CN106434923A - MTHFR gene C677T non-invasive detection kit and method - Google Patents
MTHFR gene C677T non-invasive detection kit and method Download PDFInfo
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Abstract
The invention relates to an MTHFR gene C677T non-invasive detection kit and a method. The kit comprises PCR reaction liquid, a negative control and a positive control. The kit is used for detection by means of a Taqman-MGB fluorescent probe through the PCR method, and a detection locus is an MTHFR gene C677T polymorphic site. The Taqman-MGB probe can effectively reduce background signals, is more sensitive to single base mutation and can quickly judge different genotypes according to detection result Ct values, subsequent analysis of a PCR product is not needed, the aim of non-invasive detection is achieved by means of buccal mucosal cells, and materials are easy and quick to obtain. The detection method using the kit is easy and quick to operate, consumable use is reduced, detection cost is reduced conveniently, detection results are accurate, and false positive results are reduced.
Description
Technical field
The present invention relates to field of gene detection, more particularly, to a kind of mthfr gene C677T noninvasive detection kit and side
Method.
Background technology
Methylene tetrahydrofolate reductase gene (MTHFR) is present in animal's liver.As in folic acid methylation procedure
Key enzyme, its Main Function is that 5,10- tetrahydrochysene methopterin is reduced to 5-methyltetrahydrofolate, is homocysteine conversion
There is provided methyl for methionine, and be DNA, participate in the synthesis of purine and pyrimidine in the form of one carbon unit, and for DNA, RNA and
Protein methylation provides methyl, affects DNA metabolism.The methyl of multiple bio-matrix such as protein and the neurotransmission factor supplies
Body.The internal essential amino acid of methionine behaviour, methionine biosynthesis block makes protein resulting anomaly.As methyl donor
Participate in methionine metabolism, thus maintaining homocysteine in plasma (Hcy) normal level, Hcy metabolic pathway.
After MTHFR 677C-T mutation mutation, its biologically active only has the 50% of normal enzymatic activity, and it is same with blood plasma
Type cysteine raises relevant.MTHFR C677T mutational site is the modal thermo-labile missense of the mthfr gene having now been found that
Mutation, research shows that this mutation leads to MTHFR enzymatic activity to decline and so that Hcy is methylated obstacle, leads to Hcy level to raise.
Hyperhomocysteinemiainjury is the cardiovascular disease risk factor.MTHFR is the key enzyme of Hcy metabolism, this gene
C677T is most commonly seen mutation type in Hcy metabolic pathway.The mutation of C → T makes its activity and heat resistance reduce and cause
In Hcy blood, level raises, and plasma Hcy level raises vascular endothelial cell injury that can be direct or indirect so that ET-1 synthesis increases
Plus, NO and PGI2 secretion reduces, and destruction blood vessel relaxes, the balance of the contracting factor.Do Hcy also can stimulated vascular smooth muscle cell to breed,
Reduce the compliance of vascular wall, cause vessel wall elasticity to reduce.The rising of therefore Hcy level take part in cardiovascular pathologic, physiologic mistake
Journey.Folic acid is the necessary factor that Maxillary region is developed, and may result in methylene tetrahydrofolate during folic acid metabolism after MTHFR mutation
The disappearance of reductase function makes folic acid concentration decline, thus increasing the probability that fetus suffers from non-syndromic cleft lip with cleft palate.
Premature labor is common complications of pregnancy, accounts for the 5%～15% of childbirth sum.There are about 15% premature in neonate
Phase is dead, accounts for more than the 70% of neonatal death.In the premature of survival, 10%～30% leaves the intelligence development obstacle in long term
Or the sequelae of nervous system.MTHFR mutation can make Hcy metabolism be obstructed, and accumulation in vivo causes high homotype semicanal propylhomoserin mass formed by blood stasis.
High homotype semicanal propylhomoserin mass formed by blood stasis is had with multiple miscarriage, preeclampsia, placental abruption, FGR, fetal anomaly, stillborn foetus
Close, and closely related with premature labor.In the key enzyme of impact Hcy metabolism, MTHFR is paid attention to by research the most.This be due to
There is the activity that gene mutation can reduce enzyme in MTHFR, be the important inherent cause causing Hcy concentration to raise.
The method measuring Hcy is a lot, using most have high performance liquid chromatography (HPLC), enzyme immunoassay (EIA) (EIA),
The chromatography of ions, fluorescence polarization immunoassay.Clinically using more be high performance liquid chromatography and meteorological chromatographic mass spectrometry connection
Usage.But these methods all have an experiment, and time-consuming, relatively costly, poor repeatability, complex operation, be difficult to popularize and promote etc. to lack
Point.And many in the conventional method with whole blood as sample, sampling is complex, because the shortage of folic acid and homocysteine are to very
The impact of many relevant diseases is so that the genetic polymorphism detection of MTHFR is particularly important, and the detection side that clinic is commonly used at present
There are some defects and lack foresight in method, in order to predict homocysteine level earlier and prevent and intervenes with homotype partly
The generation of disease and progress that cystine is associated it is necessary to the level of homocysteine is carried out as early as possible carry out pre-
Survey.
Content of the invention
For solving above-mentioned technical problem, the present invention provides a kind of mthfr gene C677T noninvasive detection kit and method.
The present invention designs specific primer and probe according to mthfr gene C677T loci polymorphism, by Taqman-MGB
Fluorescent PCR method, carries out Non-invasive detection with mouth desquamated cells to mthfr gene C677T site, can quickly and accurately detect
Mthfr gene C677T loci polymorphism.
According to embodiments of the invention, the present invention provides a kind of drawing for the detection of mthfr gene C677T loci polymorphism
Thing and probe, wherein：
Primer combination of probe middle and upper reaches primer for detecting mthfr gene C677T site is SEQ ID NO:1；
Primer combination of probe middle and lower reaches primer for detecting mthfr gene C677T site is SEQ ID NO:2；
For detecting that in the primer combination of probe in mthfr gene C677T site, mutated-genotype detection probe is SEQ ID
NO:3；
For detecting that in the primer combination of probe in mthfr gene C677T site, wild-type genotype detection probe is SEQ ID
NO:4；
Primer and the probe of detection mthfr gene C677T loci polymorphism is contained in reactant liquor in this described kit
Combination.
It is used in described PCR reactant liquor detecting that the primer concentration of mthfr gene C677T loci polymorphism is 200nM,
Concentration and probe concentration is 100nM.
MgCL is also included in described PCR reactant liquor2, Taq enzyme, dUTP, dATP, dCTP, dGTP, UDG enzyme, 10 ×
PCR Buffer.
Described MgCL2React final concentration of 3.5mM.
Described Taq enzyme reacts final concentration of 3U.
Described dUTP, dATP, dCTP, dGTP reacts final concentration of 0.6mM.
The described final concentration of 0.2U of UDG enzyme reaction.
Described 10 × PCR Buffer reacts final concentration of 1 × PCR Buffer.
The further present invention adopts Taqman-MGB fluorescent probe PCR technology to mthfr gene C677T loci polymorphism
Carry out double fluorescent detection.
The invention has the beneficial effects as follows：This kit and method adopt real-time fluorescence quantitative PCR to combine Taqman-MGB spy
Carry out double fluorescent detection for mthfr gene C677T loci polymorphism, the method is simple to operate, detection flux is high, quick,
Sensitivity height and high specificity, detection of drawing materials, need not carry out nucleic acid extraction to sample, can effectively shorten detection cycle and cost.
Brief description
Fig. 1 is the pattern detection amplification curve that mthfr gene C677T loci polymorphism is wild type；
Fig. 2 is the pattern detection amplification curve that mthfr gene C677T loci polymorphism is saltant type；
Fig. 3 is the pattern detection amplification curve that mthfr gene C677T loci polymorphism is heterozygote genotype.
Specific embodiment
The present invention is detected to mthfr gene polymorphism using Taqman-MGB fluorescence probe PCR method, detection site
For C677T site, positive control adopts the wild type of known mthfr gene C677T loci gene type, saltant type, heterozygosis subbase
Because of type human gene group DNA, negative control adopts aqua sterilisa to prevent system to be contaminated.
Primer combination of probe middle and upper reaches primer for detecting mthfr gene C677T site is SEQ ID NO:1；
Primer combination of probe middle and lower reaches primer for detecting mthfr gene C677T site is SEQ ID NO:2；
For detecting that in the primer combination of probe in mthfr gene C677T site, mutated-genotype detection probe is SEQ ID
NO:3；
For detecting that in the primer combination of probe in mthfr gene C677T site, wild-type genotype detection probe is SEQ ID
NO:4；
Primer and the probe of detection mthfr gene C677T loci polymorphism is contained in reactant liquor in this described kit
Combination.
It is used in described PCR reactant liquor detecting that the primer concentration of mthfr gene C677T loci polymorphism is 200nM,
Concentration and probe concentration is 100nM.
MgCL is also included in described PCR reactant liquor2, Taq enzyme, dUTP, dATP, dCTP, dGTP, UDG enzyme, 10 ×
PCR Buffer.
Described MgCL2React final concentration of 3.5mM.
Described Taq enzyme reacts final concentration of 3U.
Described dUTP, dATP, dCTP, dGTP reacts final concentration of 0.6mM.
The described final concentration of 0.2U of UDG enzyme reaction.
Described 10 × PCR Buffer reacts final concentration of 1 × PCR Buffer.
The further present invention adopts Taqman-MGB fluorescent probe PCR technology to mthfr gene C677T loci polymorphism
Carry out double fluorescent detection.
Mthfr gene C677T loci polymorphism detection of the present invention comprises the following steps：
1. fluorescent PCR augmentation detection：The PCR reactant liquor that described fluorescent PCR augmentation detection includes the present invention is treated simultaneously
Sample this (mouth desquamated cells) and positive control, negative control carry out augmentation detection.
2.PCR augmentation detection reaction condition：37℃5min；95℃5min；95 DEG C of 15S, 60 DEG C of 60S, 40cycles.
4. interpretation of result：Ct value according to amplification curve judges detected sample corresponding mthfr gene C677T site base
Because of type.
Embodiment 1
1. fluorescent PCR augmentation detection：With PCR reactant liquor to mouth desquamated cells sample, wild type, saltant type, heterozygote
Genotype positive control, negative control carries out augmentation detection, and the wherein primer concentration in PCR reactant liquor is 200nM, and probe is dense
Degree is 100nM, MgCL2React final concentration of 3.5mM, Taq enzyme reacts final concentration of 3U, and dUTP, dATP, dCTP, dGTP are anti-
Should final concentration of 0.6mM, the final concentration of 0.2U of UDG enzyme reaction, the 10 × PCR Buffer final concentration of 1 × PCR of reaction
Buffer, detect sample, wild type, saltant type, heterozygote genotype positive control, negative control all adds 10 μ l, plus go from
Sub- water supplies volume to 50 μ l.Primed probe information is as follows：
For detect mthfr gene C677T site primer combination of probe middle and upper reaches primer be 5 '-
CATCCCTATTGGCAGGTTAC-3’(SEQ ID NO:1)
For detect mthfr gene C677T site primer combination of probe middle and lower reaches primer be 5 '-
GACGATGGGGCAAGTGAT-3’(SEQ ID NO:2)
For detecting that in the primer combination of probe in mthfr gene C677T site, mutated-genotype detection probe is 5 '-FAM-
AAGACTGCGGGAGTCGATTTCATCAT-MGB-3’(SEQ ID NO:3)
For detecting that in the primer combination of probe in mthfr gene C677T site, wild-type genotype detection probe is 5 '-VIC-
AAGACTGCGGGAGCCGATTTCATCAT-MGB-3’(SEQ ID NO:4)
PCR augmentation detection reaction condition：37℃5min；95℃5min；95 DEG C of 15S, 60 DEG C of 60S, 40cycles.
3. interpretation of result：
(1) testing result can use condition：Wild type, saltant type, heterozygote genotype positive control all have corresponding detection bent
Line, that is, wild-type positive comparison VIC sense channel has detection curve and Ct value is less than 36, and knee of curve understands, FAM sense channel
No detection curve；Saltant type positive control FAM sense channel has detection curve and Ct value is less than 36, and knee of curve understands, VIC examines
Survey passage no detection curve；Heterozygote genotype positive control VIC sense channel and FAM sense channel all have detection curve and Ct
Value is less than 36, and knee of curve understands；Negative control VIC sense channel and FAM sense channel all no amplification curves, otherwise experiment is lost
Lose.
(2) different colours are arranged to different sense channels, and according to different passage augmentation detection result judgement mthfr genes
C677T loci polymorphism situation.
Fig. 1 is the pattern detection result that mthfr gene C677T loci polymorphism is wild type, and wherein VIC sense channel has
Detection curve and Ct value are less than 36, and knee of curve understands, FAM sense channel no detection curve.
Fig. 2 is the pattern detection result that mthfr gene C677T loci polymorphism is saltant type, and wherein FAM sense channel has
Detection curve and Ct value are less than 36, and knee of curve understands, VIC sense channel no detection curve.
Fig. 3 is the pattern detection result that mthfr gene C677T loci polymorphism is heterozygote genotype, and wherein VIC detects
Passage and FAM sense channel all have detection curve and Ct value is less than 36, and knee of curve understands.
It is an object of the invention to provide a kind of mthfr gene C677T noninvasive detection kit and method.Real-time fluorescence
PCR is collected by the amplification to template and to the fluorescence signal of each amplification cycles generation, thus realizing to starting template
Detection and analysis.The present invention is detected to mthfr gene C677T loci polymorphism by PCR reactant liquor, with known MTHFR
The human gene group DNA of gene C 677T loci polymorphism is positive control, and negative control adopts aqua sterilisa to prevent system dirty
Dye.
In this description, the present invention is described with reference to its specific embodiment.But it is clear that still can make
Various modifications and alterations are without departing from the spirit and scope of the present invention.Therefore, specification and drawings be considered as illustrative
And it is nonrestrictive.
Claims (9)
1. a kind of mthfr gene C677T noninvasive detection kit and method are it is characterised in that described kit includes：
Primer combination of probe middle and upper reaches primer for detecting mthfr gene C677T site is SEQ ID NO:1；
Primer combination of probe middle and lower reaches primer for detecting mthfr gene C677T site is SEQ ID NO:2；
For detecting that in the primer combination of probe in mthfr gene C677T site, mutated-genotype detection probe is SEQ ID NO:
3；
For detecting that in the primer combination of probe in mthfr gene C677T site, wild-type genotype detection probe is SEQ ID NO:
4.
2. a kind of mthfr gene C677T noninvasive detection kit and method, including PCR reactant liquor, positive control, negative control,
It is characterized in that：Described PCR reactant liquor comprises to detect the primer combination of probe of mthfr gene C677T loci polymorphism.
3. as claimed in claim 2 a kind of mthfr gene C677T noninvasive detection kit and method it is characterised in that described
Probe be Taqman-MGB probe.
4. as claimed in claim 2 a kind of mthfr gene C677T noninvasive detection kit and method it is characterised in that described
MgCL2React final concentration of 3.5mM.
5. as claimed in claim 2 a kind of mthfr gene C677T noninvasive detection kit and method it is characterised in that described
Taq enzyme react final concentration of 3U.
6. as claimed in claim 2 a kind of mthfr gene C677T noninvasive detection kit and method it is characterised in that described
DUTP, dATP, dCTP, dGTP react final concentration of 0.6mM.
7. as claimed in claim 2 a kind of mthfr gene C677T noninvasive detection kit and method it is characterised in that described
The final concentration of 0.2U of UDG enzyme reaction.
8. as claimed in claim 2 a kind of mthfr gene C677T noninvasive detection kit and method it is characterised in that PCR
Augmentation detection reaction condition is 37 DEG C of 5min；95℃5min；95 DEG C of 15S, 60 DEG C of 60S, 40cycles.
9. a kind of mthfr gene C677T noninvasive detection kit and method are it is characterised in that include following operating procedure：
(1) fluorescent PCR augmentation detection：Described double fluorescent PCR augmentation detection is to treat sample basis and sun using PCR reactant liquor
Property comparison, negative control carry out augmentation detection；
(3) interpretation of result：Judge the genotype in sample mthfr gene C677T site to be checked according to the Ct value of amplification curve.
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Cited By (7)
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|---|---|---|---|---|
| CN107267645A (en) * | 2017-07-31 | 2017-10-20 | 广州德臻生物技术有限公司 | Primer pair, probe and kit for detecting mthfr gene polymorphism |
| CN108866168A (en) * | 2018-08-13 | 2018-11-23 | 上海佰臻生物科技有限公司 | A kind of primer and probe for people's mthfr gene SNP detection |
| CN109161589A (en) * | 2018-10-07 | 2019-01-08 | 浙江数问生物技术有限公司 | A kind of people TGFBI gene mutation detection kit and its detection method |
| CN109321648A (en) * | 2018-02-14 | 2019-02-12 | 重庆京因生物科技有限责任公司 | MTHFR (C677T) genotype quick detection kit based on POCT mode |
| CN110195110A (en) * | 2018-11-21 | 2019-09-03 | 长沙金域医学检验所有限公司 | A kind of kit detecting the site mthfr gene C677T rs1801133SNP |
| CN110272987A (en) * | 2019-06-11 | 2019-09-24 | 中山大学达安基因股份有限公司 | A kind of site mthfr gene C677T rapid typing detection reagent box and method |
| CN114657243A (en) * | 2022-05-12 | 2022-06-24 | 广州知力医学诊断技术有限公司 | Primer and kit for detecting genetic anticoagulant protein deficiency and fibrinogen abnormal high-frequency gene mutation |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107267645A (en) * | 2017-07-31 | 2017-10-20 | 广州德臻生物技术有限公司 | Primer pair, probe and kit for detecting mthfr gene polymorphism |
| CN107267645B (en) * | 2017-07-31 | 2021-10-08 | 广州德臻生物技术有限公司 | Primer pair, probe and kit for detecting MTHFR gene polymorphism |
| CN109321648A (en) * | 2018-02-14 | 2019-02-12 | 重庆京因生物科技有限责任公司 | MTHFR (C677T) genotype quick detection kit based on POCT mode |
| CN108866168A (en) * | 2018-08-13 | 2018-11-23 | 上海佰臻生物科技有限公司 | A kind of primer and probe for people's mthfr gene SNP detection |
| CN109161589A (en) * | 2018-10-07 | 2019-01-08 | 浙江数问生物技术有限公司 | A kind of people TGFBI gene mutation detection kit and its detection method |
| CN110195110A (en) * | 2018-11-21 | 2019-09-03 | 长沙金域医学检验所有限公司 | A kind of kit detecting the site mthfr gene C677T rs1801133SNP |
| CN110272987A (en) * | 2019-06-11 | 2019-09-24 | 中山大学达安基因股份有限公司 | A kind of site mthfr gene C677T rapid typing detection reagent box and method |
| CN114657243A (en) * | 2022-05-12 | 2022-06-24 | 广州知力医学诊断技术有限公司 | Primer and kit for detecting genetic anticoagulant protein deficiency and fibrinogen abnormal high-frequency gene mutation |
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