CN108342473A - It is a kind of to be used to detect the kit that blood lipid metabolism related gene ABCG1 methylates - Google Patents
It is a kind of to be used to detect the kit that blood lipid metabolism related gene ABCG1 methylates Download PDFInfo
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- CN108342473A CN108342473A CN201810332363.3A CN201810332363A CN108342473A CN 108342473 A CN108342473 A CN 108342473A CN 201810332363 A CN201810332363 A CN 201810332363A CN 108342473 A CN108342473 A CN 108342473A
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- abcg1
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- methylates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
Abstract
The kit that the present invention relates to a kind of to methylate for detecting blood lipid metabolism related gene ABCG1, including ABCG1 gene methylation detection site primers and probe sequence, as shown in SEQ ID NO.1 3；Reference gene detection site primer and probe sequence, as shown in SEQ ID NO.4 6.The entire detection process of the present invention samples totally-enclosed form, avoids the possibility of cross contamination.The design and result interpretation of kit of the present invention are simple and convenient, and testing result is reliable, specific and sensitivity is high, can assist detection Dyslipidemia risk, and then prevent in advance.
Description
Technical field
The invention belongs to molecular biology and technical field of medical detection, more particularly to a kind of for detecting blood lipid metabolism phase
The kit that correlation gene ABCG1 methylates.
Background technology
Lipid-metabolism is one of basic problem of human life activity, and dyslipidemia is a kind of more typical disease, is people
The metabolic disorder of internal lipoprotein, includes mainly total cholesterol and low density lipoprotein cholesterol, triglycerides increase and/or height
Density lipoprotein-cholesterol reduction etc..Dyslipidemia is to be coronary heart disease and lack one of an important factor for leading to atherosclerosis
The independent hazard factor of courageous and upright cerebral apoplexy.In China, the incidence of dyslipidemia is high, the trend being also gradually increasing, this and China
The reasons such as the living standard of the people significantly improves, eating habit changes have substantial connection.
It is a kind of important modification of nucleic acid to methylate, and adjusts the expression and closing of gene, the various physiology work(with human body
Energy and many diseases are closely related, and DNA methylation can cause chromatin Structure, DNA conformations, DNA stability and DNA and protein
The change of interaction mode, to control gene expression.DNA methylation can close the activity of certain genes, and demethylation is then
Reactivating and expressing induction of gene.As one of most important epigenetic modification, DNA methylation can pass through regulation and control
The expression of specific gene, Adipocyte Differentiation transcription factor and transcription factor influences the growth and development of adipose tissue.DNA first
Baseization can participate in the generating process of adipose tissue growth and development and obesity, in the differentiation mechanism and lipid of adipocyte
Important regulating and controlling effect is played in building-up process.There is weight for the blood lipid metabolism situation for understanding individual to methylating to be detected
Big meaning, and new direction can be provided for the prevention and treatment of relevant disease.
A large amount of research and development find that methylating for ABCG1 genes is significantly correlated with Dyslipidemia, therefore, to ABCG1 genes
Carry out DNA methylation assay, will appreciate that risk factors of the individual in terms of blood lipid metabolism, assess the onset risk of dyslipidemia, pair with
The early diagnosis of dyslipidemia, early intervention have positive effect.
Invention content
Technical problem to be solved by the invention is to provide one kind to methylate for detecting blood lipid metabolism related gene ABCG1
Kit, be related to the detection of ABCG1 gene methylations, detection efficiency is high, with strong points.
The a kind of of the present invention is used to detect the kit that blood lipid metabolism related gene ABCG1 methylates, including：
ABCG1 gene methylation detection site primers and probe sequence, as shown in SEQ ID NO.1-3：
Forward primer：5'-TGTATTGTGATATCGACGAGAC-3',
Reverse primer：5'-ACCTCCTCGATTCTAAACGTAC-3',
Probe：5'-FAM-TTGCGGGAGTTGGACGTGG-BHQ-3'；
Reference gene detection site primer and probe sequence, as shown in SEQ ID NO.4-6：
Forward primer：5'-TGATGGAGGAGGTTTAGTAAGT-3',
Reverse primer：5'-CAATAAAACCTACTCCTCCCTTAA-3',
Probe：5'-HEX-ACCCAACACACAATAACAAACACA-BHQ-3'.
The ABCG1 gene methylations detection site is 2 islands CpG of ABCG1 genes.
The reference gene is Actin.
The kit further includes positive quality control product, negative quality-control product and blank control.
The positive quality control product is permethylated human genome DNA.
The feminine gender quality-control product is the non-human genome DNA to methylate.
The blank control is deionized water.
The detection sample of the kit is peripheral blood, saliva or Oral Mucosal Cells.
The kit can be used for early screening or the risk assessment of dyslipidemia.
Advantageous effect
Compared with prior art, kit of the present invention based on detecting ABCG1 gene methylations can be conveniently
The detection to dyslipidemia risk is realized on a molecular scale；Entire PCR processes use totally-enclosed form, avoid intersection
The possibility of pollution improves the accuracy of detection；The design of kit of the present invention is simple and convenient, is directly obtained by PCR glimmering
Optical signal judges the methylation state of gene loci so that the judgement of result is more convenient.Kit detection effect of the present invention
Rate is high, and testing result is reliable, specificity and sensitivity are high, with strong points, can assist detection Dyslipidemia risk, Jin Erti
Preceding prevention.
Description of the drawings
Fig. 1 is the ABCG1 gene methylation detection cases of positive sample in embodiment 1.
Fig. 2 is the ABCG1 gene methylation detection cases of negative sample in embodiment 1.
Specific implementation mode
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, people in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.
Embodiment 1
Utilize fluorescent quantitation detection of platform coronary heart disease tumor susceptibility gene polymorphism.
1, research object is raised：The ordinary circumstance of volunteer is investigated in the form of application form, while acquiring peripheral blood
5ml is put into EDTA anticoagulant tubes, 4 DEG C of storages.
2, genomic DNA is extracted：Use the poba gene group DNA extraction kit of Tiangeng biochemical technology Co., Ltd, production
Product number DP318, according to operating instruction extract poba gene group DNA, with 0.8% agarose gel electrophoresis determine DNA mass and
Concentration.
3, methylate conversion：Bisulfite conversion, unmethylated cytimidine (C) transformation are carried out to the DNA extracted
For uracil (U), and the cytimidine (C) to methylate is constant.DNA bisulfite conversion reagent boxes come from Tiangeng biochemical technology
(Beijing) Co., Ltd, product identification DP215.
4, gene methylation detects：Methylation sites are detected using fluorescent quantitation technology, the original substantially of this technology
Reason：The oligonucleotides (probe) that 2 kinds of ends 5' use different fluorochrome labels, two probes point are introduced in PCR reaction systems
Not Shi Bie the methylation sites of ABCG1 genes and the non-methylation sites of reference gene, the ends 3' of probe connect quenching group.Just
Often in the case of, due to probe the ends 5' fluorophor with the ends 3' quenching group together with, fluorescence is quenched.As PCR is anti-
The progress answered, probe that can be complementary with genome sample are gradually cut by the 5 prime excision enzyme activity of archaeal dna polymerase 5' → 3', cause
Fluorophor on the ends probe 5' is detached with the quenching group at the ends 3', quenching effect failure, to which fluorescent reporter group is activated,
Detecting fluorescent value by corresponding instrument can be detected；Such as can not be complementary with template, then probe can not by polymerization cleavage, because
This can't detect corresponding fluorescence signal, according to the fluorescent signals wavelengths detected, it can be determined that ABCG1 genes and reference gene
Methylation.This research carries out design of primers using Primer3 online softwares, then carries out primer synthesis, specifically draws
Object is as shown in table 1.
The PCR primer in 1 ABCG1 of table and reference gene DNA methylation assay site
5, fluorescent PCR detects：DNA is added separately in respective 50ul PCR systems after 50ng is handled, wherein each
Reaction system includes each pair of special primers of 100nmol, each pair of specific probes of 150nmol, 1X sonde method partings Mix (Tiangeng is biochemical,
China, FP211).Negative control is set simultaneously.Fluorescent PCR detector is CFX96 (biorad), and PCR response procedures are：95℃
Denaturation 3 minutes；95 DEG C are denaturalized 30 seconds, and 50 DEG C of renaturation extend 30 seconds, 35 cycles.Fluorescence signal is detected after final cycle.
6, Analysis of test results：It is detected, is made using 1 positive sample of above-mentioned reaction system pair and 1 negative sample
Gathered data is analyzed with Bio-Rad CFX Manager (biorad) software, Fig. 1, Fig. 2 are analysis result, as a result
Show：The difference of target gene Ct values and reference gene Ct values is less than 10, illustrates that ABCG1 gene methylations degree is high；Target gene
The difference of Ct values and reference gene Ct values is more than 10, illustrates that ABCG1 gene methylation degree is low, interpretation method such as table 2.
2 result interpretation of table refers to
7, kit performance verification：The detection that product precision, stability are carried out using the kit that configuration is completed, according to
The condition of above-mentioned setting carries out properties of product verification, using above-mentioned sample, if the difference of target gene Ct values and reference gene Ct values
Respectively less than 10, illustrate that ABCG1 methylations are high, Dyslipidemia risk is high；If target gene Ct values and reference gene Ct
The difference of value is all higher than 10, illustrates that ABCG1 methylations are low, Dyslipidemia risk is low.Fig. 1 is the ABCG1 of positive sample
The difference of gene methylation detection case, 4 repetitions, target gene Ct values and reference gene Ct values is respectively less than 10, illustrates ABCG1 bases
Because of methylation height；Fig. 2 be negative sample ABCG1 gene methylation detection cases, 4 repetitions, target gene Ct values with
The difference of reference gene Ct values is all higher than 10, illustrates that ABCG1 gene methylation degree is low.
Comparative example 1
Using other methylation detecting method MSP (methylation specific PCR, methylation status of PTEN promoter),
BSP (PCR is sequenced in bisulfite sequencing PCR, bisulfite), HRM (Methylation specifc-high
Resolution melting, methylation-specific high-resolution solubility curve) sample is detected, testing result and this hair
Bright kit testing result comparative situation is as shown in table 3, it is known that this kit takes shorter, moderate cost, sensibility and repetition
Property is preferable.
The different detection method comparing results of table 3
SEQUENCE LISTING
<110>Donghua University
<120>It is a kind of to be used to detect the kit that blood lipid metabolism related gene ABCG1 methylates
<130> 2018.03.05
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213>Artificial sequence
<400> 1
tgtattgtga tatcgacgag ac 22
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence
<400> 2
acctcctcga ttctaaacgt ac 22
<210> 3
<211> 19
<212> DNA
<213>Artificial sequence
<400> 3
ttgcgggagt tggacgtgg 19
<210> 4
<211> 22
<212> DNA
<213>Artificial sequence
<400> 4
tgatggagga ggtttagtaa gt 22
<210> 5
<211> 24
<212> DNA
<213>Artificial sequence
<400> 5
caataaaacc tactcctccc ttaa 24
<210> 6
<211> 24
<212> DNA
<213>Artificial sequence
<400> 6
acccaacaca caataacaaa caca 24
Claims (6)
1. a kind of for detecting the kit that blood lipid metabolism related gene ABCG1 methylates, it is characterised in that：Including ABCG1 bases
Because of DNA methylation assay site primer and probe sequence, as shown in SEQ ID NO.1-3；Reference gene detection site primer and probe
Sequence, as shown in SEQ ID NO.4-6.
2. it is according to claim 1 a kind of for detecting the kit that blood lipid metabolism related gene ABCG1 methylates, it is special
Sign is：The ABCG1 gene methylations detection site is 2 islands CpG of ABCG1 genes.
3. it is according to claim 1 a kind of for detecting the kit that blood lipid metabolism related gene ABCG1 methylates, it is special
Sign is：The reference gene is Actin.
4. it is according to claim 1 a kind of for detecting the kit that blood lipid metabolism related gene ABCG1 methylates, it is special
Sign is：The kit further includes positive quality control product, negative quality-control product and blank control.
5. it is according to claim 4 a kind of for detecting the kit that blood lipid metabolism related gene ABCG1 methylates, it is special
Sign is：The positive quality control product is permethylated human genome DNA；The feminine gender quality-control product is the non-mankind to methylate
Genomic DNA；The blank control is deionized water.
6. it is according to claim 1 a kind of for detecting the kit that blood lipid metabolism related gene ABCG1 methylates, it is special
Sign is：The detection sample of the kit is peripheral blood, saliva or Oral Mucosal Cells.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810332363.3A CN108342473A (en) | 2018-04-13 | 2018-04-13 | It is a kind of to be used to detect the kit that blood lipid metabolism related gene ABCG1 methylates |
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| CN201810332363.3A CN108342473A (en) | 2018-04-13 | 2018-04-13 | It is a kind of to be used to detect the kit that blood lipid metabolism related gene ABCG1 methylates |
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| CN108342473A true CN108342473A (en) | 2018-07-31 |
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| CN201810332363.3A Pending CN108342473A (en) | 2018-04-13 | 2018-04-13 | It is a kind of to be used to detect the kit that blood lipid metabolism related gene ABCG1 methylates |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113355412A (en) * | 2020-03-02 | 2021-09-07 | 南京腾辰生物科技有限公司 | Methylation marker and kit for auxiliary diagnosis of cancer |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090264306A1 (en) * | 2005-10-27 | 2009-10-22 | Curators Of The University Of Missouri | Dna methylation biomarkers in lymphoid and hematopoietic malignancies |
| CN103820552A (en) * | 2014-02-26 | 2014-05-28 | 东华大学 | Real-time quantification PCR chip used for detecting gene expression of mouse cholesterol metabolism |
| CN110123831A (en) * | 2013-12-13 | 2019-08-16 | 罗盖特兄弟公司 | For the composition based on methyl flamprop by increasing the treatment of HDL cholesterol levels and/or prevention disease |
-
2018
- 2018-04-13 CN CN201810332363.3A patent/CN108342473A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090264306A1 (en) * | 2005-10-27 | 2009-10-22 | Curators Of The University Of Missouri | Dna methylation biomarkers in lymphoid and hematopoietic malignancies |
| CN110123831A (en) * | 2013-12-13 | 2019-08-16 | 罗盖特兄弟公司 | For the composition based on methyl flamprop by increasing the treatment of HDL cholesterol levels and/or prevention disease |
| CN103820552A (en) * | 2014-02-26 | 2014-05-28 | 东华大学 | Real-time quantification PCR chip used for detecting gene expression of mouse cholesterol metabolism |
Non-Patent Citations (4)
| Title |
|---|
| BIO-RAD: "Real-Time PCR Application Guide", 《BIO-RAD LABORATORIES INC》 * |
| LILIANE PFEIFFER等: "DNA Methylation of Lipid-Related Genes Affects Blood Lipid Levels", 《CIRC CARDIOVASC GENET.》 * |
| 金征宇等: "《基因与纳米探针-医学分子成像理论与实践 中》", 30 November 2017, 天津科学技术出版社 * |
| 陶永光: "《肿瘤分子生物学与细胞生物学实验手册》", 31 October 2014, 湖南科学技术出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113355412A (en) * | 2020-03-02 | 2021-09-07 | 南京腾辰生物科技有限公司 | Methylation marker and kit for auxiliary diagnosis of cancer |
| CN113355412B (en) * | 2020-03-02 | 2024-02-20 | 腾辰生物科技(上海)有限公司 | Methylation markers and kits for aiding in the diagnosis of cancer |
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Application publication date: 20180731 |