCR2RFLP, find in the fat group (130 example), carry allelotrope Ala12 person weight index (BM I), hip circumference and uric acid and all be significantly higher than Pro homozygote group, and this polymorphism is independent relevant with BMI.Point out this polymorphism overweight with Chinese population and fat relevant.

Summary of the invention

" Chinese population health service gene locus authentication rules " according to the promulgation of national biological gene detection technique application evaluation authentication center, support document to the gene locus relevant with the puppy fat susceptibility, the research of molecular biology mechanism, after Chinese population suitability etc. is carried out the analysis-by-synthesis assessment, rs429358 SNP site and rs7412 SNP site on the APOE gene, rs320 SNP site on the lpl gene, on the LEP gene on rs13306517 SNP site and the PPAR γ gene rs1801282 SNP site assert by national biological gene detection technique application evaluation authentication center, can be used for detecting the assessment child fatness inheritance susceptible risk.

Polymorphism based on these 5 SNP sites on APOE, LPL, LEP, the PPAR γ gene can be used to assess on the basis of child fatness inheritance susceptible risk, the invention provides a kind of test kit that detects child fatness inheritance susceptible risk.

This test kit comprises:

Detect on the APOE gene rs429358 number with the genotypic Auele Specific Primer of rs7412 SNP polymorphism to and the dna sequencing primer;

The genotypic Auele Specific Primer of rs320 SNP polymorphism is right to reaching the specificity fluorescent probe on the detection lpl gene;

The genotypic Auele Specific Primer of rs13306517 SNP polymorphism is right to reaching the specificity fluorescent probe on the detection LEP gene;

The genotypic Auele Specific Primer of rs1801282 SNP polymorphism is right to reaching the specificity fluorescent probe on the detection PPAR γ gene;

The quantitative fluorescent PCR reaction component (comprises Taq enzyme, dNTP mixed solution, MgCl ₂Solution, quantitative fluorescent PCR reaction buffer, deionized water etc.).

The PCR reaction component (comprises Taq enzyme, dNTP mixed solution, MgCl ₂Solution, PCR reaction buffer, deionized water etc.);

PCR product purification assembly (comprising SAP enzyme, Exo I enzyme, deionized water etc.);

Dna sequencing reaction assembly (comprising BigDye mix, EDTA solution, 70% ethanolic soln, 100% ethanolic soln, HIDI solution, deionized water etc.).

Auele Specific Primer described in this test kit is to being meant at these 5 SNP sites, and the primer of dna fragmentation that can specific amplification goes out to comprise these 5 SNP sites is right.Design this class primer to being that those skilled in the art can be unlabored.Preferably, comprise in the test kit have SEQ ID NO:1 and 2, the primer of sequence shown in 3 and 4,5 and 6,7 and 8 is right.Auele Specific Primer synthesizes the synthetic technology of available routine.Those skilled in the art will appreciate that primer of the present invention is not limited to this four pairs of primers.

Specificity fluorescent probe described in this test kit designs being meant at these 3 SNP sites on LPL, LEP, the PPAR γ gene, and it is right to go out the Taqman probe of these 3 SNPs loci gene types by the fluorescent quantitative PCR technique specific detection.Designing this class probe is that those skilled in the art can be unlabored.Preferably, comprise in the test kit have SEQ ID NO:9 and 10, the Taqman probe of sequence shown in 11 and 12,13 and 14 is right.The specificity fluorescent probe synthesizes the probe synthetic technology of available routine.Those skilled in the art will appreciate that specificity fluorescent Taqman probe of the present invention to being not limited to this three pairs of probes, all can be used for probe that fluorescence quantitative PCR method detects these 3 SNPs sites all within the scope of the present invention.

Dna sequencing primer described in this test kit is meant at rs429358 SNP site on the APOE gene and rs7412 SNP site and designs, can go out the dna sequencing primer of these 2 SNPs loci gene types by dna sequencing technology specific detection.Designing this class primer is that those skilled in the art can be unlabored.Preferably, comprise dna sequencing primer in the test kit with sequence shown in the SEQ ID NO:15.The dna sequencing primer can synthesize with the synthetic technology of routine.Those skilled in the art will appreciate that dna sequencing primer of the present invention is not limited to this primer, all dna sequencing primers that can be used for these 2 SNPs sites of dna sequencing technology for detection all within the scope of the present invention.

The component and the content of test kit of the present invention comprise:

1. quantitative fluorescent PCR reacts suit

10X quantitative fluorescent PCR reaction buffer 3 μ l,

25mM dNTP mixed solution 0.3 μ l,

25mM MgCl2 solution 1.8 μ l,

5units/ μ l Taq archaeal dna polymerase 0.075 μ l,

20 μ M Auele Specific Primers are to each 0.225 μ l of every primer,

10 μ M specificity fluorescent probes are to each 0.25 μ l of every probe,

Deionized water 15.975 μ l.

2.PCR reaction suit

10X PCR reaction buffer 2.5 μ l,

25mM dNTP mixed solution 0.2 μ l,

25mM MgCl ₂Solution 1.5 μ l,

5units/ μ l Taq archaeal dna polymerase 0.125 μ l,

20 μ M APOE gene-specific primers are to each 0.25 μ l of every primer,

Deionized water 19.175 μ l.

3.PCR product purification suit

Lunits/ μ l SAP enzyme 0.75 μ l,

10units/ μ l ExoI enzyme 0.375 μ l,

Deionized water 3.875 μ l.

4. sequencing reaction suit

25％BigDye?mix?1μl，

3.2 μ M dna sequencing primer 1 μ l,

125mM EDTA solution 1 μ l,

100% ethanolic soln, 15 μ l,

70% ethanolic soln, 30 μ l,

HIDI solution 8 μ l,

Deionized water 2 μ l.

This test kit detects for a person-portion and uses, and the storage temperature of test kit is-20 ℃.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.

The use of embodiment 1. detection kit

The extraction of step 1:DNA template

Genomic dna with silica gel adsorption extracting mouth epithelial cells.

Step 2: quantitative fluorescent PCR reaction

Use the quantitative fluorescent PCR suit in the detection kit, wherein, contain following primer right with fluorescent probe:

Adopted primer 1:5 '-AGGGTGAGATTCCAAGATAA-3 ' (SEQ ID NO:1) is arranged

Antisense primer 1:5 '-CAAGCAAATGACTAAAGAGAA-3 ' (SEQ ID NO:2)

Adopted primer 2: 5 '-TCTATGTCCAAGCTGTG-3 ' (SEQ ID NO:3) is arranged

Antisense primer 2:5 '-GGGTTTTGGTGTCATC-3 ' (SEQ ID NO:4)

Adopted primer 3:5 '-AACTCTGGGAGATTCTC-3 ' (SEQ ID NO:5) is arranged

Antisense primer 3:5 '-TATCAGTGAAGGAATCG-3 ' (SEQ ID NO:6)

Band VIC fluorophor probe 1:5 '-GGATTTAAAGCgTTT-3 ' (SEQ ID NO:9)

Band FAM fluorophor probe 1:5 '-AGGATTTAAAGCtTT-3 ' (SEQ ID NO:10)

Band VIC fluorophor probe 2:5 '-ATCCAaAAAGTCCA-3 ' (SEQ ID NO:11)

Band FAM fluorophor probe 2:5 '-TCCAgAAAGTCCA-3 ' (SEQ ID NO:12)

Band VIC fluorophor probe 3:5 '-GACcCAGAAAG-3 ' (SEQ ID NO:13)

Band FAM fluorophor probe 3:5 '-GACgCAGAAAG-3 ' (SEQ ID NO:14)

Adopted primer 1 is arranged, and antisense primer 1, band VIC fluorophor probe 1, band FAM fluorophor probe 1 are specifically at detecting rs320 SNP loci polymorphism on the lpl gene;

Adopted primer 2 is arranged, and antisense primer 2, band VIC fluorophor probe 2, band FAM fluorophor probe 2 are specifically at detecting rs13306517 SNP loci polymorphism on the LEP gene;

Adopted primer 3 is arranged, and antisense primer 3, band VIC fluorophor probe 3, band FAM fluorophor probe 3 are specifically at detecting rs1801282 SNP loci polymorphism on the PPAR γ gene;

3 independently quantitative fluorescent PCR reactions are carried out in 3 SNPs sites respectively, the system of each reaction is cumulative volume 10 μ l, and comprising concentration is dna profiling 2 μ l, 1 μ l 10X quantitative fluorescent PCR reaction buffer, 0.1 μ l 25mM dNTP mixed solution, the 0.6 μ l 25mM MgCl of 20ng/ μ l ₂The band VIC fluorescent probe that adopted primer and antisense primer each 0.225 μ l, 10 μ M are arranged of solution, 0.025 μ l (5units/ μ l) Taq archaeal dna polymerase, 20 μ M and each 0.25 μ l of band FAM fluorescent probe, deionized water 5.325 μ l.

React on ABI9700 type pcr amplification instrument, reaction conditions is 50 ℃, 2 minutes, 95 ℃, 10 minutes, carries out 95 ℃ of 60 round-robin, 30 seconds, 60 ℃, 1 minute.Reaction finishes the back and read the fluorescent amount on ABI7900 type quantitative real time PCR Instrument.

Step 3:PCR amplified reaction

Use the PCR reaction suit in the detection kit, wherein, it is right to contain following primer:

Adopted primer: 5 '-TAAGCTTGCACGGCTGTCCAAGGA-3 ' (SEQ ID NO:7) is arranged

Antisense primer: 5 '-ACAGAATTCGCCCCGGCCTGGTACAC-3 ' (SEQ ID NO:8)

The system of reaction is cumulative volume 25 μ l, comprising concentration is dna profiling 1 μ l, the 10X PCR reaction buffer 2.5 μ l of 12.5ng/ μ l, 25mMdNTP mixed solution 0.2 μ l, 25mM MgCl2 solution 1.5 μ l, 5units/ μ l Taq archaeal dna polymerase 0.125 μ l, 20 μ M APOE Auele Specific Primers are to each 0.25 μ l of every primer, deionized water 19.175 μ l.

React on ABI2720 type pcr amplification instrument, reaction conditions is 94 ℃, 12 minutes, carries out 94 ℃ of 30 round-robin, 30 seconds, 60 ℃, 30 seconds, 72 ℃, 30 seconds, carries out then 72 ℃, 10 minutes.

Step 4:PCR product purification

Use the PCR product purification suit in the detection kit, reaction system is cumulative volume 25 μ l, comprises PCR product 20 μ l in the step 3,1units/ μ l SAP enzyme 0.75 μ l, 10units/ μ l ExoI enzyme 0.375 μ l, deionized water 3.875 μ l.

React on ABI2720 type pcr amplification instrument, reaction conditions is 37 ℃, 15 minutes, 72 ℃, 20 minutes.

Step 5:DNA sequencing reaction

Use the sequencing reaction suit in the detection kit, wherein, contain the dna sequencing primer of following APOE gene:

Dna sequencing primer: 5 '-TAAGCTTGCACGGCTGTCCAAGGA-3 ' (SEQ ID NO:15)

The system of reaction is cumulative volume 5 μ l, comprises PCR purified product 1 μ l in the step 4,25%BigDye mix 1 μ l, 3.2 μ M dna sequencing primers, 1 μ l, deionized water 2 μ l.

React on ABI2720 type pcr amplification instrument, reaction conditions is 98 ℃, 2 minutes, carries out 96 ℃ of 25 round-robin, 30 seconds, 55 ℃, 30 seconds, and 60 ℃, 4 minutes.

Reaction finishes the back and adds 125mM EDTA solution 1 μ l and 100% ethanolic soln, 15 μ l, and precipitation is 15 minutes under room temperature; Centrifugal 30 minutes of 4 ℃ of rotating speeds, remove supernatant liquor gently with 3650 rev/mins; Add 70% ethanolic soln, 30 μ l, with 3650 rev/mins rotating speed centrifugal 15 minutes, remove supernatant liquor gently; Room temperature is placed and is added HIDI solution 8 μ l after 20 minutes, puts into sequenator.

Step 6: gene type assay

The those skilled in the art that are familiar with fluorescent quantitative PCR technique can be by the final sample fluorescence volume that shows on the identification quantitative real time PCR Instrument, can determine the genotype in the SNP site detected according to the power of different sequence probe VIC and FAM fluorescent signal.

The those skilled in the art that are familiar with the dna sequencing technology can be by the genotype in the definite SNP site of being detected of identification dna sequencing collection of illustrative plates.

Embodiment 2. carries out the service that child fatness inheritance susceptible risk detects

Step 1:DNA extracts

Instructing the examinee to use the oral cavity sampling to wipe away by the hospital laboratory doctor carries out the mouth epithelial cells sampling, adopts silica gel adsorption to carry out the DNA extracting of mouth epithelial cells.

Step 2: genotype tests

Use test kit provided by the invention, the dna sequencing detection is carried out in rs429358 SNP site on the APOE gene and rs7412 SNP site, fluorescence quantitative PCR detection is carried out in rs1801282 SNP site on rs13306517 SNP site, the PPAR γ gene on rs320 SNP site, the LEP gene on the lpl gene of detected person's genomic dna, determine the genotype in these 5 SNPs sites.

Step 3: the analysis of juvenile's obesity in pubescence susceptible inheritance risk

By to the genotypic analysis of detected person SNPs, provide the analysis report list of child fatness inheritance susceptible risk.Describe the height of detected child fatness inheritance susceptible risk in the report in detail, and describe and understand child fatness inheritance susceptible risk analysis report list in detail to the examinee by the doctor.

Sequence table

＜110〉Shanghai Zhujian Biological Engineering Co., Ltd

＜120〉a kind of test kit that detects child fatness inheritance susceptible risk

<160>15

<210>1

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>1

AGGGTGAGAT?TCCAAGATAA 20

<210>2

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>2

CAAGCAAATG?ACTAAAGAGA?A 21

<210>3

<211>17

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>3

TCTATGTCCA?AGCTGTG 17

<210>4

<211>16

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>4

GGGTTTTGGT?GTCATC 16

<210>5

<211>17

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>5

AACTCTGGGA?GATTCTC 17

<210>6

<211>17

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>6

TATCAGTGAA?GGAATCG 17

<210>7

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>7

TAAGCTTGCA?CGGCTGTCCA?AGGA 24

<210>8

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>8

ACAGAATTCG?CCCCGGCCTG?GTACAC 26

<210>9

<211>15

<212>DNA

＜213〉artificial sequence

<220>

＜223〉probe

<400>9

GGATTTAAAG?CgTTT 15

<210>10

<211>15

<212>DNA

＜213〉artificial sequence

<220>

＜223〉probe

<400>10

AGGATTTAAA?GCtTT 15

<210>11

<211>14

<212>DNA

＜213〉artificial sequence

<220>

＜223〉probe

<400>11

ATCCAaAAAG?TCCA 14

<210>12

<211>13

<212>DNA

＜213〉artificial sequence

<220>

＜223〉probe

<400>12

TCCAgAAAGT?CCA 13

<210>13

<211>11

<212>DNA

＜213〉artificial sequence

<220>

＜223〉probe

<400>13

GACcCAGAAA?G 11

<210>14

<211>11

<212>DNA

＜213〉artificial sequence

<220>

＜223〉probe

<400>14

GACgCAGAAA?G 11

<210>15

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>15

TAAGCTTGCA?CGGCTGTCCA?AGGA 24

Claims (8)

1. test kit that detects child fatness inheritance susceptible risk is characterized in that: comprise detect Auele Specific Primer that apolipoprotein E gene (APOE) goes up rs429358 SNP site and rs7412 SNP site to and the dna sequencing primer, detect lipoprotein lipase gene (LPL) go up on rs320 SNP site, leptin (LEP) gene on the rs13306517 SNP site and peroxysome hyperplasia activated receptor γ (PPAR γ) gene the Auele Specific Primer in rs1801282 SNP site to and the specificity fluorescent probe to, Taq enzyme, dNTP mixed solution, MgCl ₂Solution, quantitative fluorescent PCR reaction buffer, PCR reaction buffer, SAP enzyme, ExoI enzyme, BigDye mix, EDTA solution, 70% ethanolic soln, 100% ethanolic soln, HIDI solution, deionized water etc.

2. test kit according to claim 1, it is characterized in that: described Auele Specific Primer is to being meant that going up rs429358 SNP site and rs7412 SNP site, lipoprotein lipase gene (LPL) at apolipoprotein E gene (APOE) goes up that on the rs13306517 SNP site and peroxysome hyperplasia activated receptor γ (PPAR γ) gene rs1801282 SNP site on rs320 SNP site, leptin (LEP) gene, and the primer of dna fragmentation that can specific amplification goes out to comprise these 5 SNP sites is right.

3. test kit according to claim 1, it is characterized in that: described specificity fluorescent probe is gone up on rs320 SNP site, leptin (LEP) gene rs1801282 SNP site on the rs13306517 SNP site and peroxysome hyperplasia activated receptor γ (PPAR γ) gene and is designed being meant at lipoprotein lipase gene (LPL), and it is right to go out the Taqman probe of these 3 SNPs loci gene types by the fluorescent quantitative PCR technique specific detection.

4. test kit according to claim 1, it is characterized in that: described dna sequencing primer is meant at rs429358 SNP site on the APOE gene and rs7412 SNP site and designs, can go out the dna sequencing primer of these 2 SNPs loci gene types by dna sequencing technology specific detection.

5. test kit according to claim 1 is characterized in that: contained Auele Specific Primer has SEQ ID NO:1 and 2 to being selected from, the primer of sequence shown in 3 and 4,5 and 6,7 and 8 is right.

6. test kit according to claim 1 is characterized in that: contained specificity fluorescent probe be selected from have SEQ ID NO:9 and 10, the Taqman probe of sequence shown in 11 and 12,13 and 14 is right.

7. test kit according to claim 1 is characterized in that: contained dna sequencing primer is selected from the primer with sequence shown in the SEQ ID NO:15.

8. test kit according to claim 1 is characterized in that the component of test kit and content comprise:

1) quantitative fluorescent PCR reaction suit: 10X quantitative fluorescent PCR reaction buffer 3 μ l, 25mM dNTP mixed solution 0.3 μ l, 25mM MgCl2 solution 1.8 μ l, 5units/ μ l Taq archaeal dna polymerase 0.075 μ l, 20 μ M Auele Specific Primers are to each 0.225 μ l of every primer, 10 μ M specificity fluorescent probes are to each 0.25 μ l of every probe, deionized water 15.975 μ l.

2) PCR reaction suit: 10X PCR reaction buffer 2.5 μ l, 25mM dNTP mixed solution 0.2 μ l, 25mM MgCl2 solution 1.5 μ l, 5units/ μ l Taq archaeal dna polymerase 0.125 μ l, 20 μ M Auele Specific Primers are to each 0.25 μ l of every primer, deionized water 19.175 μ l.

3) PCR product purification suit: 1units/ μ l SAP enzyme 0.75 μ l, 10units/ μ l ExoI enzyme 0.375 μ l, deionized water 3.875 μ l.

4) sequencing reaction suit: 25%BigDye mix 1 μ l, 3.2 μ M dna sequencing primers, 1 μ l, 125mM EDTA solution 1 μ l, 100% ethanolic soln, 15 μ l, 70% ethanolic soln, 30 μ l, HIDI solution 8 μ l, deionized water 2 μ l.

This test kit detects for a person-portion and uses, and the storage temperature of test kit is-20 ℃.