CN110195110A - A kind of kit detecting the site mthfr gene C677T rs1801133SNP - Google Patents
A kind of kit detecting the site mthfr gene C677T rs1801133SNP Download PDFInfo
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- CN110195110A CN110195110A CN201811390920.3A CN201811390920A CN110195110A CN 110195110 A CN110195110 A CN 110195110A CN 201811390920 A CN201811390920 A CN 201811390920A CN 110195110 A CN110195110 A CN 110195110A
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- 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
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Abstract
The purpose of the application is to disclose a kind of kit for detecting the site mthfr gene C677T rs1801133SNP, it is characterized in that, including the primer and probe designed according to mthfr gene C677T rs1801133SNP loci polymorphism, sequence is as follows: mthfr gene C677T forward direction amplimer: GGAGCTTTGAGGCTGACC；The reversed amplimer of mthfr gene C677T: GAGGCTGACACATTCTTCCG；Mthfr gene 677C probe: 5 '-GGAGCCGATTTCATCAT-3 '；Mthfr gene 677T probe: 5 '-GGAGTCGATTTCATCAT-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 C677T rs1801133SNP 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: the judgement link of 1. results needs gel electrophoresis, and be related to glue, point sample,
Many cumbersome links such as glue, imaging are run, while being easy error, human input is bigger；2. result is presented in the form of picture,
Interpretation process relies on subjective factor, is easy error；(3) pcr amplification product needs processing of uncapping, because DNA concentration is big at this time, easily
It causes indoor aerosols 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 examination for detecting the site mthfr gene C677T rs1801133SNP
Agent box, 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 MTHFR
The kit in the site gene C 677T rs1801133SNP practices that operating effect is bad, operation difficulty is high, is difficult to replicate operation
Technical problem.
In order to solve the above-mentioned technical problem, the invention discloses a kind of detection mthfr gene C677T rs1801133SNP
The kit in site, its technical solution is as follows:
A kind of kit detecting the site mthfr gene C677T rs1801133SNP, which is characterized in that including basis
The primer and probe of mthfr gene C677T rs1801133SNP loci polymorphism design, sequence are as follows:
Mthfr gene C677T forward direction amplimer: GGAGCTTTGAGGCTGACC；
The reversed amplimer of mthfr gene C677T: GAGGCTGACACATTCTTCCG；
Mthfr gene 677C probe: 5 '-GGAGCCGATTTCATCAT-3 '；
Mthfr gene 677T probe: 5 '-GGAGTCGATTTCATCAT-3 '.
Preferably, the fluorophor that the mthfr gene 677C probe 5 ' is held is VIC, and the quenching group at 3 ' ends is
Quencher and MGB.
Preferably, the fluorophor that the mthfr gene 677T probe 5 ' is held is FAM, and the quenching group at 3 ' ends is
Quencher and MGB.
Preferably, mthfr gene C677T primed probe mixing liquid proportional are as follows: mthfr gene C677T forward direction amplimer:
The reversed amplimer of mthfr gene C677T: mthfr gene 677C probe: mthfr gene 677T 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 667C type VIC, 677T in the mthfr gene C677T mentioned in this experiment
Type is modified (protection scope should expand, and illustrate comprising other modification modes, also within the scope of this patent) with FAM, and 3 '
Terminal modified Quencher and MGB, because Quencher is quenching group, it and fluorophor are existed simultaneously in a DNA chain
When, fluorophor cannot issue fluorescence, therefore under normal circumstances, and equipment cannot detect fluorescence signal.Only when into
When row Q-PCR reacts, it with probe specificity is integrated to SNP site, and archaeal dna polymerase included in reaction solution can be
Probe is sheared from target fragment into reaction solution, and the fluorescence in correspondent probe also divides with quenching group Quencher therewith
From, it is captured by equipment, and detect 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, strong 5. pollution risk of sample process ability is small, without processing of uncapping after PCR, will not generate aerosol, cause
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 C677T rs1801133SNP, which is characterized in that including basis
The primer and probe of mthfr gene C677T rs1801133SNP loci polymorphism design, sequence are as follows:
Mthfr gene C677T forward direction amplimer: GGAGCTTTGAGGCTGACC；
The reversed amplimer of mthfr gene C677T: GAGGCTGACACATTCTTCCG；
Mthfr gene 677C probe: 5 '-GGAGCCGATTTCATCAT-3 '；
Mthfr gene 677T probe: 5 '-GGAGTCGATTTCATCAT-3 '.
The fluorophor that the mthfr gene 677C probe 5 ' is held is VIC, the quenching groups at 3 ' ends be Quencher and
MGB。
The fluorophor that the mthfr gene 677T probe 5 ' is held is FAM, the quenching groups at 3 ' ends be Quencher and
MGB。
Mthfr gene C677T primed probe mixing liquid proportional are as follows: mthfr gene C677T forward direction amplimer: MTHFR base
Because of the reversed amplimer of C677T: mthfr gene 677C probe: mthfr gene 677T 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: it is wild that S1:C677T is mutated heterozygous, S2:C677T mutation heterozygous, S3:C677C
Type, S4: it is prominent that fixed, S5:C677C wild type, S6:C677C wild type, S7:C677T mutation heterozygous, S8:T677T homozygosis are resurveyed
Modification, S9:C677T mutation heterozygous, S10:C677C wild type, S11:T677T homozygous mutant, NTC, that is, no template control do not have
There are 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 C677T rs1801133SNP, which is characterized in that including basis
The primer and probe of mthfr gene C677T rs1801133SNP loci polymorphism design, sequence are as follows:
Mthfr gene C677T forward direction amplimer: GGAGCTTTGAGGCTGACC；
The reversed amplimer of mthfr gene C677T: GAGGCTGACACATTCTTCCG；
Mthfr gene 677C probe: 5 '-GGAGCCGATTTCATCAT-3 '；
Mthfr gene 677T probe: 5 '-GGAGTCGATTTCATCAT-3 '.
2. the kit in the detection site mthfr gene C677T rs1801133SNP according to claim 1, feature exist
In the fluorophor that the mthfr gene 677C 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 C677T rs1801133SNP according to claim 2, feature exist
In the fluorophor that the mthfr gene 677T 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 C677T rs1801133SNP according to claim 3, feature exist
In mthfr gene C677T primed probe mixing liquid proportional are as follows: mthfr gene C677T forward direction amplimer: mthfr gene
The reversed amplimer of C677T: mthfr gene 677C probe: mthfr gene 677T probe: water=10:10:4:4:192.
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| CN201811390920.3A CN110195110A (en) | 2018-11-21 | 2018-11-21 | A kind of kit detecting the site mthfr gene C677T rs1801133SNP |
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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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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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Application publication date: 20190903 |