CN101131358A - Reagent kit for detecting coronary heart disease susceptibility - Google Patents

Abstract

The invention discloses a kind of reagent box for detection of susceptibility to coronary heart disease. The reagent box comprises of specific primer pairs for the detection of the 146th SNP locus in SEQ ID NO: 1 of PON1 gene, conventional components for fluorescence quantitative PCR. The reagent box by this invention can predict individual susceptibility to coronary heart disease through the detection of individual portable type of SNP locus gene in PON1 gene.

Description

Be used to detect the kit of coronary disease susceptibility

Technical field

The present invention relates to a kind of kit that is used to detect disease susceptibility, especially a kind of kit that detects coronary disease susceptibility, (paraoxonase 1, and mononucleotide polymorphism site PON1) is predicted individual neurological susceptibility to coronary heart disease by detecting paraoxon esterase 1 gene.

Background technology

Coronary atherosclerotic heart disease (coronary atherosclerotic heart disease) refers to that coronary atherosclerosis makes the lumen of vessels stenosis or occlusion, or (with) cause myocardial ischemia-anoxemia or the downright bad heart disease that causes because of the functional change of coronary artery (spasm), general designation coronary cardiopathy (coronary heart disease), be called for short coronary heart disease (CHD), also claim ischemic heart disease (ischemic heartdisease).

Coronary atherosclerotic heart disease is the common type that atherosclerotic causes organ lesion, also is the common disease of serious harm people ' s health.This disease mostly occurred after 40 years old, and the male sex is more than the women.Developed country is common, and the U.S. has 7,000,000 people to suffer from this disease approximately, and ten thousand people die from this disease surplus in the of annual about 50, account for 1/3～1/2 of human mortality's number, account for 50%～75% of deaths from heart disease number.In China, show a rising trend in recent years.The seventies in 20th century, Beijing, Shanghai, this sick human mortality of Guangzhou were respectively 21.7/10 ten thousand populations, 15.7/10 ten thousand populations and 4.1/10 ten thousand populations; The nineties, this sick mortality ratio of China city male sex was 49.2/10 ten thousand populations, and the women is 32.2/10 ten thousand populations.

Cause of coronary heart disease increases with the growth at age, and degree also increases the weight of with the growth at age.There is data to show, from beginning in 40 years old, every increase by 10 years old, prevalence of coronary heart disease increases 1 times.The male sex 50 years old, women are after 60 years old, and the coronary sclerosis development is rapider, and the danger of same miocardial infarction also increases with advancing age.Young male patient is more than young female patient, but postclimacteric women and over 60 women, and it is dangerous just almost to have equated with the male sex, even greater than the male sex.The possibility of the elderly's heart attack is higher.

Its pathogenesis is complicated, and is comprehensive than growth process.Can reduce four steps: 1. cardiovascular endothelial cell infringement, smooth muscle cell proliferation.2. platelet adhesion reaction discharges thrombocytin behind impaired vascular wall, thromboxane A2, blood platelet IV, the VIII factor etc.3. low-density lipoprotein infiltrates subintimal smooth muscle cell by impaired endothelial cell, and is engulfed, and enters to be decomposed by lysosomal hydrolytic enzyme in the cell behind the cell and discharge free cholesterol, and the latter combines with linoleic acid, becomes important morbidity link.4. on above-mentioned pathological change basis, coronary artery fiberization, calcification take place finally.

Coronary heart disease is a kind ofly to have inherent cause and environmental factor to interact and the polygenic disease of morbidity, and the genetic mechanism of seeking coronary heart disease dependent genes and then illustrating incidence of coronary heart disease has become the focus of present research.

PON1 is paraoxonase 1 (paraoxon lipase a 1) encoding gene, and the paraoxon lipase 1 in the serum can be used as antioxidant, and biomacromolecules such as low-density lipoprotein LPL are hydrolyzed and reduce oxidation level.Thereby reduce endovascular oxidation potential, play protective effect blood vessel.After the gene generation defective, causing is hydrolyzed and reduces oxidation level biomacromolecules such as low-density lipoprotein LPL descends, and causes the damage to vascular wall, brings the disease of cardiovascular and cerebrovascular.

SNP (single nucleotide polymorphism), promptly the single nucleotide polymorphism mark is the dna polymorphism that a class causes based on single nucleotide variation, is called third generation genetic marker by hereditary educational circles.Mainly be meant the dna sequence polymorphism that causes by the variation on the genome nucleotide level, comprise single base conversion, transversion, and the insertion/disappearance of single base etc.It is a class polymorphism mark that the most extensively exists in the genome, accounts for about 90%.The variation of these genome sequences can cause the difference of phenotype between individuality and Different Individual to disease, particularly the neurological susceptibility of complex disease and to the difference of environmental factor, drug response.Studies show that, the polymorphism (A/G) in the rs662 SNP site on the PON1 gene (shown in the SEQ ID NO:1 in the sequence 146) can cause that the 192nd amino acid of gene coded protein becomes Arg by Gln, and patients with coronary heart disease has the frequency of Arg apparently higher than normal population, and the Gln192Ar8 polymorphism of PON1 gene and coronary heart disease seriousness, coronary atherosclerosis progress degree, blood lipid level are relevant with the blood lipid-lowering medicine result of treatment.A large amount of association analysis both domestic and external shows, the Gln of PON1 gene polymorphic (the SNP loci gene type is A) can reduce coronary heart disease neurological susceptibility, delay coronary atherosclerosis progress degree.The polymorphism in this SNPs site can be used for assessing individual neurological susceptibility to coronary heart disease, has passed through the authentication of biological gene detection technique application evaluation authentication center of country of the Chinese Academy of Sciences at present.

Summary of the invention

Can be used to assess on the basis of individuality to the neurological susceptibility of coronary heart disease based on the rs662 SNP site on the PON1 gene, the invention provides a kind of kit that is used to detect coronary disease susceptibility.

This kit comprises: the Auele Specific Primer that detects the 146th SNP among the PON1 genes of SEQ ID NO:1 is to reaching specificity fluorescent probe, Taq enzyme, dNTP mixed liquor, MgCl ₂Solution, quantitative fluorescent PCR reaction buffer, deionized water.

Auele Specific Primer described in this kit designs being meant at the 146th SNP site among the SEQ ID NO:1, and it is right that the energy specific amplification goes out the primer of the dna fragmentation that comprises the 146th SNP site among the SEQ ID NO:1.Design this class primer to being that those skilled in the art can be unlabored.Preferably, it is right to comprise the primer with sequence shown in SEQ ID NO:2 and 3 in the kit.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 two pairs of primers.

Specificity fluorescent probe described in this kit is meant the NO at SEQ ID; The 146th SNP site in 1 and designing can go out the probe of this SNP site coronary heart disease tumor susceptibility gene type by the fluorescent quantitative PCR technique specific detection.Designing this class probe is that those skilled in the art can be unlabored.Preferably, comprise Taqman probe in the kit with sequence shown in the SEQ ID NO:4.The specificity fluorescent probe can synthesize with the synthetic technology of routine.Those skilled in the art will appreciate that specificity fluorescent probe of the present invention is not limited to this probe, all probes that can be used for the 146th SNP site among the fluorescence quantitative PCR detection SEQ ID NO:1 all within the scope of the present invention.

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

1 μ l 10X quantitative fluorescent PCR reaction buffer,

0.1 μ l 25mM dNTP mixed liquor,

0.6 μ l 25mM MgCl ₂Solution,

0.025 μ l (5units/ μ l) Taq archaeal dna polymerase,

20 μ M Auele Specific Primers are to (two) each 0.225 μ l,

10 μ M specificity fluorescent probes, 0.25 μ l,

Deionized water 5.575 μ l.

This kit detects for a person-portion and uses, and the storage temperature of 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

Step: the extraction of dna profiling

Genomic DNA with silica gel adsorption extracting mouth epithelial cells.

Step 2: quantitative fluorescent PCR reaction

Use can detect the fluorescent quantificationally PCR detecting kit of coronary disease susceptibility, wherein, contain following primer to and fluorescence probe:

It is 56 ℃ that adopted primer: 5 '-GGACCTGAGCACTTTTATG-3 ' (SEQ ID NO:2) Tm value is arranged

Antisense primer: 5 '-TTGGACTATAGTAGACAACA-3 ' (SEQ ID NO:3) Tm value is 54 ℃

Fluorescence probe: 5 '-CGATCCTGGGAGATGTATTTGG-3 ' (SEQ ID NO:4) Tm value is 64 ℃

The quantitative fluorescent PCR reaction system 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 liquor, the 0.6 μ l 25mM MgCl of 20ng/ μ l ₂The fluorescence probe 0.25 μ l 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, deionized water 5.575 μ l.

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

Step 3:SNP genotyping

The final sample fluorescence volume and the normal control that show on the quantitative real time PCR Instrument are compared, fluorescence signal and normal control that fluorescence probe is final are more or less the same, illustrate that the rs662 SNP loci gene type on the PON1 gene of detected DNA carries the type into A, non-coronary heart disease susceptible type.

Embodiment 2. instructs people initiatively to prevent the service of coronary heart disease

Carried out this service in Shanghai Zhujian Biological Engineering Co., Ltd at present.

Step 1:DNA extracts

The detected person is served preceding consulting, sign Informed Consent Form, fill in living and diet custom questionnaire by the detected person.Using the oral cavity sampling to wipe away according to the indication in the sampling box carries out the mouth epithelial cells sampling, adopts silica gel adsorption to carry out the DNA extracting of mouth epithelial cells.

Step 2: Genotyping detects

Use kit provided by the invention, fluorescence quantitative PCR detection is carried out in the rs662 SNP site on the PON1 gene of detected person DNA, determine the genotype in this SNP site.

Step 3: instruct people initiatively to prevent coronary heart disease

By to the genotypic analysis of detected person SNP, provide the report of Genotyping examining report and detected person's individuation health guidance.The genetic test report describes the height of detected person's coronary heart disease susceptible degree and ill probability size in detail.The report of individuation health guidance is based on detected person's Genotyping testing result, in conjunction with its living and diet custom survey result, the relative risk of assessment detected person coronary heart disease inheritance susceptible.Make the personalized healthy action scheme that is directed to the detected person simultaneously, scheme is included in the recommendation on improvement on eating habit, the life style etc., and popularizes the health knowledge of prevention coronary heart disease for the detected person.

Sequence table

＜110〉Shanghai Zhujian Biological Engineering Co., Ltd

＜120〉be used to detect the kit of coronary disease susceptibility

<160>4

<210>1

<211>300

<212>DNA

＜213〉Genus Homo, and ethnic group (Homo sapiens, human)

<400>1

ccagttaata?atcctgtaat?gttcaatacc?ttcaccttat?atattatgtg?tgtatgtttt 60

aattgcagtt?tgaatgatat?tgttgctgtg?ggacctgagc?acttttatgg?cacaaatgat 120

cactattttc?ttgaccccta?cttacaatcc?tgggagatgt?atttgggttt?agcgtggtcg 180

tatgttgtct?actatagtcc?aagtgaagtt?cgagtggtgg?cagaaggatt?tgattttgct 240

aatggaatca?acatttcacc?cgatggcaag?tatgtgaact?ctctgaaatg?tagtggattt 300

<210>2

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>2

ggacctgagc?acttttatg 19

<210>3

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>3

ttggactata?gtagacaaca 20

<210>4

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉probe

<400>4

cgatcctggg?agatgtattt?gg 22。

Claims (6)

1. a kit that is used to detect coronary disease susceptibility is characterized in that: comprise that the Auele Specific Primer that detects the 146th SNP site among the PON1 genes of SEQ ID NO:1 is to reaching specificity fluorescent probe, Taq enzyme, dNTP mixed liquor, MgCl ₂Solution, quantitative fluorescent PCR reaction buffer, deionized water.

2. kit according to claim 1, it is characterized in that: described Auele Specific Primer designs being meant at the 146th SNP site among the SEQ ID NO:1, and it is right that the energy specific amplification goes out to comprise the primer of the dna fragmentation in the 146th SNP site among the SEQ ID NO:1.

3. kit according to claim 1, it is characterized in that: described specificity fluorescent probe is meant at the 146th SNP site among the SEQ ID NO:1 and designs, can go out the probe of this SNP site coronary heart disease tumor susceptibility gene type by the fluorescent quantitative PCR technique specific detection.

4. kit according to claim 1 is characterized in that: contained Auele Specific Primer is right to being selected from the primer with sequence shown in SEQ ID NO:2 and 3.

5. kit according to claim 1 is characterized in that: contained specificity fluorescent probe is selected from the Taqman probe with sequence shown in the SEQ ID NO:4.

6. kit according to claim 1 is characterized in that: the component of kit and content comprise 1 μ l, 10 X quantitative fluorescent PCR reaction buffers, 0.1 μ l 25mM dNTP mixed liquor, 0.6 μ l 25mM MgCl ₂Solution, 0.025 μ l (5units/ μ l) Taq archaeal dna polymerase, 20 μ M Auele Specific Primers are to (two) each 0.225 μ l, 10 μ M specificity fluorescent probes, 0.25 μ l, deionized water 5.575 μ l.This kit detects for a person-portion and uses, and the storage temperature of kit is-20 ℃.