CN109554465A - Noninvasive rs1801133 genotype rapid typing detection reagent box - Google Patents
Noninvasive rs1801133 genotype rapid typing detection reagent box Download PDFInfo
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Abstract
The present invention provides a kind of noninvasive rs1801133 genotype rapid typing detection reagent box, be related to medicine detection technique, biotechnology and clinical molecular diagnosis technical field, which includes: (1) forward and reverse primer: the forward primer is the nucleotide chain or its complementary strand of sequence shown in MTHFR-CUT-F；The reverse primer is the nucleotide chain or its complementary strand of sequence shown in MTHFR-CUT-A；Forward primer MTHFR-CUT-F, sequence: 5 '-GCCTCTCCTGACTGTCATCC-3 '；Reverse primer MTHFR-CUT-A, sequence: 5 '-AGGACGGTGCGGTGAGAGTG-3 '；(2) reaction reagent；(3) plasmid comprising rs1801133 CC, CT and TT genotype, as positive control and negative control: deionized water.The present invention judges genotype by real time fluorescent quantitative detection with a polymerase chain reaction.The present invention only needs general primer, is not necessarily to probe, and lower production costs are conducive to marketing and industrialization.
Description
Technical field
The present invention relates to medicine detection technique, biotechnology and clinical molecular diagnosis technical fields, specifically a kind of
Noninvasive 5,10-CH2-THFA reductase (MTHFR) rs1801133 genotype rapid typing detection reagent box, specifically relates to
And take a blood sample without wound, it can be detected 5,10-CH2-THFA reductase gene simultaneously using samples such as mouth epithelial cells
The detection kit and its clinical application of parting.
Background technique
5,10-CH2-THFA reductase (5,10-methylenetetrahydrofolatereductase,
MTHFR), main function is to convert 5,10-CH2-THFA to biological function in folic acid metabolism access
5-methyltetrahydrofolate.5-methyltetrahydrofolate can travel further into methyl transmission path, pass through the weight of homocysteine
It is connected in DNA methylation between new methylation procedure and protein methylation provides methyl and makes the homocysteine water in blood
It is flat to be maintained at a lower level.Furthermore the mesostate of folic acid also has important work in nucleotide synthesis process
With, by one carbon unit be metabolized as purine ring formation provide carbon atom.
MTHFR is folic acid metabolism rate-limiting enzyme, and catalysis 5,10-CH2-THFA is converted into 5-methyltetrahydrofolate, from
And it plays an important role in folic acid metabolism, DNA methylation and in repairing.The reduction of MTHFR enzymatic activity caused by mthfr gene makes a variation can
Suppress homocysteine to be converted into methionine, lead to low folic acid mass formed by blood stasis and hyperhomocysteinemiainjury, to increase new
The generation of the diseases such as the raw risks such as youngster's birth defect risk or spontaneous abortion and cancer, the cardiovascular disease of adult.
The rs1801133 (C677T) of MTHFR is a kind of C > T missense mutation, the protein for causing mthfr gene to encode
222 amino acid become Val from Ala, and enzymatic activity and thermal stability is caused to decline.This mutation, institute very common in asian population
With Clinical significance of detecting with higher.If with its MTHFR activity when individual rs1801133CC genotype for 100%, CT genotype
Activity then for CC genotype less than 65%, TT genotype only less than 30%.Therefore, rs1801133 and folic acid metabolism phase
Related disorders are closely related.Rs1801133T allele carrier needs because folic acid metabolism ability is weaker (especially TT genotype)
Reduction birth defect and hypertensive patient's apoplexy, myocardial infarction and mortality risk can effectively be reached by increasing folic acid taking dose.
Wilson etc. and Moslev's etc. studies have shown that folic acid deficiency is neural tube defect (neural in gravid woman's body
Tube defects, NTDs) an important risk factor.Mother augments leaf in the critical period that gestational period Foetal organ is formed
Acid can effectively reduce the risk that NTDs occurs for fetus.Furthermore, the defect of mthfr gene can cause nerve channel to lack Pregnant women
A variety of diseases such as sunken, congenital heart disease, harelip, hypertensive disorder in pregnancy, spontaneous abortion, cancer, cardiovascular and cerebrovascular disease
Disease
Folic acid belongs to B family vitamin, and human body cannot synthesize folic acid, it is necessary to rely on exogenous supply.Folic acid in human body with
The form of tetrahydrofolic acid participates in the formation and metabolism of many important compounds such as the mutual conversion of amino acid, DNA synthesis, is cell
Substance necessary to growth and tissue repair, indispensable nutrient even more in embryo development procedure.The difference of genetic constitution
Body is resulted in the difference of folic acid Utilization ability, therefore, folic acid supplementary behavior should vary with each individual, and augment excessive or very few be all unfavorable for
Foetus health and pregnant woman health.From another point of view, folic acid supplementary behavior should not be only confined in women, and male's Supplement of folic acid has on an equal basis
Important meaning.Research has shown that, folic acid is lacked in males, will lead to sperm quality reduction, the dyeing that sperm carries in sperm
Body quantity is abnormal, causes Newborn Birth-defects.Body folate level is low, and causes infertility, spontaneous abortion
One of the major reasons.Therefore, before newly-married couple prepares pregnancy, folic acid metabolism genetic test should be at least done, is more advantageous in this way
It is excellent pregnant.Therefore, by folic acid metabolism genetic test, it can find that level is absorbed and utilized to folic acid in Different Individual as early as possible, thus
Screening goes out to easily cause the people at highest risk of folic acid deficiency, realizes personalized supplement folic acid.
Summary of the invention
Technical assignment of the invention is to solve the deficiencies in the prior art, provides a kind of noninvasive 5,10-CH2-THFA
Reductase (MTHFR) rs1801133 genotype rapid typing detection reagent box.
CAPs (cleaved amplification polymorphism sequence-tagged sites) technology,
Claiming PCR-RFLP, is to use PCR amplification target DNA, amplified production is cut into different size segment with specificity endonuclease digestion again,
The DNA sequence dna detection technique directly differentiated in gel electrophoresis.The restriction enzyme site of iso-allele is not distributed different, is produced
The DNA fragmentation band of raw different length.Technique substantially increases the content and relative specificity of target DNA, and method
Simplicity, the parting time is short, and the instrument and equipment needed is relatively inexpensive, it is crucial that, the present invention is explored without blood sampling, need to only use mouth
Chamber swab scraping mouth epithelial cells can be detected, and substantially increase materials simplicity, accomplish real whole people's detection.
Based on the advantage of CAPs technology, the present invention explores the method to carry out the rs1801133 (C677T) of MTHFR inspection
It surveys and succeeds, it is therefore an objective to be developed into kit, detecting the rs1801133 (C677T) of MTHFR becomes efficient, great Rong
The detection project of amount and low cost is preferably applied to clinic.
The present invention provides a kind of folic acid metabolism related gene MTHFR hereditary variation detection kit and its application, can be effective
Ground detects MTHFR hereditary variation rs1801133, facilitates clinician and takes correct folic acid taking dose, folic acid metabolism phase
The onset risk of related disorders.
The technical scheme is that realize in the following manner, noninvasive rs1801133 genotype of the invention is quickly divided
Type detection kit, which includes:
(1) forward and reverse primer: the forward primer is nucleotide chain or its complementation of sequence shown in MTHFR-CUT-F
Chain；The reverse primer is the nucleotide chain or its complementary strand of sequence shown in MTHFR-CUT-A；
Forward primer MTHFR-CUT-F, sequence: 5 '-GCCTCTCCTGACTGTCATCC-3 '；
Reverse primer MTHFR-CUT-A, sequence: 5 '-AGGACGGTGCGGTGAGAGTG-3 '；
(2) reaction reagent:
A: oral epithelium DNA extracting solution；
B:PCR reaction solution: MTHFR MIX main reaction liquid；
C:CUT reaction solution: Hinf I enzyme and its buffer；
D:TBE solution: electrophoretic buffer；
E: rubber powder: Ago-Gel；
F: loading concentrate: the Loading Buffer 6X of bromophenol blue configuration；
G: instruction band: DNA Maker (1000-100bp)；
H: color developing agent: Gold-View double-stranded DNA coloring agent；
(3) include rs1801133CC, CT and TT genotype plasmid, as positive control and negative control: go from
Sub- water.
The PCR reaction system of the detection kit is 20 μ L, including 15ul MTHFR MIX main reaction liquid, genome
5 μ L of DNA profiling；Endonuclease reaction system is 20ul, including 10ul PCR reaction product, 10ul CUT reaction solution；Loading system
6ul, including CUT product 5ul, loading concentrate 1ul；Gel preparation method is the TBE solution of 0.5X, is configured to 2% concentration
Ago-Gel contains color developing agent.
The condition of the PCR reaction of the detection kit are as follows:
95 DEG C initial denaturation 5 minutes；
95 DEG C are annealed 20 seconds for denaturation 20 seconds, 65 DEG C, and 72 DEG C extend 20 seconds, 35 circulations；
72 DEG C of terminals extend 5 minutes；
4 ° of preservations.
The program of the endonuclease reaction of CUT reaction solution are as follows: 37 ° 2 hours, 4 ° preservation.
D:TBE solution: electrophoretic buffer；Electrophoresis and the program of analysis are as follows:
120V voltage, 50mA, electrophoresis 30min or so, until bromophenol blue instruction band moves on in the middle part of gel；Gel is put into solidifying
Glue Image analysis system, experimental result should be similar shown in Fig. 1, and wherein CC profile bar band is 282bp, TT profile bar band be 176bp and
106bp, CT profile bar band are 282bp, 176bp and 106bp.
The testing result of the detection kit, to effective amplification and yin and yang attribute control meet it is pre- at that time, sample detection
Credible result, otherwise test experience needs to repeat；Sample amplification is abnormal, and digestion is abnormal, need to exclude manual operation and instrument failure
The problems such as after re-start test experience.
A kind of primer pair for folic acid metabolism related gene MTHFR hereditary variation detection, the primer pair are drawn including forward direction
Object and reverse primer, the nucleotide chain or its complementary strand of sequence shown in the forward primer MTHFR-CUT-F；The reverse primer
For the nucleotide chain or its complementary strand of sequence shown in MTHFR-CUT-A；
Forward primer MTHFR-CUT-F, sequence: 5 '-GCCTCTCCTGACTGTCATCC-3 '；
Reverse primer MTHFR-CUT-A, sequence: 5 '-AGGACGGTGCGGTGAGAGTG-3 '.
A kind of positive quality control for folic acid metabolism related gene MTHFR hereditary variation detection, extension increasing sequence are connected to
In empty plasmid vector, and gene sequencing is carried out, comprising wild, the right-on plasmid of two kinds of sequences of mutation and it is mixed
Close object.
The noninvasive rs1801133 genotype rapid typing detection reagent box, preparation prevention birth defect product with
And the application in preventing hypertension patient's apoplexy product.
The noninvasive rs1801133 genotype rapid typing detection reagent box is high for screening folic acid deficiency in preparation
Application in the product of danger crowd.
Generated beneficial effect is the present invention compared with prior art:
Noninvasive 5,10-CH2-THFA reductase (MTHFR) rs1801133 genotype fast typing inspection of the invention
Test agent box, the present invention is with a polymerase chain reaction, and forward and reverse primer comprising specificity in reaction system passes through
Real time fluorescent quantitative detects to judge genotype.The present invention only needs general primer, is not necessarily to probe, and lower production costs are conducive to
Marketing and industrialization.
The present invention has the advantages such as accurate quickly, the easy interpretation of result of easy to operate, parting, can be used for clinically with folic acid generation
The detection and screening for thanking to related gene MTHFR hereditary variation rs1801133, enable clinician predict pregnant woman, standby pregnant Mr. and Mrs or
Hypertensive patient's folic acid metabolism capabilities might, assists a physician and selects the folic acid of suitable dose, to prevent birth defect and high blood
Press patient's apoplexy.
Detailed description of the invention
Attached drawing 1 is the gel electrophoresis figure in the embodiment of the present invention.
Specific embodiment
With reference to the accompanying drawing to noninvasive 5,10-CH2-THFA reductase (MTHFR) rs1801133 base of the invention
Because type rapid typing detection reagent box is described in detail below.
Noninvasive 5,10-CH2-THFA reductase (MTHFR) rs1801133 genotype fast typing inspection of the invention
Test agent box, for identifying rs1801133 genotype, which contains the kit:
(1) forward and reverse primer: the forward primer is nucleotide chain or its complementation of sequence shown in MTHFR-CUT-F
Chain；The reverse primer is the nucleotide chain or its complementary strand of sequence shown in MTHFR-CUT-A；
(2) reaction reagent:
A: oral epithelium DNA extracting solution
B:PCR reaction solution: MTHFR MIX main reaction liquid；
C:CUT reaction solution: Hinf I enzyme and its buffer；
D:TBE solution: electrophoretic buffer；
E: rubber powder: Ago-Gel；
F: loading concentrate: the Loading Buffer 6X of bromophenol blue configuration；
G: instruction band: DNA Maker (1000-100bp)；
H: color developing agent: Gold-View double-stranded DNA coloring agent；
(3) comprising the plasmid (positive control) and negative control (deionization of rs1801133CC, CT and TT genotype
Water).
As currently preferred technical solution, the PCR reaction system of the detection kit is 20 μ L, including 15ul
MTHFR MIX main reaction liquid, 5 μ L of genomic DNA template；Endonuclease reaction system is 20ul, including 10ul PCR reaction product,
10ul CUT reaction solution；Loading system 6ul, including CUT product 5ul, loading concentrate 1ul；Gel preparation method is 0.5X's
TBE solution is configured to the Ago-Gel of 2% concentration (containing color developing agent).
As currently preferred technical solution, the condition of the PCR reaction of the detection kit are as follows:
95 DEG C initial denaturation 5 minutes；
95 DEG C are annealed 20 seconds for denaturation 20 seconds, 65 DEG C, and 72 DEG C extend 20 seconds, 35 circulations；
72 DEG C of terminals extend 5 minutes；
4 ° of preservations
The program of endonuclease reaction are as follows:
37 ° 2 hours
4 ° of preservations
Electrophoresis and the program of analysis are as follows:
120V voltage, 50mA, electrophoresis 30min or so, until bromophenol blue instruction band moves on in the middle part of gel；Gel is put into solidifying
Glue Image analysis system, experimental result should be similar shown in Fig. 1, and wherein CC profile bar band is 282bp, TT profile bar band be 176bp and
106bp, CT profile bar band are 282bp, 176bp and 106bp.
As currently preferred technical solution, the testing result of the detection kit, to effective amplification and yin and yang attribute
Control meet it is pre- at that time, sample detection credible result, otherwise test experience needs to repeat；Sample amplification is abnormal, and digestion is abnormal
Deng re-starting test experience after the problems such as need to excluding manual operation and instrument failure.
In another aspect of the invention, provide it is a kind of for folic acid metabolism related gene MTHFR hereditary variation detection
Primer pair, the primer pair include forward primer and reverse primer, the nucleotide of sequence shown in the forward primer MTHFR-CUT-F
Chain or its complementary strand；The reverse primer is the nucleotide chain or its complementary strand of sequence shown in MTHFR-CUT-A.
In another aspect of the invention, provide it is a kind of for folic acid metabolism related gene MTHFR hereditary variation detection
Positive quality control, extension increasing sequence are connected in empty plasmid vector, and have carried out gene sequencing, include wild, two kinds of sequences of mutation
Arrange right-on plasmid and its mixture.
In another aspect of the invention, the application of above-mentioned detection kit is provided, preparation prevention birth defect is included in
Application in product and preventing hypertension patient's apoplexy product.
First part: the composition of kit and interpretation of result
1. human gene group DNA extracts:
The suitable sample of this kit includes being not limited to whole blood, saliva or buccal swab equal samples type, and genomic DNA is taken out
It mentions and the DNA in tissue is extracted and purified using commercialized kit.
Also the DNA extracting solution that the offer of this reagent can be used extracts mouth epithelial cells DNA.
2. the formula and composition of kit:
1 reaction system formula of table
| Constituent and concentration | Volume |
| 2x PCR MIX (contains Dye) | 10ul |
| Forward primer (1 μM) | 2ul |
| Reverse primer (1 μM) | 2ul |
| Genomic DNA (10-100ng/ μ L) | 5ul |
| ddH2O | 1ul |
2 CUT of table reacts formula of liquid
| Constituent and concentration | Volume |
| 10x Cut Buffer H | 2ul |
| Hinf I(6000U/ul) | 1ul |
| PCR reaction solution | 10ul |
| ddH2O | 7ul |
3 primer sequence of table
3. kit tests response procedures:
4 PCR program of table and analysis program
5 endonuclease reaction program of table
| Stage | Recurring number (a) | Temperature (DEG C) | Time (hour) | Remarks |
| Digestion | 1 | 37 | 2-3 | |
| It is cooling | 1 | 4 | For a long time |
5. interpretation of result
With reference to Fig. 1, the rs1801133 genotype in sample is judged according to pillar location and number.
The wherein premise of credible result are as follows:
A, sample strip band is normal and naked eyes are visible, bright.
B, negative quality-control product is without amplification.
C, three kinds of positive quality control product experimental results should be similar shown in Fig. 1, and wherein CC profile bar band is 282bp, TT profile bar band
For 176bp and 106bp, CT profile bar band is 282bp, 176bp and 106bp.
If ABC meets the requirements simultaneously, it is CC type that sample, which meets wild type Quality Control band, meets saltant type Quality Control
Band is TT type, and meeting heterozygous Quality Control band is CT type.
It is not inconsistent if ABC has, needs after excluding operation, kit or sample problem, re-start experiment.
Second part: the clinical application of testing result
It takes basal dose in conjunction with the folic acid of rs1801133 genotype and is shown in Table 4, for being associated with of other high risk factors
Body can on this basis augment folate dose.
The suggestion amount that 6 folic acid of table is taken
Claims (10)
1. noninvasive rs1801133 genotype rapid typing detection reagent box, it is characterised in that the kit includes:
(1) forward and reverse primer: the forward primer is nucleotide chain or its complementation of sequence shown in MTHFR-CUT-F
Chain；The reverse primer is the nucleotide chain or its complementary strand of sequence shown in MTHFR-CUT-A；
Forward primer MTHFR-CUT-F, sequence: 5 '-GCCTCTCCTGACTGTCATCC-3 '；
Reverse primer MTHFR-CUT-A, sequence: 5 '-AGGACGGTGCGGTGAGAGTG-3 '；
(2) reaction reagent:
A: oral epithelium DNA extracting solution；
B:PCR reaction solution: MTHFR MIX main reaction liquid；
C:CUT reaction solution: Hinf I enzyme and its buffer；
D:TBE solution: electrophoretic buffer；
E: rubber powder: Ago-Gel；
F: loading concentrate: the Loading Buffer 6X of bromophenol blue configuration；
G: instruction band: DNA Maker(1000-100bp)；
H: color developing agent: Gold-View double-stranded DNA coloring agent；
(3) plasmid comprising rs1801133 CC, CT and TT genotype, as positive control and negative control: deionization
Water.
2. noninvasive rs1801133 genotype rapid typing detection reagent box according to claim 1, it is characterised in that: institute
The PCR reaction system for stating detection kit is 20 μ L, including 15ul MTHFR MIX main reaction liquid, 5 μ L of genomic DNA template；
Endonuclease reaction system is 20ul, including 10ul PCR reaction product, 10ul CUT reaction solution；Loading system 6ul, including CUT are produced
Object 5ul, loading concentrate 1ul；Gel preparation method is the TBE solution of 0.5X, is configured to the Ago-Gel of 2% concentration, containing aobvious
Toner.
3. noninvasive rs1801133 genotype rapid typing detection reagent box according to claim 1, it is characterised in that: institute
State the condition of the PCR reaction of detection kit are as follows:
95 DEG C initial denaturation 5 minutes；
95 DEG C are annealed 20 seconds for denaturation 20 seconds, 65 DEG C, and 72 DEG C extend 20 seconds, 35 circulations；
72 DEG C of terminals extend 5 minutes；
4 ° of preservations.
4. noninvasive rs1801133 genotype rapid typing detection reagent box according to claim 1, it is characterised in that: CUT
The program of the endonuclease reaction of reaction solution are as follows: 37 ° 2 hours, 4 ° preservation.
5. noninvasive rs1801133 genotype rapid typing detection reagent box according to claim 1, it is characterised in that: D:
TBE solution: electrophoretic buffer；Electrophoresis and the program of analysis are as follows:
120V voltage, 50mA, electrophoresis 30min or so, until bromophenol blue instruction band moves on in the middle part of gel；Gel be put into gel at
As analysis system, experimental result should be similar shown in Fig. 1, and wherein CC profile bar band is 282bp, TT profile bar band be 176bp and
106bp, CT profile bar band are 282bp, 176bp and 106bp.
6. noninvasive rs1801133 genotype rapid typing detection reagent box according to claim 1, it is characterised in that: institute
The testing result for stating detection kit, to effective amplification and yin and yang attribute control meet it is pre- at that time, sample detection credible result is no
Then test experience needs to repeat；After the problems such as sample amplification is abnormal, and digestion is abnormal, need to exclude manual operation and instrument failure again
Carry out test experience.
7. a kind of primer pair for folic acid metabolism related gene MTHFR hereditary variation detection, which includes forward primer
And reverse primer, the nucleotide chain or its complementary strand of sequence shown in the forward primer MTHFR-CUT-F；The reverse primer is
The nucleotide chain of sequence shown in MTHFR-CUT-A or its complementary strand；
Forward primer MTHFR-CUT-F, sequence: 5 '-GCCTCTCCTGACTGTCATCC-3 '；
Reverse primer MTHFR-CUT-A, sequence: 5 '-AGGACGGTGCGGTGAGAGTG-3 '.
8. a kind of positive quality control for folic acid metabolism related gene MTHFR hereditary variation detection, extension increasing sequence are connected to sky
In plasmid vector, and gene sequencing is carried out, has included wild, the right-on plasmid of two kinds of sequences of mutation and its mixing
Object.
9. noninvasive rs1801133 genotype rapid typing detection reagent box described in claim 1 prevents birth defect in preparation
Application in product and preventing hypertension patient's apoplexy product.
10. noninvasive rs1801133 genotype rapid typing detection reagent box described in claim 1 is used for screening leaf in preparation
Application in the product of acid heat people at highest risk.
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| CN201811649183.4A CN109554465A (en) | 2018-12-30 | 2018-12-30 | Noninvasive rs1801133 genotype rapid typing detection reagent box |
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| CN201811649183.4A CN109554465A (en) | 2018-12-30 | 2018-12-30 | Noninvasive rs1801133 genotype rapid typing detection reagent box |
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