CN101173310A - Reagent kit for detecting hyperlipemia disease susceptibility with LPL - Google Patents

Abstract

The invention discloses a kit detecting hyperlipidemia susceptibility, comprising a probe detecting the specific primers and the specific fluorescence of the one hundred and eighteen SNP site in CETP gene SEQ ID NO: 1, and a normal component detecting the fluorescent quantification PCR. The invented kit predicts the susceptibility of an individual to hyperlipidemia by detecting and analyzing the SNP site genotype on the individual CETP gene.

Description

Detect the test kit of hyperlipemia disease susceptibility by LPL

Technical field

The present invention relates to the test kit that a kind of disease susceptibility detects usefulness, especially a kind of test kit that detects hyperlipemia disease susceptibility, (lipoprotein lipase, individual susceptibility to hyperlipidaemia is predicted in single nucleotide polymorphism LPL) (SNP) site by detecting lipoprotein lipase gene.

Background technology

Normally be called hyperlipidaemia (hyperlipidemia) because metabolism of fat or running make in the blood plasma one or more lipids be higher than unusually, can show as hypercholesterolemia (hyperchoesterolemia), hypertriglyceridemia (hypertriglyceridemia) or both have (combined hyperlipidemia familial) concurrently.Lipid is insoluble or be slightly soluble in water, must exist with the lipoprotein form with protein bound, could turn round in blood circulation, and therefore, hyperlipidaemia often is the reflection of hyperlipoproteinemia (hyperlipoproteinemia).Owing to recognize that gradually it also is a kind of metabolism disorder of blood lipid that blood plasma middle-high density lipoprotein reduces, thereby it is more comprehensive, accurate to be referred to as hyperlipemia (dyslipidemia).WHO is divided into five big classes with hyperlipoproteinemia at present, is mainly the phenotypic classification that carries out according to the degree difference of plasma lipoprotein rising: familial hyperchylomicronemia (familial hypertriglyceridemia), familial hypercholesterolemia (familial hyperbetalipoproteinemia), familial type 3 hyperlipoproteinemia, hyper pre-, mix mode hypertriglyceridemia (mix mode hyperlipidaemia).

The protein part of lipoprotein is a kind of special albumen, because of combine the function of undertaking in blood plasma running lipid with lipid, so be called lipophorin (apoprotein).Lipophorin forms water-soluble substances except combining with lipid, becomes beyond the carrier of transhipment lipid, also has other specific function, and especially participate in the enzyme active and regulate, and the identification and the association reaction that participate in lipoprotein and cell-membrane receptor.Lipoprotein in the blood plasma is microgranular, and core is mainly triglyceride level and cholesteryl ester, and skin is made of phosphatide, cholesterol, lipophorin.Water-soluble enzyme can enter internal layer through the top layer and work.The metabolism of lipoprotein has two approach: 1. exogenous metabolism approach: be meant cholesterol and triglyceride level synthetic CM and metabolic process thereof in small intestine that diet is taken in; 2. endogenous metabolism approach: be meant by liver synthetic VLDL and change IDL and LDL into, and LDL is by liver or the metabolic process of other organs.Wherein chylomicron CM can not enter in the arterial wall, general atherogenicity not, but easily bring out pancreatitis.It is the Hazard Factor of coronary heart disease that blood plasma vldl VLDL level raises.Low-density lipoprotein LDL enters arterial wall and oxidized modification easily, is main atherogenic factor.The rising of high-density lipoprotein (HDL) HDL level helps peripheral tissues and removes cholesterol, prevents that atherosclerosis from taking place.

Hyperlipidaemia (hyperlipoproteinemia) can be divided into two classes clinically: 1. primary belongs to heredity disorders of lipid metabolism disease; 2. Secondary cases is common in the bad diabetes of control, drinks, hypothyroidism, nephrotic syndrome, dialysis, renal transplantation, biliary obstruction, oral contraceptive etc.Hyperlipemia and cardiovascular diseases especially with the generation of coronary heart disease with develop closely relatedly, are one of moietys of metabolic syndrome.

The known portions Primary hyperlipemia is by due to the congenital genetic flaw, can cause familial hypercholesterolemia as the ldl receptor gene defective.

And the common Secondary cases hyperlipidaemia cause of disease has following several:

1, hypercholesterolemia: diabetes, nephrotic syndrome, hypothyroidism, Cushing syndromes;

2, hypertriglyceridemia: diabetes (control), nephrotic syndrome, uremia (during dialysis), obesity, estrin treatment, glycogen storage disease (I type), drink, systemic lupus erythematous, unusual gamma globulin mass formed by blood stasis, gout;

3, high dyslipoproteinemia: the liver that a variety of causes causes inside and outside obstruction of biliary tract, cholestasis liver and bladder disease, comprise intrahepatic cholangiolitic hepatitis, cholehepatocirrhosis.

Population of China blood fat mean level (ML) is lower than developed country, but its rising amplitude is very surprising.China is to the 9 groups of crowds in different areas, from early 1980s to the end of the nineties 20 in the period of carried out the investigation of 3 comparabilities, the result shows serum total cholesterol (TC)＞5.20mmol/L (200mg/d1), the male sicken rate rises to 33% continuously by 17%, and women's morbidity rises to 32% continuously by 9%.Therefore, actively detecting, prevent and controlling hyperlipemia becomes one of main contents of developed area cardiovascular diseases prevention work.

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 susceptibility of complex disease and to the difference of environmental factors, drug reaction.

The LPL protein coding gene is positioned at chromosomal 8p22 section, full length gene 28.0kb, mRNA total length 3550bp No. 8, have 10 exons, encoded protein contains 476 amino acid, encoding apolipoprotein lipase, and this enzyme is expressed in heart, muscle and fatty tissue.LPL is with the proteic form functionating of homodimer, and its molecular biological effect comprises two portions, and 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 can cause I type hyperlipoproteinemia under serious situation, light degree then causes the proteometabolic disorder of body lactones.Rs320 site on the lpl gene (shown in the SEQ ID NO:1 in the sequence 118) is be positioned at No. 8 No. 8 intron of lipoprotein lipase LPL on the karyomit(e) 8p22 polymorphic, to the change of G original HindIII restriction enzyme site is disappeared by T.The HindIII site that studies show that LPL both at home and abroad is relevant with blood lipid level.The association analysis of the Chinese population of large sample (totally 3000 many cases) result shows that the individuality that has the H-haplotype has the high density lipoprotein cholesterol level of lower triglyceride level and Geng Gao, may be the individual haplotype that is not subjected to hyperlipemia harm of protection.

A large amount of association analysis both domestic and external shows, can the triglyceride reducing level when rs320 SNP loci gene type is for G on the lpl gene, and the individual harm that is not subjected to hyperlipemia of protection.The polymorphism in this SNP site can be used for assessing individual susceptibility to hyperlipidaemia, 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

Polymorphism based on rs320 SNP site on the lpl gene can be used for assessing on the basis of individuality to the susceptibility of hyperlipidaemia, the invention provides a kind of test kit that detects hyperlipemia disease susceptibility.

This test kit comprises: the Auele Specific Primer that detects the 118th SNP among the lpl gene SEQ ID NO:1 is to reaching specificity fluorescent probe, Taq enzyme, dNTP mixed solution, MgCl ₂Solution, quantitative fluorescent PCR reaction buffer, deionized water.

Auele Specific Primer described in this test kit designs being meant at the 118th 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 118th 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 test 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 to primer.

Specificity fluorescent probe described in this test kit is meant at the 118th SNP site among the SEQ ID NO:1 and designs, can go out the probe of this SNP site hyperlipidaemia 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 test 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 118th 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 test kit of the present invention comprise:

1 μ l 10X quantitative fluorescent PCR reaction buffer,

0.1 μ l 25mM dNTP mixed solution,

0.5 μ l 25mM MgCl ₂Solution,

0.02 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 5.68 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 can detect the fluorescent quantificationally PCR detecting kit of hyperlipemia disease susceptibility, wherein, contain following primer to and fluorescent probe:

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

Antisense primer: 5 '-CAAGCAAATGACTAAAGAGAA-3 ' (SEQ ID NO:3) Tm value is 56 ℃

Fluorescent probe: 5 '-CGCTATAGGATTTAAAGCTTTTATA-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 solution, the 0.5 μ l 25mM MgCl of 20ng/ μ l ₂The fluorescent probe 0.25 μ l that adopted primer and antisense primer each 0.225 μ l, 10 μ M are arranged of solution, 0.02 μ l (5units/ μ l) Taq archaeal dna polymerase, 20 μ M, deionized water 5.68 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:SNP gene type assay

The final sample fluorescence volume and the normal control that show on the quantitative real time PCR Instrument are compared, the final fluorescent signal of fluorescent probe is apparently higher than normal control, illustrate that rs320 SNP loci gene type carries the T type on the lpl gene of detected DNA, be hyperlipidaemia susceptible type.Embodiment 2. instructs the initiatively service of preventing hyperlipidemia of people

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: genotype tests

Use test kit provided by the invention, fluorescence quantitative PCR detection is carried out in rs320 SNP site on the lpl gene of detected person DNA simultaneously, determine the genotype in this SNP site.

Step 3: instruct initiatively preventing hyperlipidemia of people

By to the genotypic analysis of detected person SNP, provide genotype tests report and the report of detected person's individuation health guidance.The gene test report describes the height of detected person's hyperlipidaemia susceptible degree and ill probability size in detail.The report of individuation health guidance is based on detected person's genotype tests result, in conjunction with its living and diet custom survey result, the relative risk of assessment detected person hyperlipidaemia 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 food habits, the mode of life etc., and popularizes the health knowledge of preventing hyperlipidemia for the detected person.

Sequence table

＜110〉Shanghai Zhujian Biological Engineering Co., Ltd

＜120〉detect the test kit of hyperlipemia disease susceptibility by LPL

<160>4

<210>1

<211>300

<212>DNA

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

<400>1

tcaaaccaac?ctcttcaaga?agggtgagat?tccaagataa?tctcaacctg?tctccgcagc 60

cccacccatg?tgtacccata?aaatgaatta?cacagagatc?gctataggat?ttaaagcttt 120

tatactaaat?gtgctgggat?tttgcaaact?atagtgtgct?gttattgtta?atttaaaaaa 180

actctaagtt?aggattgaca?aattatttct?ctttagtcat?ttgcttgtat?caccaaagaa 240

gcaaacaaac?aaacaaaaaa?aaaaagaaaa?agatcttggg?gatggaaatg?ttataaagaa 300

<210>2

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>2

agggtgagat?tccaagataa 20

<210>3

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>3

caagcaaatg?actaaagaga?a 21

<210>4

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉probe

<400>4

cgctatagga?tttaaagctt?ttata 25

Claims (6)

1. test kit that detects hyperlipemia disease susceptibility comprises: the Auele Specific Primer that detects the 118th SNP site among the lpl gene SEQ ID NO:1 to and specificity fluorescent probe, Taq enzyme, dNTP mixed solution, MgCl ₂Solution, quantitative fluorescent PCR reaction buffer, deionized water.

2. test kit according to claim 1, it is characterized in that: described Auele Specific Primer designs being meant at the 118th 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 118th SNP site among the SEQ ID NO:1.

3. test kit according to claim 1, it is characterized in that: described specificity fluorescent probe is meant at the 118th SNP site among the SEQ ID NO:1 and designs, can go out the probe of this SNP site hyperlipidaemia tumor susceptibility gene type by the fluorescent quantitative PCR technique specific detection.

4. test 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. test 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. test kit according to claim 1 is characterized in that: the component of test kit and content comprise 1 μ l 10X quantitative fluorescent PCR reaction buffer, 0.1 μ l 25mM dNTP mixed solution, 0.5 μ l 25mM MgCl ₂Solution, 0.02 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 5.68 μ l.This test kit detects for a person-portion and uses, and the storage temperature of test kit is-20 ℃.