CN110205368A - A kind of kit detecting the site mthfr gene A1298C rs1801131SNP - Google Patents
A kind of kit detecting the site mthfr gene A1298C rs1801131SNP Download PDFInfo
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Abstract
The purpose of the application is to disclose a kind of kit for detecting the site mthfr gene A1298C rs1801131SNP, it is characterized in that, including the primer and probe designed according to mthfr gene A1298C rs1801131SNP loci polymorphism, sequence is as follows: mthfr gene A1298C forward direction amplimer: GGAGCTGCTGAAGATGTGG；The reversed amplimer of mthfr gene A1298C: CCTCTCGGGAGAACCAAAC；Mthfr gene 1298A probe: 5 '-TGAAGAAAGTGTCT-3 '；Mthfr gene 1298C probe: 5 '-TGAAGCAAGTGTCT-3 '；Easy to operate, replicability is strong, and as a result accurately, good compatibility, pollution risk is small.
Description
Technical field
The invention belongs to field of water quality detection, in particular to detection mthfr gene A1298C rs1801131SNP a kind of
The kit of point.
Background technique
Methylenetetrahydrofolate reductase (Methylenetetrahydrofolate reductase, MTHFR) is first sulphur
Key enzyme in propylhomoserin-folic acid metabolism system can make 5,10-CH2-THFA be reduced to 5-methyltetrahydrofolate, and one
The indirect donor that aspect can be used as methyl participates in internal purine, the synthesis of pyrimidine and the methylation of DNA, RNA, protein；Separately
On the one hand, under the catalysis of methionine synthetase, using vitamin B12 as coenzyme, occur that homocysteine in blood again
Methylation production methionine, to maintain normal homocysteine level in vivo.Mthfr gene mutation can lead to its volume
The folic acid metabolism key enzyme activity of code reduces, and causes folate metabolism disorder, causes folate level reduction and homocysteine
Mass formed by blood stasis.Homocysteine can make blood vessel endothelium injury and dysfunction, and stimulated vascular smooth muscle cell hyperplasia destroys body
Blood coagulation and fibrinolytic system make body be in prethrombotic state, and the raising of chronic intracellular homocysteine level can be led
Cause DNA hypomethylation, chromosome abnormality.Discovered in recent years HyperhomocysteinemiaInduced with include cardiovascular and cerebrovascular disease, birth
A variety of sickness such as defect, pregnancy related disorder, diabetes are closely related.
There are two types of the common mutation of mthfr gene: the c.677 position in NCBI single nucleotide polymorphism database (db SNP)
Point C/T polymorphism (SNP ID:rs1801133) and c.1298 site A/C polymorphism (SNP ID:rs1801131).Wherein,
C.677 C/T polymorphism in site is to be found for the first time by Frosst equal to nineteen ninety-five, and the MTHFR up to the present found is most
For common mutational site.Studies have shown that the alanine of coding is substituted by valine after c.677 site C becomes T, cause
The thermal stability and enzymatic activity of MTHFR enzyme reduce, and according to the literature, the frequency which is mutated in asian population is up to
40%.In addition, glutamic acid is replaced by alanine after c.1298 site A becomes C, equally declines the enzymatic activity of MTHFR, cause
The raising of Plasma Homocysteine and the reduction of folate level, the frequency which is mutated in asian population are up to
19%.
It is 400 micro- grams/day that pregnancy period magnitude of recruitment is recommended in China, and people normal for mthfr gene absorbs the leaf of recommended amounts
Acid can significantly reduce the birth defect rate of infant；For the people of mthfr gene exception, MTHFR enzymatic activity is substantially reduced, leaf
Acid metabolic obstacle causes the onset risk of the diseases such as newborn's neural tube defect, Down's syndrome and harelip obviously to increase, this
Class people, which requires supplementation with more folic acid, can be only achieved expected effect.
The method of detection folic acid metabolism ability reported at present, has:
1. a kind of " detection PCR amplification of the detection site rs1801133 of folic acid metabolism capability evaluation of CN104774943A-
A kind of primer and Single base extension primer-disclosure " " detection site of folic acid metabolism capability evaluation of CN104789669A-
Detection PCR amplification primer and Single base extension primer-disclosure of rs1801131 ".
After the amplification of the method based on PCR and Single base extension, the parting of SNP genotype is carried out with MassARRAY, but is had
Have the disadvantage that: (1) equipment is expensive, and general testing agency is difficult to be equipped with, restricted application；(2) PCR amplification and single base
After extension, it is required to wheel digestions, the investment of complex steps, manpower and reagent consumptive material is big；(3) pcr amplification product needs
Processing of uncapping because DNA concentration is big at this time easily causes indoor aerosols to pollute, and influences the accuracy of result；Therefore have certain
Limitation.
2. " CN103184269B- detect homocysteine metabolism associated SNP positions kit and its amplification method and
Detection method-authorization " utilizes two forward primers and a reverse primer, after PCR amplification, see under ultraviolet light through gel electrophoresis
To the product band distinguished according to clip size, the genotype of target SNP site is judged according to the presence or absence of band.
This method is limited in that: (1) the judgement link of result needs gel electrophoresis, and is related to glue, point
Sample runs many cumbersome links such as glue, imaging, and while being easy error, human input is bigger；(2) result is in the form of picture
It presents, interpretation process relies on subjective factor, is easy error；(3) pcr amplification product needs are uncapped processing, because of DNA concentration at this time
Greatly, easily indoor aerosols is caused to pollute, influences the accuracy of result；Therefore there is certain limitation.
Summary of the invention
The main problem that the application solves is to provide a kind of detection mthfr gene A1298C rs1801131SNP site
Kit, easy to operate, easy to use, objectivity is strong, it is not easy to malfunction, it is good to practice operating effect, to solve a kind of detection
The kit in the site mthfr gene A1298C rs1801131SNP practices that operating effect is bad, operation difficulty is high, is difficult to replicate
The technical issues of operation.
In order to solve the above-mentioned technical problem, the invention discloses a kind of detection mthfr gene A1298C rs1801131SNP
The kit in site, its technical solution is as follows:
A kind of kit detecting the site mthfr gene A1298C rs1801131SNP, which is characterized in that including basis
The primer and probe of mthfr gene A1298C rs1801131SNP loci polymorphism design, sequence are as follows:
Mthfr gene A1298C forward direction amplimer: GGAGCTGCTGAAGATGTGG；
The reversed amplimer of mthfr gene A1298C: CCTCTCGGGAGAACCAAAC；
Mthfr gene 1298A probe: 5 '-TGAAGAAAGTGTCT-3 '；
Mthfr gene 1298C probe: 5 '-TGAAGCAAGTGTCT-3 '.
Preferably, the fluorophor that the mthfr gene 1298A probe 5 ' is held is VIC, and the quenching group at 3 ' ends is
Quencher and MGB.
Preferably, the fluorophor that the mthfr gene 1298C probe 5 ' is held is FAM, and the quenching group at 3 ' ends is
Quencher and MGB.
Preferably, mthfr gene A1298C primed probe mixing liquid proportional are as follows: the amplification of mthfr gene A1298C forward direction is drawn
Object: the reversed amplimer of mthfr gene A1298C: mthfr gene 1298A probe: mthfr gene 1298C probe: water=10:
10:4:4:192。
This experiment makes amplified fragments that is, by respectively setting a primer in purpose SNP site upstream and downstream using Q-PCR principle
In 50~150bp, while two genotype of SNP site are directed to, separately design the probe of two specific bindings.The 5 ' of probe
End is modified by special fluorophor, such as 1298A type VIC in the mthfr gene A1298C mentioned in this experiment,
1298C type is modified with FAM, 3 ' terminal modified Quencher and MGB, because Quencher is quenching group, it and fluorophor are same
When being present in a DNA chain, fluorophor cannot issue fluorescence, therefore under normal circumstances, and equipment cannot detect
To fluorescence signal.Only when carrying out Q-PCR reaction, it with probe specificity is integrated to SNP site, and is wrapped in reaction solution
The archaeal dna polymerase contained can probe from target fragment shearing into reaction solution, and the fluorescence in correspondent probe also therewith with
Quenching group Quencher separation, is captured by equipment, and detects the specific genotype of purpose SNP.
QPCR method used by this method has the advantage that 1. is simple, a QPCR, react end when
It waits, can directly carry out result interpretation；2. convenience, related equipment price is cheap, and most of mechanism can be equipped with；3. result
It presents in digital form, objectivity is strong, it is not easy to malfunction；4. it is compatible strong, it as a result can directly enter in corresponding table, carry out
Quick interpretation, sample process ability are strong；5. pollution risk is small, without processing of uncapping after PCR, aerosol will not be generated, is made
At false positive.
Specific embodiment
As used some vocabulary to censure specific components in the specification and claims.Those skilled in the art answer
It is understood that hardware manufacturer may call the same component with different nouns.This specification and claims are not with name
The difference of title is as the mode for distinguishing component, but with the difference of component functionally as the criterion of differentiation.Specification
Subsequent descriptions be implement the application better embodiment, so it is described description be for the purpose of the rule for illustrating the application,
It is not intended to limit the scope of the present application.The protection scope of the application is as defined by the appended claims.
Embodiment one:
A kind of kit detecting the site mthfr gene A1298C rs1801131SNP, which is characterized in that including basis
The primer and probe of mthfr gene A1298C rs1801131SNP loci polymorphism design, sequence are as follows:
Mthfr gene A1298C forward direction amplimer: GGAGCTGCTGAAGATGTGG；
The reversed amplimer of mthfr gene A1298C: CCTCTCGGGAGAACCAAAC；
Mthfr gene 1298A probe: 5 '-TGAAGAAAGTGTCT-3 '；
Mthfr gene 1298C probe: 5 '-TGAAGCAAGTGTCT-3 '.
The fluorophor that the mthfr gene 1298A probe 5 ' is held is VIC, the quenching groups at 3 ' ends be Quencher and
MGB。
The fluorophor that the mthfr gene 1298C probe 5 ' is held is FAM, the quenching groups at 3 ' ends be Quencher and
MGB。
Mthfr gene A1298C primed probe mixing liquid proportional are as follows: mthfr gene A1298C forward direction amplimer: MTHFR
The reversed amplimer of Gene A 1298C: mthfr gene 1298A probe: mthfr gene 1298C probe: water=10:10:4:4:
192。
Specific experiment process is as follows:
1. reaction system of table
| Reagent | Reaction system |
| TaqProbe 2X qPCR Mastermix | 10.0ul |
| Primer&Probe Mix | 2.0ul |
| nuclease free water | 3.0ul |
| Template | 5.0ul |
Table 2.Primer&Probe Mix system
3. response procedures of table
As a result when interpretation, CT > 32 or be Undetermined be to be not detected the fluorescence signal, the spy is not detected in expression
The corresponding genotype of needle；CT < 29 are to detect the fluorescence signal, and expression detects the corresponding genotype of the probe；29≦CT≦
32, belong to critical value, then needs to redeterminate, such as:
4. example results of table
That is the genotype of sample are as follows: S1:A1298A wild type, S2:A1298C mutation heterozygous, S3:A1298A wild type,
S4:T1298C is resurveyed fixed, S5:A1298A wild type, S6:A1298A wild type, S7:A1298C mutation heterozygous, S8:A1298C
Heterozygous, S9:A1298A wild type, S10:A1298A wild type, S11:A1298C mutation heterozygous, NTC are mutated i.e. without template
Compare no probe signals.
Above description shows and describes several preferred embodiments of the present application, but as previously described, it should be understood that the application
Be not limited to forms disclosed herein, should not be regarded as an exclusion of other examples, and can be used for various other combinations,
Modification and environment, and the above teachings or related fields of technology or knowledge can be passed through in application contemplated scope described herein
It is modified.And changes and modifications made by those skilled in the art do not depart from spirit and scope, then it all should be in this Shen
It please be in the protection scope of appended claims.
Claims (4)
1. a kind of kit for detecting the site mthfr gene A1298C rs1801131SNP, which is characterized in that including basis
The primer and probe of mthfr gene A1298C rs1801131SNP loci polymorphism design, sequence are as follows:
Mthfr gene A1298C forward direction amplimer: GGAGCTGCTGAAGATGTGG；
The reversed amplimer of mthfr gene A1298C: CCTCTCGGGAGAACCAAAC；
Mthfr gene 1298A probe: 5 '-TGAAGAAAGTGTCT-3 '；
Mthfr gene 1298C probe: 5 '-TGAAGCAAGTGTCT-3 '.
2. the kit in the detection site mthfr gene A1298C rs1801131SNP according to claim 1, feature
It is, the fluorophor that the mthfr gene 1298A probe 5 ' is held is VIC, and the quenching group at 3 ' ends is Quencher and MGB.
3. the kit in the detection site mthfr gene A1298C rs1801131SNP according to claim 2, feature
It is, the fluorophor that the mthfr gene 1298C probe 5 ' is held is FAM, and the quenching group at 3 ' ends is Quencher and MGB.
4. the kit in the detection site mthfr gene A1298C rs1801131SNP according to claim 3, feature
It is, mthfr gene A1298C primed probe mixing liquid proportional are as follows: mthfr gene A1298C forward direction amplimer: MTHFR base
Because of the reversed amplimer of A1298C: mthfr gene 1298A probe: mthfr gene 1298C probe: water=10:10:4:4:192.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111593097A (en) * | 2020-04-23 | 2020-08-28 | 长沙金域医学检验实验室有限公司 | Primer composition, reagent, detection method and system for realizing accurate typing of vitamin D receptor Fok1 locus based on probe method |
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| CN103757106A (en) * | 2014-01-07 | 2014-04-30 | 河南科技大学 | Kit and method for detecting gene polymorphism of human MTHFR (methylene tetrahydrofolate reductase) based on Taqman-MGB (Minor Groove Binder) probe |
| CN104109714A (en) * | 2014-06-27 | 2014-10-22 | 湖北维达健基因技术有限公司 | Primer for detecting folic acid metabolism related gene SNP, fluorescent probe and application |
| CN105002275A (en) * | 2015-07-20 | 2015-10-28 | 武汉友芝友医疗科技有限公司 | Specific sequence specific primers-polymerase chain reaction primers and kit for human MTHFR and MTRR gene polymorphism detection |
| CN107988353A (en) * | 2017-12-08 | 2018-05-04 | 益善生物技术股份有限公司 | A kind of mankind MTHFR and/or MTRR genetic polymorphism detection kit |
| CN108456727A (en) * | 2018-04-19 | 2018-08-28 | 深圳会众生物技术有限公司 | Mthfr gene polymorphic detection probe, primer, kit and detection method |
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2018
- 2018-11-21 CN CN201811391611.8A patent/CN110205368A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103757106A (en) * | 2014-01-07 | 2014-04-30 | 河南科技大学 | Kit and method for detecting gene polymorphism of human MTHFR (methylene tetrahydrofolate reductase) based on Taqman-MGB (Minor Groove Binder) probe |
| CN104109714A (en) * | 2014-06-27 | 2014-10-22 | 湖北维达健基因技术有限公司 | Primer for detecting folic acid metabolism related gene SNP, fluorescent probe and application |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111593097A (en) * | 2020-04-23 | 2020-08-28 | 长沙金域医学检验实验室有限公司 | Primer composition, reagent, detection method and system for realizing accurate typing of vitamin D receptor Fok1 locus based on probe method |
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