CN104059990A - Detection method and kit for coronary heart disease susceptibility loci rs1801133 - Google Patents
Detection method and kit for coronary heart disease susceptibility loci rs1801133 Download PDFInfo
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Abstract
The invention relates to a detection method and a kit for coronary heart disease susceptibility loci. The method comprises the following steps: (1) extracting a genome deoxyribonucleic acid (DNA) of a sample, and amplifying a region containing rs1801133 loci in a methylene tetrahydrofolate reductase (MTHFR) gene exon of the sample, so as to obtain an amplification product; (2) detecting the genetype of single nucleotide polymorphism (SNP) loci rs1801133 in the amplification product by using a high resolution melting (HRM) analysis technology. The genetype analysis is carried out on the region containing the rs1801133 loci in the amplified MTHFR gene by adopting the HRM analysis technology. The method is simple and quick to operate, low in use cost and accurate in result, and can achieve batch inspection; whether the tested people carry with coronary heart disease susceptibility genes can be clearly judged by using the kit, the coronary heart disease susceptibility people can be screened out from people, the poor living habit is improved, and the target of prevention is achieved.
Description
[technical field]
The present invention relates to a kind of method and test kit that detects coronary disease susceptibility, say more specifically single nucleotide polymorphism (the single nucleotide polymorphism with coronary heart disease dependent genes Methylene tetrahydrofolate reductase (MTHFR) gene by mensuration, SNP) prediction experimenter is for the susceptibility of coronary heart disease, the method can be used for auxiliary diagnosis, treatment and the new drug development of disease, belongs to molecular biology and medical field.
[background technology]
Coronary heart disease is coronary artery generation atherosclerotic lesion and cause lumen of vessels stenosis or occlusion, causes myocardial ischemia, anoxic or heart trouble downright bad and that cause.It is relevant with the dangerous increase of atherosclerotic vascular disease that a large amount of epidemiological investigations shows that Plasma Homocysteine raises, and between homocysteine in plasma and atherosclerotic vascular disease, exist a kind of strong dose-dependence, this relation does not rely on the risk factor of traditional atherogenicity vascular disease.In human body, the production of homocysteine in plasma and metabolism keep balance under normal circumstances.A variety of causes causes homocysteine in plasma generation to increase or metabolic disturbance, all can make homocysteine in plasma accumulate in vivo, forms hyperhomocysteinemiainjury.Cause the reason of homocysteine to be substantially divided into heredity metabolism and the large class of acquired metabolic disturbance two, wherein a class is inherited genetic factors, it is mainly the key enzyme producer sudden change in homocysteine in plasma metabolic process, cause enzymic activity to decline, make methionine metabolism obstacle, cause homocysteine in plasma to be accumulated in vivo.
In research, MTRR sudden change is to cause the avitaminotic Etiological of folic acid/methyl, is also homocysteine, the abnormal one of the main reasons of folic acid metabolism at present.And homocysteine, folic acid metabolism are relevant to numerous disease (Down syndromes-mongolism, neurocele disease, cardiovascular disorder etc.).MTHFR is 5,10-methylenetetrahydrofolate reductase (NADPH), Methylene tetrahydrofolate reductase protein coding gene, Main Function is 5,10-CH2-THFA to be converted into the 5-methyltetrahydrofolate with biological function in folic acid metabolism path.5-methyltetrahydrofolate can further enter methyl transmission path, the process that again methylates by homocysteine indirectly provides methyl and makes the homocysteine level in blood remain on a lower level for DNA methylation and protein methylate, the homocysteine of high density can damage the endotheliocyte of blood vessel, becomes important cardiovascular and cerebrovascular paathogenic factor.In addition the mesostate of folic acid also has important effect in Nucleotide building-up process, and the formation that is purine skeleton by one carbon unit metabolism provides carbon atom.The defect of mthfr gene will cause the disorder of a plurality of basic biochemistry processes of body, comprises that cell cycle regulating, DNA replication dna, DNA and protein methylate to modify etc., and and then causes the various disease conditions such as neural tube defect, cancer, cardiovascular and cerebrovascular diseases.Have now found that mthfr gene has various mutations type, different gene mutation types produces different impacts to the enzymic activity of MTHFR and thermostability, shows various enzymic activity and thermostability.In general, the sudden change that relates to the upper conserved sequence of evolving can cause the sharply decline of enzymic activity, and hazardness is larger; On the contrary, the sudden change in non-conservative sequence is less to the activity influence of enzyme, and hazardness is also less.Rs1801133 is a SNP site MTHFR C677T in mthfr gene, is a common thermo-labile missense mutation.Mthfr gene the 677th bit codon C is replaced by T, thereby makes the L-Ala of a high conservative become α-amino-isovaleric acid, has produced a HInfI and TaqI restriction enzyme site simultaneously.C677T is positioned at MTHFR catalysis region, and this sudden change directly affect activity and the thermotolerance of Methylene tetrahydrofolate reductase, shows as enzymic activity reduction in various degree and with the thermotolerance reduction of enzyme.
In sum, for the final treatment coronary heart disease that realizes, this area is in the urgent need to seeking the coronary heart disease genetic risk factor, and exploitation detects method and the test kit of the coronary heart disease genetic risk factor.
[summary of the invention]
Main purpose of the present invention is to provide a kind of method of auxiliary diagnosis coronary heart disease, and the method is simple to operate, quick, and use cost is low, and result is accurate, can realize batch detection.
Another object of the present invention is to provide a kind of test kit for detection of coronary heart disease susceptibility loci, and this test kit comprises PCR primer and the test kit that contains this primer, and it can be realized amplification and analyze with HRM.
In order to address the above problem, the technical solution used in the present invention is: a kind of method that detects coronary heart disease susceptibility loci, comprise step: (1) extracts the genomic dna of sample, the region that comprises rs1801133 site in the mthfr gene exon of amplification sample, obtains amplified production; (2) use HRM analytical technology to detect the genotype of SNP site rs1801133 in amplified production.
Further, the genotype in the rs1801133 site of mthfr gene is during with TT, and under this mthfr gene, the tester of sample does not have the tester of TT for the susceptibility of coronary heart disease higher than genotype.
Further, use the region that comprises rs1801133 site in the mthfr gene of primer amplified sample, this Auele Specific Primer refers to for rs1801133 SNP site on mthfr gene and designs, and can specific amplification goes out to comprise the primer pair of the DNA fragmentation in above-mentioned SNP site.
Further, described Auele Specific Primer has the nucleotide sequence shown in SEQ ID NO:2 and SEQ ID NO:3.
Further, use the genotype of the primer pair of the DNA fragmentation that comprises above-mentioned SNP site after the amplification of HRM analytical technology direct-detection.
Further, the primer pair of the DNA fragmentation that comprises above-mentioned SNP site after amplification is transferred to analytical equipment, then uses HRM analytical technology to analyze the genotype of this primer pair.
Further, the detection method of the described coronary heart disease susceptibility loci application of the susceptibility of non-diagnostic assays coronary heart disease in vitro.
In order to address the above problem, another technical scheme that the present invention adopts is: a kind of test kit for detection of coronary heart disease susceptibility loci, increases and HRM technical Analysis to the region that comprises rs1801133 site in mthfr gene exon.
Further, described test kit comprises the Auele Specific Primer in the region that comprises rs1801133 site in specific amplification mthfr gene.
Further, described Auele Specific Primer has the nucleotide sequence shown in SEQ ID NO:2 and SEQ ID NO:3.
Further, the component of test kit and content comprise 5ul2X LightCycler480HighResolution Melting Master Mix, 1.2ul25mM MgCl2 solution, 1ul2 μ M PrimerMix, 1ul5ng/ μ l left and right DNA, 1.8ul H2O, have the Auele Specific Primer of the nucleotide sequence shown in SEQ ID NO:2 and SEQ ID NO:3.
Further, the application of described test kit aspect predicting susceptibility of coronary heart disease.
Further, described method and the test kit application aspect detection coronary heart disease genetic risk factor mononucleotide polymorphism site.
Compared with prior art, the present invention has the following advantages: gene type assay is carried out in the region that comprises rs1801133 site in the mthfr gene after adopting HRM analytical technology to amplification, and the method is simple to operate, quick, and use cost is low, result is accurate, can realize batch detection.
[accompanying drawing explanation]
Fig. 1 is the HRM detected result of mthfr gene rs1801133 loci gene type;
Fig. 2 is Taqman probe somatotype result sectional drawing, has shown the genotypic Taqman probe of a kind of SNP result of rs1801133.
[embodiment]
Below in conjunction with specific embodiment, further set forth the present invention.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition as the people such as Sambrook, molecular cloning, laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The invention provides a kind of method and test kit that detects coronary heart disease susceptibility loci, described method comprises step: (1) extracts the genomic dna of sample, the region that comprises rs1801133 site in Methylene tetrahydrofolate reductase (MTHFR) gene extron of amplification sample, obtains amplified production; (2) use high-resolution fusion curve (high-resolution melt, HRM) analytical technology to detect the genotype of single nucleotide polymorphism (SNP) site rs1801133 in amplified production.The detection method of this coronary heart disease susceptibility loci and test kit also can be used for predicting the susceptibility of coronary heart disease.
Described rs1801133 is arranged in the exon (fragment contig contig:NT_021937.19 position 7861110G > A) of mthfr gene, wherein, thymus nucleic acid (DNA) sequence numbering: the nucleotide sequence of rs1801133 position based on shown in SEQ ID NO:1; The nucleotide sequence of primer 1 based on shown in SEQ IDNO:2; The nucleotide sequence of primer 2 based on shown in SEQ ID NO:3; The nucleotide sequence of amplified production based on shown in SEQ ID NO:4.
Closely related through having studies have shown that for many years mthfr gene mononucleotide polymorphism site rs1801133 and coronary heart disease, and found its new function: the genotypic change that mthfr gene mononucleotide polymorphism site rs1801133 is arranged in the exon of mthfr gene will cause the onset risk of coronary heart disease to raise, wherein association study result shows, there is significant difference (p < 0.05) in the distribution at mthfr gene rs1801133C → T in case and control group, this SNP polymorphism can change transcription factor binding site point.
The nucleotide sequence that the detailed sequence of mthfr gene can be rs1801133 referring to accession number (can referring to network address http://www.ncbi.nlm.nih.gov/).
Embodiment 1: fluorescent PCR detects
One, experiment material
LightCycler480 quantitative real time PCR Instrument is purchased from Switzerland's Roche (Roche) company, and pcr reaction solution (LightCycler480High Resolution Melting Master) is synthetic by the customization of Switzerland's Roche (Roche) company.
Two, primer and probe design and synthetic:
The partial sequence of mthfr gene exon of take is template, uses Primer Premier5 software analysis primer, and synthetic by the customization of Shanghai Sheng Gong biotechnology company limited.
Detection primer:
Mthfr gene rs1801133 upstream primer sequence: 5 '-
ACCTGAAGCACTTGAAGGAGAA-3′(SEQ ID NO2)
Mthfr gene rs1801133 downstream primer sequence: 5 '-CAAAGCGGAAGAATGTGTCAG-3 ' (SEQ ID NO3)
Three, pattern detection:
Experiment detects 50 routine coronary heart disease cases and 50 routine normal control crowds altogether, and every example is collected the about 2ml of blood sample, and with phenol/chloroform drawer genomic dna, micro-ultraviolet/visible light spectrophotometer for extracting result (Themo Fisher company) detects.
By following system, carry out fluorescent PCR amplification, finally with roche LC480software1.5 scanning cluster analysis
| 384 orifice plates (ul) | |
| LightCycler480High Resolution Melting Master Mix(2X) | 5 |
| MgCl2(25mM) | 1.2 |
| Primer Mix(2μM) | 1 |
| DNA (5ng/ μ l left and right) | 1 |
| H2O | 1.8 |
| Reaction cumulative volume | 10 |
Four, type detected result:
The detected result of genome DNA extraction:
The genomic dna of all samples meets testing requirement (260/280 > 1.8, concentration > 10ng/ul)
Embodiment 2:Taqman method detects the genotype in coronary heart disease rs1801133 site
By Taqman method, detect the rs1801133 site of coronary heart disease genetic risk gene M THFR gene.Select each 10 examples of above-mentioned coronary heart disease case-control group sample and carry out the experiment of Taqman somatotype, the genotype of judgement rs1801133.
One, experimental technique
Taqman detecting probe method is used the Taqman probe of Applied biosystems, the Taqman Mix of Suzhou new marine life Science and Technology Ltd..
By following system, carry out pcr amplification, finally with roche LC480software1.5 scanning cluster analysis
| 384 orifice plates (ul) | |
| Taqman Mix | 5 |
| Taqman Probe | 0.1 |
| DNA (5ng/ μ l left and right) | 1 |
| H2O | 3.9 |
| Reaction cumulative volume | 10 |
Two, experimental result
Taqman somatotype result result sectional drawing as shown in Figure 2.
Finally, the Taqman somatotype result of 20 examples and the HRM analytical results of LightCycler480 fluorescent PCR are in full accord.
Three, the association analysis of mthfr gene rs1801133 genotype and coronary heart disease susceptible
The relatively employing RxC χ2-test,chi-square test that mthfr gene rs1801133 distributes in patients with coronary artery disease and contrast. with SPSS software, carry out statistical study, the SPSS software analysis result of detected result is as shown in the table:
Embodiment 3: the detection kit of coronary heart disease susceptibility loci
Detection kit provided by the invention can increase and HRM technical Analysis to the region that comprises rs1801133 site in mthfr gene exon, this detection kit can detect the susceptibility of coronary heart disease, it includes can amplify the Auele Specific Primer that comprises rs1801133 site in mthfr gene exon, and other PCR-HRM corresponding reagent, pcr amplification is identical with the component concentration that HRM analyzes.
Detection kit detects application for 50 person-portions, keeps in Dark Place in-20 ℃, and its component and content comprise:
5ul2X LightCycler480High Resolution Melting Master Mix
1.2ul25mM MgCl2 solution
1ul2μM Primer Mix
1ul5ng/ μ l left and right DNA
1.8ul H2O
Each 0.2uM of Auele Specific Primer with the nucleotide sequence shown in SEQ ID NO:2 and SEQ ID NO:3.
The present invention has the illustration of practicality:
Detect the genotype in rs1801133 site in this individual mthfr gene, whether the onset risk that judges this individuality trouble coronary heart disease with this is higher than general population.
In mthfr gene, the polymorphism in rs1801133 site can be directly used in the personalized treatment of coronary heart disease.
The genotype that detects rs1801133 site in mthfr gene also can be used for diagnosis of coronary heart disease.
All documents of mentioning in the present invention are all quoted and are made reference in this application, just as each piece of document, are solely quoted as a reference.
Technology contents of the present invention and technical characterstic disclose as above, yet those of ordinary skill in the art still may carry out all replacement and modifications that does not deviate from spirit of the present invention based on teaching of the present invention and announcement.Therefore, protection scope of the present invention should be not limited to the content that embodiment discloses, and comprises various do not deviate from replacement of the present invention and modifications, is present patent application claim and contains.
Claims (10)
1. a detection method for coronary heart disease susceptibility loci, is characterized in that: comprise step:
(1) extract the genomic dna of sample, the region that comprises rs1801133 site in Methylene tetrahydrofolate reductase (MTHFR) gene extron of amplification sample, obtains amplified production; With
(2) use high-resolution fusion curve (high-resolution melt, HRM) analytical technology to detect the genotype of single nucleotide polymorphism (SNP) site rs1801133 in amplified production.
2. the method for claim 1, is characterized in that: the genotype in the rs1801133 site of mthfr gene is during with TT, and under this mthfr gene, the tester of sample does not have the tester of TT for the susceptibility of coronary heart disease higher than genotype.
3. the method for claim 1, it is characterized in that: use the region that comprises rs1801133 site in the mthfr gene of primer amplified sample, this Auele Specific Primer refers to for rs1801133 SNP site on mthfr gene and designs, and can specific amplification goes out to comprise the primer pair of the DNA fragmentation in above-mentioned SNP site.
4. method as claimed in claim 3, is characterized in that: described Auele Specific Primer has the nucleotide sequence shown in SEQ IDNO:2 and SEQ ID NO:3.
5. method as claimed in claim 3, is characterized in that: the genotype of using the primer pair of the DNA fragmentation that comprises above-mentioned SNP site after the amplification of HRM analytical technology direct-detection.
6. method as claimed in claim 3, is characterized in that: the primer pair of the DNA fragmentation that comprises above-mentioned SNP site after amplification is transferred to analytical equipment, then uses HRM analytical technology to analyze the genotype of this primer pair.
7. for detection of a test kit for coronary heart disease susceptibility loci, it is characterized in that: to comprising in mthfr gene, increase in the region in rs1801133 site and HRM technical Analysis.
8. test kit as claimed in claim 7, is characterized in that: described test kit comprises the Auele Specific Primer in the region that comprises rs1801133 site in specific amplification mthfr gene.
9. test kit as claimed in claim 8, is characterized in that: described Auele Specific Primer has the nucleotide sequence shown in SEQID NO:2 and SEQ ID NO:3.
10. test kit as claimed in claim 9, is characterized in that: the component of test kit and content comprise 5ul2X LightCycler480High Resolution Melting Master Mix, 1.2ul25mMMgCl2 solution, 1ul2 μ M Primer Mix, 1ul5ng/ μ l left and right DNA, 1.8ul H2O, have the Auele Specific Primer of the nucleotide sequence shown in SEQ ID NO:2 and SEQ ID NO:3.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104928378A (en) * | 2015-06-15 | 2015-09-23 | 广州金域医学检验中心有限公司 | Primer combination used for MTHFR-C677T genetic locus polymorphic detection, MTHFR-C677T genetic locus polymorphic detection method and application |
| CN105803107A (en) * | 2016-05-30 | 2016-07-27 | 上海核盾生物科技有限公司 | Folate metabolism related gene MTHFR heritable variation detection kit and application thereof |
| CN106011282A (en) * | 2016-07-22 | 2016-10-12 | 苏州康吉诊断试剂有限公司 | Primers and probes for detecting MTHFR (methylene tetrahydrofolate reductase) enzymatic activity or folic acid metabolic capability and application of primers and probes |
| CN107974495A (en) * | 2017-05-04 | 2018-05-01 | 光瀚健康咨询管理(上海)有限公司 | Coronary heart disease genetic risk factor detection kit |
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| CN108949966A (en) * | 2018-08-24 | 2018-12-07 | 山东德诺生物科技有限公司 | For detecting the primed probe group and its application of rs1801133 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103468781A (en) * | 2012-06-06 | 2013-12-25 | 解码（上海）生物医药科技有限公司 | Coronary artery heart disease susceptibility gene non-invasive detection kit |
| CN103667440A (en) * | 2013-09-05 | 2014-03-26 | 谢小冬 | Application of SNP loci of GATA4 genes |
-
2014
- 2014-07-10 CN CN201410331666.5A patent/CN104059990B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103468781A (en) * | 2012-06-06 | 2013-12-25 | 解码（上海）生物医药科技有限公司 | Coronary artery heart disease susceptibility gene non-invasive detection kit |
| CN103667440A (en) * | 2013-09-05 | 2014-03-26 | 谢小冬 | Application of SNP loci of GATA4 genes |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104928378A (en) * | 2015-06-15 | 2015-09-23 | 广州金域医学检验中心有限公司 | Primer combination used for MTHFR-C677T genetic locus polymorphic detection, MTHFR-C677T genetic locus polymorphic detection method and application |
| CN105803107A (en) * | 2016-05-30 | 2016-07-27 | 上海核盾生物科技有限公司 | Folate metabolism related gene MTHFR heritable variation detection kit and application thereof |
| CN106011282A (en) * | 2016-07-22 | 2016-10-12 | 苏州康吉诊断试剂有限公司 | Primers and probes for detecting MTHFR (methylene tetrahydrofolate reductase) enzymatic activity or folic acid metabolic capability and application of primers and probes |
| CN107974495A (en) * | 2017-05-04 | 2018-05-01 | 光瀚健康咨询管理(上海)有限公司 | Coronary heart disease genetic risk factor detection kit |
| CN108034710A (en) * | 2017-12-19 | 2018-05-15 | 上海派森诺医学检验所有限公司 | For detecting the primer, fluorescence probe and application of folic acid metabolism related gene SNP |
| CN108949966A (en) * | 2018-08-24 | 2018-12-07 | 山东德诺生物科技有限公司 | For detecting the primed probe group and its application of rs1801133 |
| CN109852683A (en) * | 2018-12-30 | 2019-06-07 | 济南齐鲁医学检验有限公司 | The efficient parting detecting reagent of rs1801133 genotype |
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