CN107760769A - Mthfr gene polymorphic detection system and its kit - Google Patents
Mthfr gene polymorphic detection system and its kit Download PDFInfo
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Abstract
The present invention relates to a kind of mthfr gene polymorphic detection system and its kit, kit includes PCR detection liquid and SYBR GreenI mixed liquors；PCR detection liquid includes being used for the upstream and downstream primer and peptide nucleic acid for detecting mthfr gene C677T sites；The nucleotide sequence of peptide nucleic acid and the site complete complementary of wild type 677.C.677 polymorphic detection system and its kit sensitivity and specificity are high for the mthfr gene of the present invention, and sample requirements are few, can carry out mthfr gene polymorphic detection exactly with rapid and convenient.
Description
Technical field
The present invention relates to a kind of method for detecting mthfr gene polymorphism and detection product, belong to biological technical field.
Background technology
Genomic DNA changes on a certain specific nucleotide position, transversion, insertion or missing, and it is in group
Frequency >=1% occurred in body, referred to as SNP (SNP).MTHFR full name 5,10-
Methylenetetrahydrofolate reductase (5,10-CH2-THFA reductase), positioned at No.1 chromosome
Lp36.3 positions.Methylenetetrahydrofolate reductase is a highly important enzyme in human body folic acid metabolism.Folic acid and its generation
Thank to intermediate product in organism physiology biochemical reaction to play an important roll, once folate deficient or folic acid metabolism enzyme occur for body
The defects of, it may result in that the cell cycle is abnormal, and DNA, protein methylation abnormal reaction, DNA base can not be synthesized normally
A variety of biochemical reactions are abnormal, so as to directly or indirectly cause the cancer of neonate or Foetus neural tube defect and adult,
The generation of angiocardiopathy.The polymorphism in the sites of MTHFR 677 is to influence a key factor of the enzymatic activity, causes enzymatic activity
Decline with heat endurance.If its MTHFR activity is 100% when carrying 677CC genotype with individual, the activity of CT genotype is carried
It is then the 71% of CC, genotype is only the 34% of TT types.At present mainly using generation sequencing, FLP, the widow of allele specific
The technologies such as nucleotide hybridization, the PCR of allele specific and genetic chip, but operating process is cumbersome, it is necessary to change reaction tube
And easily pollute, need electrophoresis and multistep to post-process more, influence the accuracy of testing result.Or instrument and equipment is expensive, it is difficult to
Large-scale promotion application.
Therefore, a kind of product for detecting mthfr gene polymorphism is developed, to realize more high sensitivity, more inexpensive, more square
Just efficiently mthfr gene polymorphic detection is necessary.
The content of the invention
High the technical problem to be solved in the present invention is to provide a kind of sensitivity and specificity, sample requirements are few, can be fast
Speed easily and accurately carries out the mthfr gene polymorphic detection system and its kit of mthfr gene polymorphic detection.
The present invention is to solve a kind of technical scheme that above-mentioned technical problem proposes to be：A kind of mthfr gene polymorphic detection
System, including for detecting the upstream and downstream primer in mthfr gene C677T sites, fluorescent dye SYBR GreenI, and peptide core
Acid；
The nucleotide sequence of the sense primer is as shown in SEQ ID No.1, and the nucleotide sequence of the anti-sense primer is such as
Shown in SEQ ID No.2；
The nucleotide sequence of the peptide nucleic acid and MTHFR wild type gene C677T site complete complementaries and nucleotide sequence
As shown in SEQ ID No.3.
Above-mentioned upstream and downstream primer has carried out LNA modifications.
Ultimate density of the above-mentioned upstream and downstream primer in reaction system is 0.1~0.3 μM/L.
Ultimate densities of the above-mentioned sense primer A in reaction system is 0.17 μM/L, and the anti-sense primer B is in reaction system
In ultimate density be 0.25 μM/L.
Ultimate density of the above-mentioned peptide nucleic acid in reaction system is 1~1.5 μM/L.
Ultimate density of the above-mentioned peptide nucleic acid in reaction system is 1.25 μM/L.
The present invention is to solve a kind of technical scheme that above-mentioned technical problem proposes to be：It is a kind of using above-mentioned detection architecture
Mthfr gene polymorphic detection product.
The present invention is to solve a kind of technical scheme that above-mentioned technical problem proposes to be：A kind of mthfr gene polymorphic detection
Kit, including PCR detection liquid and SYBR GreenI mixed liquors；
The PCR detections liquid includes being used for the sense primer, anti-sense primer and peptide core for detecting mthfr gene C677T sites
Acid；
The nucleotide sequence of the sense primer is as shown in SEQ ID No.1, and the nucleotide sequence of the anti-sense primer is such as
Shown in SEQ ID No.2,
The nucleotide sequence of the peptide nucleic acid and MTHFR wild type gene C677T site complete complementaries and nucleotide sequence
As shown in SEQ ID No.3.
Above-mentioned upstream and downstream primer has carried out LNA modifications；Ultimate density of the upstream and downstream primer in reaction system is 0.1
~0.3 μM/L；Ultimate densities of the peptide nucleic acid B in reaction system is 1~1.5 μM/L.
Above-mentioned mthfr gene polymorphic detection kit, in addition to positive control A, positive control B and negative control；
The positive control solution A is that the wild plasmid comprising mthfr gene C677T sites that concentration is 100ng/ μ L is molten
Liquid；
The positive control solution B is that the mutant plasmids comprising mthfr gene C677T sites that concentration is 100ng/ μ L are molten
Liquid；
The negative controls are free from the Genomic DNA solution of mthfr gene.
The present invention has positive effect：
(1) present invention clamps down on real-time quantitative PCR detection method using PNA, it is only necessary to 1 pair of primer and 1 PNA, it is bent by melting
Line analysis, you can distinguish mthfr gene saltant type with wild type.By single step reaction, can solve PCR amplifications, mutation richness
Collection and quantitative whole process, without subsequent steps such as electrophoresis, hybridization, digestions, enormously simplify experimentation, whole reaction
Process is completed in seal pipe, reduces the possibility of pollution to greatest extent, reduces false positive rate.
(2) mthfr gene pleiomorphism detecting method and its kit of the invention, can detect C677T homozygotes simultaneously and dash forward
Change and heterozygous mutation, high sensitivity.Sensitivity can reach 0.1%~0.5%, and compared with the concentration of Standard PCR, the present invention holds
Change places the genotype in the C677T sites for detecting very low concentration sample, overcome TaqMan probe method and need to use specificity
The shortcomings that probe is detected, meet the requirement of large sample size Clinical screening.
(3) present invention selection SYBR GreenI are cheap as fluorescent dye, substantially reduce testing cost.
Brief description of the drawings
Fig. 1 detects positive control A wild type C677T locus specificity products peak Tm values using the kit of embodiment.
Fig. 2 is the saltant type C677T locus specificity products peak Tm using the kit detection positive control B of embodiment
Value.
Embodiment
The present invention is specifically described below by embodiment, it is necessary to it is pointed out here that be that following examples are only used
It is further described in the present invention, it is impossible to be interpreted as limiting the scope of the invention, person skilled in art can
So that some nonessential modifications and adaptations are made to the present invention according to the invention described above content.In following embodiments, if not specially
Show, reagent used is that analysis is pure, and agents useful for same can obtain from commercial channel.The experiment of unreceipted actual conditions in text
Method, what the Science Press generally write according to normal condition such as J. Pehanorm Brookers etc. published for 2002《Molecular cloning is real
Test guide》Condition described in one book, or according to the condition proposed by manufacturer.Unless otherwise defined, the institute used in text
There is specialty identical with meaning known to one skilled in the art with scientific words.In addition, it is any similar to described content or
Impartial method and material all can be applied in the present invention.
First, the composition of kit.
The mthfr gene polymorphic detection kit of the present embodiment, including PCR detection liquid, SYBR GreenI mixed liquors,
Negative control, positive control A and positive control B.Each composition of kit is as shown in table 1.
The Kit components table of table 1
Reagent constituents is described as follows in above-mentioned table 1：
1) main component of SYBR GreenI mixed liquors includes archaeal dna polymerase, ultra-pure water, dNTPs mixed liquors, SYBR
GreenI, buffer solution etc. (are provided, article No. by Bioer companies：B255l1).
2) PCR detects liquid and included:For detect mthfr gene C677T sites upstream and downstream primer and with wild type C677T
The peptide nucleic acid of site complete complementary.
The present embodiment has carried out LNA modifications to upstream and downstream primer.A LNA base is introduced i.e. in oligonucleotide chain, and
The specificity of optimal fusing point (Tm) and hybridization is adjusted by adjusting position of the LNA bases in primer.Drawing after modification
Thing has higher Tm values, more preferably can be combined with mutagenesis template, and the real-time quantitative PCR primer after modification, length is shorter, can be with
Increase the flexibility of design.
The present embodiment adds a small amount of peptide nucleic acid (PNA) in PCR detects liquid, is not enough to completely inhibit wild type gene expansion
Increase, now the Tm values of wild type are unchanged, and amplification efficiency substantially reduces when not adding PNA, and expand nothing to mutated genes
Influence, but its Tm declines substantially, the Tm differences increase of the two, can now be analyzed by solubility curve by two kinds of gene regions
Separate.
3) positive control solution A is the solution for the C677T of mthfr gene containing the wild type sites plasmid that concentration is 100ng/ μ l.
4) positive control solution B is the solution for the C677T of mthfr gene containing the saltant type sites plasmid that concentration is 100ng/ μ l.
5) negative controls are free from mthfr gene C677T Genomic DNA solution.
Wild plasmid is retrieved as wild plasmid routine construction step：Design is located at the primer of mutational site both sides,
The wild type product containing corresponding site is expanded, is built into plasmid, selects simultaneously sequence verification.
Mutant plasmids are retrieved as homozygous mutation plasmid routine construction step：The corresponding polymorphic position of one covering of design
Point simultaneously will wait bit base to be incorporated into special primer in primer sequence, and corresponding upstream or the pairing of downstream wild primers,
The purpose fragment product containing corresponding site is expanded, is built into plasmid, selects simultaneously sequence verification.
2nd, primer, peptide nucleic acid design.
The primer length that the present invention designs is 18~22bp, and 200~220bp. of product length is directed to the spy of wild-type sequence
Different in nature peptide nucleic acid PNA fragments and wherein mthfr gene C677T site sequences complete complementary, length are 15~20bp.Such as the institute of table 2
Show.
The primer peptide nucleic acid sequence table of table 2
| Position | Detect classification | Seq No. | Sequence(5’—3’) |
| C677T | Sense primer A | 1 | TGCTGAGAACATTGCCTATG |
| C677T | Anti-sense primer B | 2 | CATTAGGCAGTGACTCGATGA |
| C677T | Peptide nucleic acid A | 3 | TGGTGTCACAGGAAGTGAT |
Primer and peptide nucleic acid are synthesized by the Li Ge Bioisystech Co., Ltd of Shanghai hundred, are diluted according to primer specification by dry powder
It is configured to detect working solution, primer and peptide nucleic acid mixed liquor by mother liquor addition sterilizing ultra-pure water into working stocks, then by mother liquor
Dilution forms.
Concentration of the peptide nucleic acid PNA in reaction system is most important in the present invention, 0.25~8.0 μm of ol/L concentration range
It is interior, when PNA concentration is less than 3.75 μm of ol/L, there is wild specific amplification peak on the melting curve of wild-type template, show
Wild type gene background does not completely inhibit, can not now judge whether mutator；When PNA concentration is in 3.75~4.0 μ
During mol/L, melting curve of the saltant type template after PCR is expanded can be observed to be mutated peak, and wild type only has primer dimer
Peak, i.e. wild-type template are suppressed can not expand completely；With the increase of PNA concentration, it can be observed either that wild type is still
The amplification efficiency of saltant type template gradually reduces, when PNA concentration is higher than 40 times of primer, during up to 8.0 μm of ol/L, and all templates
It is suppressed and without any amplified peak exist.
The application method of three kits.
1st, sample DNA extracts
Sample blood sample is extracted using kit (Axygen Multisource Genomic DNA Miniprep Kit)
This DNA, or use buccal swab genome DNA extracting reagent kit (article No. of the health for century bio tech ltd：
CW0530S mucous membrane of mouth sample DNA) is extracted, is specifically operated referring to reagent kit product specification.
2nd, sample DNA quality testing.
After obtaining sample DNA, concentration is determined by Thermo-Fisher nucleic acid-proteins quantitative instrument (NanoDrop 2000)
And purity, sample quality is controlled, ultimately joins the sample in reaction system.OD260/OD280 ratio is between 1.8~2.0
Obtain peak optimization reaction result, concentration dilution is 5~20ng/ μ l.
3rd, PCR reacts.
Detected using the mthfr gene polymorphic detection kit examination of the present embodiment, the volume of reaction system is 10 μ
2 μ l, SYBR GreenI mixed liquors of l, wherein PCR detection liquid 5 μ l, the μ l of template DNA 1, the μ l of ultra-pure water 2.(the volume of reaction system
20 μ l are can also be, when preparing that the component in 10 μ l reaction systems is double).
1) 10 μ l detection PCR reaction systems are prepared：
Detect PCR reaction systems：Take respectively 2 μ l PCR detect liquid, 5 μ l SYBR GreenI mixed liquors, 2 μ l ultra-pure waters, 1
μ l templates (if DNA sample, 5~20ng/ of concentration μ l), and repetition is set.
Template in reaction system refers to corresponding sample DNA, positive control, negative control respectively.
2) PCR response procedures.
Each reaction system carries out real-time fluorescence PCR reaction on ABI real-time fluorescence quantitative PCRs amplification instrument (stepone), most
Excellent response procedures are as shown in table 3.
The PCR response procedures tables of table 3
Preferably, the condition of PCR amplifications is：95 DEG C of the condition in pre-degeneration stage, 1min；Circulate 0 circulation of Isosorbide-5-Nitrae, condition
For 60 DEG C of 95 DEG C of 15s, 70 DEG C of PNA combination 10s, primer annealing and elongating temperature, time 1min；；2 conditions are circulated as 4 DEG C of guarantors
Temperature.
Reaction carries out solubility curve analysis immediately after terminating：95 DEG C of lasting 15s, 65 DEG C of holding 1min are cooled to, and with 0.3
DEG C/s speed is gradually heating to 95 DEG C of lasting 15s.Instrument software automatically analyzes out Ct values, i.e. SYBR Green I detection fluorescence
Signal is significantly higher than period during background signal.
4th, PCR result judgements.
1) judgement of kit validity.
Positive control A：All amplification curves of positive control FAM signalling channels have obvious Exponential growth stage, and Ct values
Positive≤30；Solubility curve is analyzed, and the Tm values of PCR primer are (88.8 ± 0.5) DEG C.
Positive control B:All amplification curves of positive control FAM signalling channels have obvious Exponential growth stage, and Ct values
Positive≤30；Solubility curve is analyzed, and the Tm values of PCR primer are (77.9 ± 0.6) DEG C.
Negative control：All amplification curves of negative control FAM signalling channels are bent without obvious Exponential growth stage, or dissolving
Without specific product peak on line.
2) judgement of polymorphism result.
By above step, under the premise of determining that experimental result is effective, then the result of sample is judged.According to sample
The difference that solubility curve Tm values compare solubility curve Tm values with wild-type positive carries out interpretation to pattern detection result, determines sample
Genotype.
If the amplification curve for A. detecting the FAM signalling channels of liquid detection has obvious Exponential growth stage, and | Tm(positive A)-
Tm(sample)|≤6, product peak Tm values are similar to shown in Fig. 1, then the mthfr gene C677T sites of detection sample are wild type.
If the amplification curve for B. detecting the FAM signalling channels of liquid detection has obvious Exponential growth stage, and | Tm(positive A)-
Tm(sample)| >=6, product peak Tm values are similar to shown in Fig. 2, then the mthfr gene C677T sites of detection sample are saltant type.
Obviously, above-described embodiment is only intended to clearly illustrate example of the present invention, and is not to the present invention
The restriction of embodiment.For those of ordinary skill in the field, it can also be made on the basis of the above description
Its various forms of changes or variation.There is no necessity and possibility to exhaust all the enbodiments.And these belong to this hair
Among the obvious changes or variations that bright spirit is extended out is still in protection scope of the present invention.
Sequence table
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tgctgagaac attgcctatg 20
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Claims (10)
- A kind of 1. mthfr gene polymorphic detection system, it is characterised in that：Including for detecting mthfr gene C677T sites Upstream and downstream primer, fluorescent dye SYBRGreen, and peptide nucleic acid；The nucleotide sequence of the sense primer is as shown in SEQ ID No.1, the nucleotide sequence such as SEQ of the anti-sense primer Shown in ID No.2；The nucleotide sequence of the peptide nucleic acid and MTHFR wild type gene C677T site complete complementaries and nucleotide sequence such as SEQ Shown in ID No.3.
- 2. mthfr gene polymorphic detection system according to claim 1, it is characterised in that：The upstream and downstream primer is entered LNA modifications are gone.
- 3. mthfr gene polymorphic detection system according to claim 1, it is characterised in that：The upstream and downstream primer exists Ultimate density in reaction system is 0.1~0.3 μM/L.
- 4. mthfr gene polymorphic detection system according to claim 3, it is characterised in that：The sense primer A is anti- It is 0.17 μM/L to answer the ultimate density in system, and ultimate densities of the anti-sense primer B in reaction system is 0.25 μM/L.
- 5. mthfr gene polymorphic detection system according to claim 1, it is characterised in that：The peptide nucleic acid is reacting Ultimate density in system is 1~1.5 μM/L.
- 6. mthfr gene polymorphic detection system according to claim 5, it is characterised in that：The peptide nucleic acid is reacting Ultimate density in system is 1.25 μM/L.
- A kind of 7. mthfr gene polymorphic detection product using detection architecture as claimed in claim 1.
- A kind of 8. mthfr gene polymorphic detection kit, it is characterised in that：Liquid and SYBR Green are detected including PCRMixing Liquid；The PCR detections liquid includes being used for the sense primer, anti-sense primer and peptide nucleic acid for detecting mthfr gene C677T sites；The nucleotide sequence of the sense primer is as shown in SEQ ID No.1, the nucleotide sequence such as SEQ of the anti-sense primer Shown in ID No.2,The nucleotide sequence of the peptide nucleic acid and MTHFR wild type gene C677T site complete complementaries and nucleotide sequence such as SEQ Shown in ID No.3.
- 9. mthfr gene polymorphic detection kit according to claim 8, it is characterised in that：The upstream and downstream primer LNA modifications are carried out；Ultimate density of the upstream and downstream primer in reaction system is 0.1~0.3 μM/L；The peptide nucleic acid B Ultimate density in reaction system is 1~1.5 μM/L.
- 10. mthfr gene polymorphic detection kit according to claim 8, it is characterised in that：Also include positive control A, positive control B and negative control；The positive control solution A is the wild plasmid solution for including mthfr gene C677T sites that concentration is 100ng/ μ L；The positive control solution B is the mutant plasmids solution for including mthfr gene C677T sites that concentration is 100ng/ μ L；The negative controls are free from the Genomic DNA solution of mthfr gene.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1975248A1 (en) * | 2007-03-28 | 2008-10-01 | Kabushiki Kaisha Toshiba | Nucleotide primer set and nucleotide probe for detecting genotype of methylene tetrahydrofolate reductase (MTHFR) |
| CN104531852A (en) * | 2014-12-12 | 2015-04-22 | 广东省妇幼保健院 | Probe and primer sensitized by locking nucleic acid and used for detecting C677T mutation of MTHFR gene, kit and detection method |
| CN104962653A (en) * | 2015-07-28 | 2015-10-07 | 上海睿玻生物科技有限公司 | Kit and detection method for polymorphism detection of methylenetetrahydrofolate reductase gene |
| CN105296621A (en) * | 2015-10-27 | 2016-02-03 | 智海生物工程（北京）有限公司 | Primer pair, fluorescence probe and kit for detecting polymorphism of MTHFR gene |
| CN107267621A (en) * | 2017-07-05 | 2017-10-20 | 上海赛安生物医药科技股份有限公司 | MDR1 genetic polymorphism detections system and its kit |
-
2017
- 2017-11-09 CN CN201711094014.4A patent/CN107760769A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1975248A1 (en) * | 2007-03-28 | 2008-10-01 | Kabushiki Kaisha Toshiba | Nucleotide primer set and nucleotide probe for detecting genotype of methylene tetrahydrofolate reductase (MTHFR) |
| CN104531852A (en) * | 2014-12-12 | 2015-04-22 | 广东省妇幼保健院 | Probe and primer sensitized by locking nucleic acid and used for detecting C677T mutation of MTHFR gene, kit and detection method |
| CN104962653A (en) * | 2015-07-28 | 2015-10-07 | 上海睿玻生物科技有限公司 | Kit and detection method for polymorphism detection of methylenetetrahydrofolate reductase gene |
| CN105296621A (en) * | 2015-10-27 | 2016-02-03 | 智海生物工程（北京）有限公司 | Primer pair, fluorescence probe and kit for detecting polymorphism of MTHFR gene |
| CN107267621A (en) * | 2017-07-05 | 2017-10-20 | 上海赛安生物医药科技股份有限公司 | MDR1 genetic polymorphism detections system and its kit |
Non-Patent Citations (1)
| Title |
|---|
| 林寒等: ""应用肽核酸钳制PCR技术检测胰腺癌K-ras基因突变"", 《第二军医大学学报》, vol. 30, no. 7, pages 762 - 766 * |
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