CN101240320A - Kit for detecting cardiovascular disease incidence inheritance risk - Google Patents

Abstract

The invention discloses a agent box for detecting individual cardiovascular disease pathogenesis heredity risks. The agent box comprises electrophoresis detection primer pair for detecting number rs4646994 SNP polymorphism gene type on detection angiotensin conversion enzyme gene (ACE), specificity primer pair and specificity fluorescent detecting probe pair for detecting number re5186 SNP site on detection angiotensin II-1type receptor gene (AGTR1), number rs5443 SNP site on G albumen 3 second unit gene (GNB3), and number rs1799983 SNP site on under bark type nitric oxide synthetase gene (NOS3), fluorescent definite quantity PCR general component, PCR reaction component etc.. The agent box of the invention assesses individual cardiovascular disease pathogenesis heredity risks by detecting synchronously mononucleotide polymorphism site gene type on ACE, AGTR1, GNB3 and NOS3 genes correlative closely to individual cardiovascular disease pathogenesis heredity risks.

Description

A kind of test kit that detects cardiovascular disease incidence inheritance 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 cardiovascular disease incidence inheritance risk, assess individual cardiovascular disease incidence inheritance risk by the mononucleotide polymorphism site genotype that detects simultaneously on atp binding cassette transporter body A1 gene (ABCA1), apolipoprotein B gene (APOB), apolipoprotein E gene (APOE) and the lipoprotein lipase gene (LPL).

Background technology

Cardiovascular disorder seizes 1,200 ten thousand people's life every year, near 1/4 of the total death toll of world population, becomes the No.1 formidable enemy of human health.Although over nearly 30 years, the most countries of cardiovascular disorder mortality ratio except that East Bloc all has decline in various degree, but majority state still high ranking first in the deaths in men reason more than 45 years old, then be second cause of the death that is only second to tumour in the women, having a strong impact on human life expectancy and life quality.Along with China steps into aging society, cardiovascular and cerebrovascular diseases is the serious problems that entire society faces.

The expert of WHO points out: although cardiovascular disorder is No.1 killer, if actively develop prevention, can save 6,000,000 people's life every year.No matter be developing country or developed country, prevention is the way of most realistic minimum cost, is the method for keeping fit without medicine and living.And available research achievements shows, the correlative factor of cardiovascular disordeies such as hypertension, coronary heart disease all is subjected to the inherited genetic factors regulation and control.The cardiovascular disease risk difference takes place in the people of different genetic constitutions, and required precautionary approach also there are differences.Therefore, understand the hereditary situation and very necessary of self to the influence of cardiovascular health, also could really accomplish only so with positive, have purpose and effectively intervention means keep cardiovascular systems health, improve quality of life.

Cardiovascular disorder is genetic predisposition and environmental factors results of interaction.It is generally acknowledged that on ratio, inherited genetic factors accounts for 40%, environmental factors accounts for 60%.The inheritance susceptible gene of cardiovascular disorder is mainly angiotensin I converting enzyme gene (ACE), Angiotensin II-1 receptor gene (AGTR1), G albumen 3 subunit genes (GNB3), endothelial type nitric oxide synthase gene (NOS3).

The I/D that studies show that ACE in the crowd of Tibet is polymorphic all to have significant correlation with Tibet women's essential hypertension morbidity, the numerical value of DBP, MAP, and does not find obviously relevant with the male sex's morbidity.In Chinese han population, carried out the Meta analysis to the I/D of ACE is polymorphic, altogether 1612 cases in 18 research are analyzed 1710 contrasts, found that having the genotypic individuality of DD is 1.37 (95%CI=1.15-1.63 to other individual relative risks, P＜0.01), therefore think the I/D of ACE polymorphic be the initiation potential factor of essential hypertension in Chinese han population.Find that in 116 researchs of routine essential hypertension in Chinese Wuhan the DD genotype had remarkable influence (P＜0.05) to the morbidity and the elevation of blood pressure of the male sex's essential hypertension during the I/D of ACE was polymorphic to 91 routine normal controls.Research has in addition shown that also the morbidity with essential hypertension is relevant among a plurality of crowds of the polymorphic each department in China of the I/D of ACE, and wherein the DD genotype can be used as the independent risk factor of essential hypertension.

Angiotensin II-1 receptor receives much attention as the co-channel that the renin-angiotensin system cascade reaction acts on target cell at last.Angiotensin II-1 receptor gene A 1166C polymorphic position is replaced and is formed by C by base A in 5 ' end of 3 ' end non-translational region.This site of this gene is the research focus in hypertension and forward position, coronary heart disease field always, mainly concentrates on aspects such as side effect, myocardial hypertrophy, heart failure such as restenosis after AGTR1 gene polymorphic A1166C and hypertension, coronary heart disease and the operation thereof at present.By studies show that in a large number, AGTR1 gene A C genotype is relevant with essential hypertension, and C allelotrope is hypertensive risk factor in Chinese han population.

The first-generation children of 187 hypertension familys of Daqing Area and 189 non-hypertension familys are carried out the mensuration of indexs such as fasting plasma glucose, fasting insulin, blood fat and Fibrinogen, inquire into the relation of GNB3C825T polymorphism and blood pressure, insulin resistant with multiplicity.The result shows that GNB3C825T genotype and insulin sensitivity, blood pressure level and weight index are related.GNB3C825T allelotrope increases the boosting of insulin resistant, and under similar insulin resistant condition, CT/TT genotype person blood pressure level is higher.

Endothelial type nitric oxide synthase (eNOS) mainly is distributed in the inner membrance of coronary vasodilator and chambers of the heart face, and major function is to participate in arginine and proline(Pro) metabolism, and catalysis generates nitrogen protoxide (NO).NO is a kind of endothelium relaxation factor, has vasodilator, regulates blood flow, suppresses vascular smooth muscle cell proliferation, suppresses critical functions such as thrombocyte and leukocyte adhesion, has participated in the pathophysiological process of multiple disease.192 hypertensive patients and 122 healthy people are studied, find that the genotypic frequency of G894G significantly is lower than normal population among the hyperpietic, illustrate that the G894G genotype has reduced hypertensive susceptibility.

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, to with the support document in cardiovascular disease incidence inheritance risk genes involved site, the research of molecular biology mechanism, after Chinese population suitability etc. is carried out the analysis-by-synthesis assessment, angiotensin I converting enzyme gene (ACE) goes up rs4646994 SNP site, Angiotensin II-1 receptor gene (AGTR1) is gone up rs5186 SNP site, G albumen 3 subunit genes (GNB3) are gone up rs5443 SNP site, endothelial type nitric oxide synthase (NOS3) is gone up rs1799983 SNP site and is assert by national biological gene detection technique application evaluation authentication center, can be used for detecting assessing cardiovascular disease incidence inheritance risk.

Can be used to assess on the basis of individual cardiovascular disease incidence inheritance risk based on the polymorphism in these 4 SNP sites on ACE, AGTR1, GNB3, the NOS3 gene, the invention provides a kind of test kit that detects individual cardiovascular disease incidence inheritance risk.

This test kit comprises:

The genotypic electrophoresis detection primer of rs4646994 SNP polymorphism is right on the detection ACE gene;

The genotypic Auele Specific Primer of rs5186 SNP polymorphism is right to reaching the specificity fluorescent probe on the detection AGTR1 gene;

The genotypic Auele Specific Primer of rs5443 SNP polymorphism is right to reaching the specificity fluorescent probe on the detection GNB3 gene;

The genotypic Auele Specific Primer of rs1799983 SNP polymorphism is right to reaching the specificity fluorescent probe on the detection NOS3 gene;

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

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

Electrophoresis detection primer described in this test kit designs being meant at rs4646994 SNP site on the ACE gene, and it is right to detect the electrophoresis detection primer of this SNP loci gene type by the electrophoretic technique specific amplification.Design this class primer to being that those skilled in the art can be unlabored.Preferably, it is right to comprise the electrophoresis detection primer with sequence shown in SEQ ID NO:1 and 2 in the test kit.The electrophoresis detection primer synthesizes the synthetic technology of available routine.Those skilled in the art will appreciate that electrophoresis detection primer of the present invention to being not limited to this to primer, all can be used for electrophoresis detection primer that electrophoretic technique detects this SNP site to all within the scope of the present invention.

Auele Specific Primer described in this test kit is to being meant at these 3 SNP sites, rs1799983 SNP site on rs5443 SNP site, the NOS3 gene on rs5186 SNP site, the GNB3 gene on the AGTR1 gene, and the primer of dna fragmentation that can specific amplification goes out to comprise these 3 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:3 and 4, the primer of sequence shown in 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 three pairs of primers.

Specificity fluorescent probe described in this test kit designs being meant at these 3 SNP sites, rs1799983 SNP site on rs5443 SNP site, the NOS3 gene on rs5186 SNP site, the GNB3 gene on the AGTR1 gene, and it is right to go out the Taqman probe of these 3 SNP 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 SNP sites 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. electrophoresis detection 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 ACE gene electrophoresis detection primers are to each 0.25 μ l of every primer,

Deionized water 19.175 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: electrophoresis detection somatotype

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

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

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

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 ACE gene electrophoresis detection 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.

Utilize electrophoretic technique then, carry out agarose gel electrophoresis, observe band quantity.

Step 3: 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 '-TGCAGCACTTCAC-3 ' (SEQ ID NO:3) is arranged

Antisense primer 1:5 '-TCCTTCAATTCTGA-3 ' (SEQ ID NO:4)

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

Antisense primer 2:5 '-GGCGGCCACTGAGGG-3 ' (SEQ ID NO:6)

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

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

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

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

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

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

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

Band FAM fluorophor probe 3:5 '-GAtCCCCCAGAAC-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 rs5186 SNP loci polymorphism on the AGTR1 gene;

Adopted primer 2 is arranged, and antisense primer 2, band VIC fluorophor probe 2, band FAM fluorophor probe 2 are specifically at detecting rs5443 SNP loci polymorphism on the GNB3 gene;

Adopted primer 3 is arranged, and antisense primer 3, band VIC fluorophor probe 3, band FAM fluorophor probe 3 are specifically at detecting rs1799983 SNP loci polymorphism on the NOS3 gene;

3 independently quantitative fluorescent PCR reactions are carried out in 3 SNP 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 50C, 2 minutes, and 95C, 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 4: gene type assay

The those skilled in the art that are familiar with the electrophoresis detection technology can determine the genotype in the SNP site detected by the quantity of DNA band on the identification electrophorogram.

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.

2. couples of people of embodiment carry out the service that individual cardiovascular disease incidence inheritance 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, rs4646994 SNP on the ACE gene of detected person's genomic dna is carried out electrophoresis detection in the site, rs1799983 SNP on rs5443 SNP site, the NOS3 gene on rs5186 SNP site, the GNB3 gene on the AGTR1 gene is carried out fluorescence quantitative PCR detection in the site, determine the genotype in these 4 SNP sites.

Step 3: the analysis of individual cardiovascular disease incidence inheritance risk

By to the genotypic analysis of detected person SNP, provide the analysis report list of individual cardiovascular disease incidence inheritance risk.Describe the height of detected person's cardiovascular disease incidence inheritance risk in the report in detail, and describe and understand individual cardiovascular disease incidence inheritance 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 cardiovascular disease incidence inheritance risk

<160>14

<210>1

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>1

CTGGAGACCA?CTCCCATCCT TTCT 24

<210>2

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>2

GATGTGGCCA?TCACATTCGT?CAGAT 25

<210>3

<211>13

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>3

TGCAGCACTT?CAC 13

<210>4

<211>14

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>4

TCCTTCAATT?CTGA 14

<210>5

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>5

AGAGCATCAT?CTGCGGCAT 19

<210>6

<211>15

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>6

GGCGGCCACT?GAGGG 15

<210>7

<211>12

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>7

CCCTGCTGCT?GC 12

<210>8

<211>13

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>8

GGGGCAGAAG?GAA 13

<210>9

<211>15

<212>DNA

＜213〉artificial sequence

<220>

＜223〉probe

<400>9

TGAGCaTTAG?CTACT 15

<210>10

<211>14

<212>DNA

＜213〉artificial sequence

<220>

＜223〉probe

<400>10

TGAGCcTTAG?CTAC 14

<210>11

<211>12

<212>DNA

＜213〉artificial sequence

<220>

＜223〉probe

<400>11

ACGTCcGTGG?CC 12

<210>12

<211>13

<212>DNA

＜213〉artificial sequence

<220>

＜223〉probe

<400>12

ACGTCtGTGG?CCT 13

<210>13

<211>12

<212>DNA

＜213〉artificial sequence

<220>

＜223〉probe

<400>13

AgCCCCCAGA?AC 12

<210>14

<211>13

<212>DNA

＜213〉artificial sequence

<220>

＜223〉probe

<400>14

GAtCCCCCAG?AAC 13

Claims (8)

1. test kit that detects individual cardiovascular disease incidence inheritance risk is characterized in that: comprise detect angiotensin I converting enzyme gene (ACE) go up the genotypic electrophoresis detection primer of rs4646994 SNP polymorphism to, detect Angiotensin II-1 receptor gene (AGTR1) go up rs5186 SNP site, G albumen 3 subunit gene genes (GNB3) go up the Auele Specific Primer of rs5443 SNP site, endothelial type nitric oxide synthase (NOS3) going up rs1799983 SNP site to and the specificity fluorescent probe to, Taq enzyme, dNTP mixed solution, MgCl ₂Solution, quantitative fluorescent PCR reaction buffer, PCR reaction buffer, deionized water etc.

2. test kit according to claim 1, it is characterized in that: described electrophoresis detection primer designs being meant at rs4646994 SNP site on the ACE gene, and it is right to detect the electrophoresis detection primer of this SNP loci gene type by the electrophoretic technique specific amplification.

3. test kit according to claim 1, it is characterized in that: described Auele Specific Primer is to being meant at these 3 SNP sites, rs1799983 SNP site on rs5443 SNP site, the NOS3 gene on rs5186 SNP site, the GNB3 gene on the AGTR1 gene, and the primer of dna fragmentation that can specific amplification goes out to comprise these 3 SNP sites is right.

4. test kit according to claim 1, it is characterized in that: described specificity fluorescent probe designs being meant at these 3 SNP sites, rs1799983 SNP site on rs5443 SNP site, the NOS3 gene on rs5186 SNP site, the GNB3 gene on the AGTR1 gene, and it is right to go out the Taqman probe of these 3 SNP loci gene types by the fluorescent quantitative PCR technique specific detection.

5. test kit according to claim 1 is characterized in that: contained electrophoresis detection primer is right to being selected from the primer with sequence shown in SEQ ID NO:1 and 2.

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

7. 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.

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 0.75 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.

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