CN108048560A - A kind of kit, application method and application for detecting T1 gene rs1047896 polymorphic sites - Google Patents
A kind of kit, application method and application for detecting T1 gene rs1047896 polymorphic sites Download PDFInfo
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Abstract
The present invention discloses a kind of kit, application method and application for detecting T1 gene rs1047896 polymorphic sites, belongs to field of biomedicine technology.It finds that rs1047896 polymorphic sites influence T1 protein expressions and related to coronary heart disease for the first time, a kind of kit of detection T1 gene rs1047896 polymorphic sites is provided according to this discovery, including specific primer to, specific alleles fluorescence probe etc..The kit is using the genomic DNA of detected sample cell as template, the primer and probe sequence provided using kit, it determines the genotype of rs1047896 polymorphic sites, the risk of coronary heart disease, prevention and auxiliary diagnosis available for coronary heart disease is then predicted according to the value-at-risk of offer.
Description
Technical field
The present invention relates to a kind of kit, application method and applications for detecting T1 gene rs1047896 polymorphic sites, belong to
Field of biomedicine technology.
Background technology
Angiocardiopathy is to be only second to No. second killer of malignant tumour, is murderous one of the main reasons.At present
There are about 2.3 hundred million people in China to suffer from the cardiovascular diseases such as coronary heart disease, cerebral apoplexy, heart failure and hypertension.It is annual to die of cardiovascular patient
Nearly 3,000,000 people.Coronary heart disease is main angiocardiopathy, finds the early warning marker of this kind of disease for angiocardiopathy
Prevention it is significant.
Association study of the mononucleotide polymorphic (SNP) between coronary heart disease is the pre- of searching predicting susceptibility of coronary heart disease at present
One of main method of alert marker.SNP refers to DNA sequence polymorphism caused by the horizontal single nucleotide acid variation of genomic DNA.
SNP is widely present in normal population.The mankind race on genomic dna sequence more than 99% sequence be all it is identical,
SNP is the main performance of the hereditary difference between individual.SNP can be in chromosome blotting, DNA modification, RNA secondary structures, albumen
Expression and the activity of gene are influenced in multiple levels such as space structure.In addition SNP genetic stabilities are good, are easy to analyze, therefore SNP
As the outstanding feature object of Disease Warning Mechanism.At present find more with the relevant SNP of coronary heart disease, for the individual of following coronary heart disease
Changing medical, accurate medicine, it is necessary to act on.
Recent study find brain-derived neurotrophic factor and its receptor tyrosine kinase receptor B (TrkB) accesses except
It plays a significant role in the existence, growth and maintenance of maincenter and peripheral neurons outer, also expresses and play in cardiovascular system
Effect.Research shows that the access can promote myocardial viability, promotes the angiogenesis in embryonic development and the heart after adult heart infarction
Intramuscular angiogenesis.Our research finds TrkB great expressions in the aorta inner skin of people and mouse, by promoting endothelium
The synthesis of cadherin maintains endothelium complete.Our previous studies also found the blood plasma brain source property god of patients with unstable angina
Control group is substantially less than through nutrition factor concentration, and high blood plasma brain-derived neurotrophic factor concentration indicates the low acute heart
Vascular events incidence and the low death rate prompt the access to play a significant role in the morbidity and prognosis of coronary heart disease.We
This result cause U.S. cardiopulmonary blood research institute Framingham cardiac studies Chief of Centre Vasan S.Ramachandran
Interest, then he Mendel's randomized clinical of large sample has been led to study (Kaess BM, et al.J Am Heart
Assoc.2015；4:E001544), our conclusion, i.e. brain-derived neurotrophic factor access and coronary heart disease are further demonstrated
There are causalities.
T1 albumen is the truncation expression-form of nerves within the body tyrosine kinase receptor 2 (NTRK2), is brain-derived neurotrophy
Another receptor of the factor.T1 and TrkB possesses identical extracellular domain, but has different Intracellular domains, lacks the intracellular of TrkB
Kinases area, therefore can but be unable to molecule in activating cell with the extracellular brain-derived neurotrophic factor of TrkB competitive bindings and lead to
Road.The nao-yi-an granule of TrkB albumen high expression during being embryonic development；And in manhood, TrkB albumen tables
It is substantially reduced up to compared to during embryonic development, and T1 albumen becomes the main expression of receptor form of brain-derived neurotrophic factor.
Future for coronary heart disease Personalized medicine there is an urgent need to largely with the relevant SNP of coronary heart disease, our early-stage studies it has been found that
It is related to coronary heart disease to influence a polymorphic site of TrkB protein expression levels, but at present still not on T1 polymorphic sites and hat
The associated research report of worry.
The content of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of reagents for detecting T1 gene rs1047896 polymorphic sites
Box, application method and application, are found that the function polymorphic site of an influence T1 protein expression for the first time, which is
Rs1047896 polymorphic sites, positioned at the 3 ' non-translational regions (UTR) of T1mRNA, and the polymorphic site is related to coronary heart disease, can use
In the neurological susceptibility of prediction coronary heart disease and the Personalized medicine of following coronary heart disease.
Technical scheme is as follows：
A kind of kit for detecting T1 gene rs1047896 polymorphic sites, including following reagent：10 × PCR buffer solutions,
10mMdNTP mixed liquors, 5U/ μ L Taq DNA polymerases, specific primer is to T1-F, T1-R, T allele fluorescence probes T1-
T, C allele fluorescence probe T1-C, ddH2O；
Wherein, specific primer designs the upstream and downstream respectively in T1 gene rs1047896 polymorphic sites, amplification production
Object corresponds to the nucleotide sequence of genetic fragment as shown in SEQ ID NO.1, the nucleotide sequence such as SEQ ID of sense primer T1-F
Shown in NO.2, the nucleotide sequence of anti-sense primer T1-R is as shown in SEQ ID NO.3；
Wherein, allele fluorescence probe special target rs1047896 polymorphic sites, T allele fluorescence probes T1-T
Fluorescent reporter group for FAM, the fluorescent reporter group of quenching group TAMRA, C allele fluorescence probe T1-C is HEX,
Quenching group is TAMRA；The nucleotide sequence of T allele fluorescence probes T1-T is as shown in SEQ ID NO.4, C allele
The nucleotide sequence of fluorescence probe T1-C is as shown in SEQ ID NO.5.
10 × PCR buffer solutions, dNTP mixed liquors, Taq DNA polymerase are commercial product in mentioned reagent box.
Preferred according to the present invention, the kit forms of the detection T1 gene rs1047896 polymorphic sites are as follows：
100 μ L 10 × PCR buffer solutions；
100 μ L 10mMdNTP mixed liquors；
50 μ L Taq DNA polymerases, concentration 5U/ μ L；
50 μ L T1-F primers, concentration are 10 μM；
50 μ L T1-R primers, concentration are 10 μM；
50 μ L probe T1-T, concentration are 10 μM；
50 μ L probe T1-C, concentration are 10 μM；
500μL ddH2O。
A kind of application method for detecting T1 gene rs1047896 polymorphic site kits, includes the following steps：
(1) genomic DNA of detected sample cell is extracted；
(2) using the kit of detection T1 gene rs1047896 polymorphic sites, reaction system is prepared, using real-time quantitative
PCR combination Taqman probe techniques detect FAM passages and HEX channel fluorescence values；
(3) the FAM passages of detection in step (2) and HEX channel fluorescences value are substituted into Excel and makes scatter diagram, wherein
It is TT genotype along X-axis distribution, is CC genotype along Y-axis distribution, it is CT heterozygotes to be diagonally distributed.
Preferred according to the present invention, detected sample is body fluid in above-mentioned steps (1), including peripheral blood, aqueous humor, and amniotic fluid,
Ascites, pericardial fluid and pleural effusion.
Preferred according to the present invention, the reaction system in above-mentioned steps (2) is：10 × PCR buffer solutions 2 μ L, 10mMdNTP
2 μ L, 5U/ μ L Taq DNA polymerases of mixed liquor 1 μ L, 10 μM of T1-F primers 1 μ L, 10 μM of T1-R primers 1 μ L, 10 μM of probe T1-
T 1 μ L, 10 μM of 1 μ L of probe T1-C, genomic DNA 2 μ L, ddH2O complements to 20 μ L；Real-time quantitative PCR detects program：95
DEG C pre-degeneration 30 seconds；95 DEG C are denatured 0 second, and 60 DEG C are annealed 5 seconds, and 72 DEG C extend 10 seconds, repeat 35 Xun Huans, and Xun Huan is terminated each time
Detect fluorescence；Fluorescence is detected after total overall reaction, with color compensation correction to program fluorescent value.
Application of the mentioned reagent box in predicting susceptibility of coronary heart disease, includes the following steps：
(1) detected sample is collected, extracts the genomic DNA of sample cell；
(2) kit of application detection T1 gene rs1047896 polymorphic sites, prepares 20 μ L reaction systems, using real-time
Quantitative PCR combination Taqman probe techniques detect FAM passages and HEX channel fluorescence values, determine the base of rs1047896 polymorphic sites
Because of type；
(3) coronary disease susceptibility is predicted：The genotype TT homozygotes of rs1047896 polymorphic sites are miscellaneous compared with CC homozygotes and CT
The risk that zygote suffers from coronary heart disease dramatically increases, relative risk 1.56.
Preferred according to the present invention, detected sample is body fluid in above-mentioned steps (1), it may include peripheral blood, aqueous humor, sheep
Water, ascites, pericardial fluid and pleural effusion.
Preferred according to the present invention, 20 μ L reaction systems of the real-time quantitative PCR in above-mentioned steps (2) are：10 × PCR delays
2 μ L, 10mM dNTP mixed liquors of fliud flushing, 2 μ L, 5U/ μ L Taq DNA polymerases 1 μ L, 10 μM of T1-F primers 1 μ L, 10 μM of T1-R draw
Object 1 μ L, 10 μM of probe T1-T 1 μ L, 10 μM of 1 μ L of probe T1-C, genomic DNA 2 μ L, ddH2O complements to 20 μ L；It is fixed in real time
Amount PCR amplification program be：95 DEG C of pre-degenerations 30 seconds；95 DEG C are denatured 0 second, and 60 DEG C are annealed 5 seconds, and 72 DEG C extend 10 seconds, repeat 35
A cycling cycles detection of end fluorescence each time；Fluorescence is detected after total overall reaction, with color compensation correction to program fluorescence
Value.
Preferred according to the present invention, the definite method of the genotype of rs1047896 polymorphic sites is in above-mentioned steps (2)：
The FAM passages of detection in step (2) and HEX channel fluorescences value are substituted into Excel and make scatter diagram, wherein being distributed along X-axis
It is TT genotype, is CC genotype along Y-axis distribution, it is CT heterozygotes to be diagonally distributed.
Preferred according to the present invention, the genotype TT homozygotes of rs1047896 polymorphic sites are pure compared with CC in above-mentioned steps (3)
The risk that zygote and CT heterozygotes suffer from coronary heart disease dramatically increases, relative risk 1.56.
Advantageous effect：
1st, the present invention provides a kind of detection T1 bases for the function polymorphic site rs1047896 for influencing T1 protein expressions
Because of the kit of rs1047896 polymorphic sites, rs1047896 polymorphic sites are located at the 3 ' non-translational regions of T1mRNA, the kit
Including being used to expand the specific primer comprising rs1047896 polymorphic sites and the equipotential base for marking different genotype
Because of fluorescence probe, the genotype of T1 gene rs1047896 polymorphic sites can be fast and accurately detected.
2nd, due to influencing the function polymorphic site rs1047896 of T1 protein expressions and the correlation of coronary heart disease, institute of the present invention
State the kit of detection T1 gene rs1047896 polymorphic sites, prevention, auxiliary diagnosis available for coronary heart disease.
Description of the drawings:
Fig. 1 is that the allele of rs1047896 polymorphic sites differentiates collection of illustrative plates；
Wherein：It is TT genotype along X-axis distribution, is CC genotype along Y-axis distribution, is diagonally distributed
For CT heterozygotes；
Fig. 2 is the influence of luciferase reporter vector schematic diagram and rs1047896 polymorphic sites to protein expression.
Specific embodiment
Technical scheme is further described with reference to embodiment and Figure of description, but is simultaneously not only limited
In this, if wherein the experimental method used without specified otherwise is conventional method.
Agents useful for same and drug of the present invention are ordinary commercial products.Wherein, 10 × PCR buffer solutions, 10mMdNTP mixing
Liquid and Taq DNA polymerase are purchased from Dalian treasured biotech firm；Roche high-fidelity Taq enzyme is purchased from Roche Holding Ag of the U.S., Dual-Luciferase
Reporting system kit is purchased from Promega companies, and liposome 2000 is purchased from Invitrogen companies.
Embodiment 1：A kind of kit for detecting T1 gene rs1047896 polymorphic sites
A kind of kit for detecting T1 gene rs1047896 polymorphic sites, including following reagent：10 × PCR buffer solutions,
10mMdNTP mixed liquors, the Taq DNA polymerase of 5U/ μ L, specific primer is to T1-F, T1-R, T allele fluorescence probes T1-
T, C allele fluorescence probe T1-C, ddH2O；
Wherein, T1 gene rs1047896 polymorphic sites to be detected, positioned at the 3 ' non-translational regions (UTR) of T1mRNA, PCR
The nucleotide sequence (SEQ ID NO.1) that amplified production corresponds to genetic fragment is as follows, is rs1047896 polymorphic sites in box：
attaaaa ttgacctgca aagttaaaaa aaaattaaag ttgagaacag gtataagtgc
acactgaata gtctaatcta catgtaacac atattttagt tgattttct atactctaat cagcactgaa
ttcagagggt ttgacttttt catctataac acagtgacta aaagagttaa gggtatatat accatcactt
tgggacttgg t
Specific primer is as follows to sequence：
Sense primer T1-F：5'-ACCAAGTCCCAAAGTGAT-3'(SEQ ID NO.2),
Anti-sense primer T1-R：5'-ATTAAAATTGACCTGCAAAG-3'(SEQ ID NO.3)；
Allele fluorescence probe sequence is as follows：
T allele T1-T：FAM-GAAAATCAtACTAAAATAT-TAMRA (SEQ ID NO.4),
C allele T1-C：HEX-GAAAATCAcACTAAAATAT-TAMRA (SEQ ID NO.5),
Wherein, FAM is the fluorescent reporter group of T allele fluorescence probes T1-T, and TAMRA is quenching group, HEX C
The fluorescent reporter group of allele fluorescence probe T1-C, TAMRA are quenching group.
Embodiment 2：A kind of application for detecting T1 gene rs1047896 polymorphic site kits
CHD group：Blood sample number is 1460, non-CHD group：Blood sample number is 1640.
DNA extraction method：Take 1 milliliter of blood sample, EDTA anti-freezings；10000rpm centrifuges 5min；It is inhaled with pasteur pipet
Leukocytic cream is taken into a new EP pipes；0.2%NaCl to 900 μ L is added in, overturns mixing；10000rpm centrifuges 5min；It goes
Clearly, precipitation is resuspended in 10mM Tris-HCI and the 10mM EDTA for adding in 100 μ L；300 μ L write cell lysis buffers are added in (containing 10%
SDS, 25mg/mL Proteinase K and 10mg/mL RNaseA), mixing is overturned, 37 DEG C of water-baths are stayed overnight；With chloroform 2 times, water intaking
Phase；1/3 volume 3M sodium acetate solutions are added in, 2 times of cold absolute ethyl alcohols of volume overturn mixing, precipitate DNA；It is heavy to be washed with 70% ethyl alcohol
It forms sediment 2 times；With TE buffer solution DNA, then with spectrophotometric determination DNA concentration；The DNA concentration for adjusting all samples is
30ng/ μ L, -20 DEG C of preservations.
Using detection T1 gene rs1047896 polymorphic site kits, rs1047896 polymorphic sites in above-mentioned sample are detected
Genotype is as follows：
1st, according to above-mentioned sample size, according to following table system, overall reaction liquid is configured, is then dispensed into Roche capillary,
The 2 μ L of genomic DNA of the above-mentioned sample of extraction are separately added into, is closed the lid, is centrifuged and reaction solution is thrown to tube bottom, it is glimmering with Roche
The fluorescent value of Fluorescent Quantitative PCR instrument, upper machine testing FAM passages and HEX passages；
1 real-time quantitative PCR reaction system of table
2nd, PCR detections program is：
95 DEG C of pre-degenerations 30 seconds；95 DEG C are denatured 0 second, and 60 DEG C are annealed 5 seconds, and 72 DEG C extend 10 seconds, repeat 35 Xun Huans, each
Secondary Xun Huan detection of end fluorescence；Fluorescence is detected after total overall reaction, with color compensation correction to program fluorescent value.
3rd, the FAM passages detected and HEX channel fluorescences value are substituted into Excel and makes scatter diagram, generate allele
Differentiate collection of illustrative plates, as shown in Fig. 2, three kinds of different genotypes are located at different position in a coordinate system, wherein it is TT to be distributed along X-axis
Genotype is CC genotype along Y-axis distribution, and it is TC heterozygotes (Fig. 1) to be diagonally distributed.
Genotype frequency and Gene frequency distribution are as shown in table 2：
2 genotype frequency of table and Gene frequency distribution situation
All genotype frequencies of the results show all meet the Hardy-Weinberg laws of genetic equilibrium, and statistical efficiency is
100%.The gene frequency and genotype frequency of rs1047896 gene polymorphics in CHD group and non-CHD group significantly not
Together, the more non-CHD group of TT genotype individuals substantially increases (P in CHD group<0.05).
Embodiment 3：The correlation detection of rs1047896 gene polymorphics and coronary heart disease
Statistical analysis：The comparison of the gene frequency of case-control and genotype frequency difference and genetic equilibrium in table 2
It examines using Chi-square Test.It evaluates the relation of genotype and coronary heart disease and likelihood when 95% is calculated using logistic regression analyses
Credibility interval.The group difference of continuous variable is examined with Student t.Classified variable compares using Chi-square Test.All statistics
Detection sets bilateral P<0.05 is with significant difference.All 15.0 softwares of statistical analysis application SPSS (SPSS,
Chicago,IL)。
Statistical result：
1) the basic clinical data of control-case is as shown in table 3：
Table 3 compares the-basic clinical data of case
2) rs1047896 gene polymorphics and coronary heart disease are significantly correlated
Logistic regression analysis is the result shows that eliminating gender, age and body mass index, blood pressure, blood fat and blood
After the influence of the Confounding factors such as sugar, the polymorphic TT genotype of rs1047896 is an independent hazard factor of coronary heart disease, TT homozygotes
It compares the risk that CC homozygotes and CT heterozygotes suffer from coronary heart disease to dramatically increase, relative risk is 1.56 (1.28-1.91), such as table
Shown in 4：
Table 4
Embodiment 4：Influence of the rs1047896 polymorphic sites to T1 protein expression levels
Using Roche high-fidelity Taq enzyme, the 3 ' UTR areas in PCR amplification T1 genes gene polymorphic containing rs1047896 site.PCR
Amplified production generates pGL3-T1UTR plasmids into pGL3 plasmid vectors through digestion rear clone.Direct mutagenesis generation is carried out respectively to contain
The reporter plasmid of rs1047896 sites C bases and the T bases of site containing rs1047896.
Renilla luciferase control plasmid and purpose plasmid co-transfection correct transfection efficiency between different specimens as internal reference
Difference；Transfected respectively with liposome 2000 pGL3-T1UTR-C and pGL3-T1UTR-T reporter plasmids to HeLa cells 48 it is small when
Afterwards, luciferase substrate is added in and with the chemiluminescence Function detection result of multi-function microplate reader.Experimental result use weighs three times
The average value tested again.
The result shows that the uciferase activity of pGL3-T1UTR-C groups is substantially less than pGL3-T1UTR-T in HeLa cells
Group (Fig. 2), illustrates that pGL3-T1UTR-C is compared with pGL3-T1UTR-T, and the expression of T1 albumen significantly reduces.
SEQUENCE LISTING
<110>Shandong Qilu Hospital
<120>A kind of kit, application method and application for detecting T1 gene rs1047896 polymorphic sites
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 208
<212> DNA
<213> Homo sapiens
<400> 1
attaaaattg acctgcaaag ttaaaaaaaa attaaagttg agaacaggta taagtgcaca 60
ctgaatagtc taatctacat gtaacacata ttttagtgtg attttctata ctctaatcag 120
cactgaattc agagggtttg actttttcat ctataacaca gtgactaaaa gagttaaggg 180
tatatatacc atcactttgg gacttggt 208
<210> 2
<211> 18
<212> DNA
<213>It is artificial synthesized
<400> 2
accaagtccc aaagtgat 18
<210> 3
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 3
attaaaattg acctgcaaag 20
<210> 4
<211> 19
<212> DNA
<213>It is artificial synthesized
<400> 4
gaaaatcata ctaaaatat 19
<210> 5
<211> 19
<212> DNA
<213>It is artificial synthesized
<400> 5
gaaaatcaca ctaaaatat 19
Claims (10)
1. a kind of kit for detecting T1 gene rs1047896 polymorphic sites, including following reagent：10 × PCR buffer solutions,
10mMdNTP mixed liquors, 5U/ μ L Taq DNA polymerases, specific primer is to T1-F, T1-R, T allele fluorescence probes T1-
T, C allele fluorescence probe T1-C, ddH2O；
Wherein, specific primer designs the upstream and downstream respectively in T1 gene rs1047896 polymorphic sites, amplified production pair
The nucleotide sequence of genetic fragment is answered as shown in SEQ ID NO.1, the nucleotide sequence such as SEQ ID NO.2 of sense primer T1-F
Shown, the nucleotide sequence of anti-sense primer T1-R is as shown in SEQ ID NO.3；
Wherein, allele fluorescence probe special target rs1047896 polymorphic sites, T allele fluorescence probes T1-T's is glimmering
Light reporter group is FAM, and the fluorescent reporter group of quenching group TAMRA, C allele fluorescence probe T1-C is HEX, is quenched
Group is TAMRA；The nucleotide sequence of T allele fluorescence probes T1-T is as shown in SEQ ID NO.4, C allele fluorescence
The nucleotide sequence of probe T1-C is as shown in SEQ ID NO.5.
2. kit as described in claim 1, which is characterized in that 10 × PCR buffer solutions, dNTP mixed liquors, TaqDNA polymerizations
Enzyme is commercial product.
3. kit as described in claim 1, which is characterized in that the examination of the detection T1 gene rs1047896 polymorphic sites
Agent box composition is as follows：
100 μ L 10 × PCR buffer solutions；
100 μ L 10mMdNTP mixed liquors；
50 μ L Taq DNA polymerases, concentration 5U/ μ L；
50 μ L T1-F primers, concentration are 10 μM；
50 μ L T1-R primers, concentration are 10 μM；
50 μ L probe T1-T, concentration are 10 μM；
50 μ L probe T1-C, concentration are 10 μM；
500μL ddH2O。
4. a kind of application method for detecting T1 gene rs1047896 polymorphic site kits, includes the following steps：
(1) genomic DNA of detected sample cell is extracted；
(2) using the kit of detection T1 gene rs1047896 polymorphic sites, reaction system is prepared, using real-time quantitative PCR knot
Close Taqman probe techniques detection FAM passages and HEX channel fluorescence values；
(3) the FAM passages of detection and HEX channel fluorescences value it will substitute into Excel and make scatter diagram in step (2), wherein along X
Axis distribution is TT genotype, is CC genotype along Y-axis distribution, it is CT heterozygotes to be diagonally distributed.
5. application method as claimed in claim 4, which is characterized in that detected sample is body fluid in step (1), including periphery
Blood, aqueous humor, amniotic fluid, ascites, pericardial fluid and pleural effusion.
6. application method as claimed in claim 4, which is characterized in that the reaction system in step (2) is：10 × PCR is buffered
2 μ L, 10mMdNTP mixed liquor of liquid, 2 μ L, 5U/ μ L Taq DNA polymerases 1 μ L, 10 μM of 1 μ L of T1-F primers, 10 μM of T1-R primers 1
μ L, 10 μM of probe T1-T 1 μ L, 10 μM of 1 μ L of probe T1-C, genomic DNA 2 μ L, ddH2O complements to 20 μ L；Real-time quantitative
PCR detects program：95 DEG C of pre-degenerations 30 seconds；95 DEG C are denatured 0 second, and 60 DEG C are annealed 5 seconds, and 72 DEG C extend 10 seconds, repeat 35 and follow
Ring cycles detection of end fluorescence each time；Fluorescence is detected after total overall reaction, with color compensation correction to program fluorescent value.
7. application of the kit described in claim 1 in predicting susceptibility of coronary heart disease, includes the following steps：
(1) detected sample is collected, extracts the genomic DNA of sample cell；
(2) kit of application detection T1 gene rs1047896 polymorphic sites, prepares 20 μ L reaction systems, using real-time quantitative
PCR combination Taqman probe techniques detect FAM passages and HEX channel fluorescence values, determine the gene of rs1047896 polymorphic sites
Type；
(3) coronary disease susceptibility is predicted：The genotype TT homozygotes of rs1047896 polymorphic sites are compared with CC homozygotes and CT heterozygotes
The risk for suffering from coronary heart disease dramatically increases, relative risk 1.56.
8. the use as claimed in claim 7, which is characterized in that detected sample is body fluid in step (1), it may include periphery
Blood, aqueous humor, amniotic fluid, ascites, pericardial fluid and pleural effusion.
9. the use as claimed in claim 7, which is characterized in that 20 μ L reaction systems of the real-time quantitative PCR in step (2)
For：10 × PCR buffer solutions, 2 μ L, 10mM dNTP mixed liquors, 2 μ L, 5U/ μ L Taq DNA polymerases 1 μ L, 10 μM of 1 μ of T1-F primers
L, 10 μM of T1-R primers 1 μ L, 10 μM of probe T1-T 1 μ L, 10 μM of 1 μ L of probe T1-C, genomic DNA 2 μ L, ddH2O is supplied
To 20 μ L；
The amplification program of real-time quantitative PCR is：95 DEG C of pre-degenerations 30 seconds；95 DEG C are denatured 0 second, and 60 DEG C are annealed 5 seconds, 72 DEG C of extensions 10
Second, 35 Xun Huans are repeated, cycle detection of end fluorescence each time；Fluorescence is detected after total overall reaction, with color compensation program
Correct fluorescent value；
The definite method of the genotype of rs1047896 polymorphic sites is：The FAM passages of detection and HEX passages in step (2) is glimmering
Light value substitutes into Excel and makes scatter diagram, is CC genes along Y-axis distribution wherein being TT genotype along X-axis distribution
Type, it is CT heterozygotes to be diagonally distributed.
10. the use as claimed in claim 7, which is characterized in that the genotype TT of rs1047896 polymorphic sites in step (3)
Homozygote is dramatically increased compared with the risk that CC homozygotes and CT heterozygotes suffer from coronary heart disease, relative risk 1.56.
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