CN108315409A - A kind of Primer composition and kit for detecting folic acid metabolism related gene - Google Patents
A kind of Primer composition and kit for detecting folic acid metabolism related gene Download PDFInfo
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- CN108315409A CN108315409A CN201810393250.4A CN201810393250A CN108315409A CN 108315409 A CN108315409 A CN 108315409A CN 201810393250 A CN201810393250 A CN 201810393250A CN 108315409 A CN108315409 A CN 108315409A
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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Abstract
The present invention relates to a kind of Primer compositions and kit for detecting folic acid metabolism related gene, and Primer composition includes MTHFR primer sets；Kit includes above-mentioned Primer composition.Compared with prior art, the present invention uses the method that ARMS technologies are combined with SYBR dyestuffs, obtaining one kind can be quick, kit that is sensitive and easily detecting folic acid metabolism related gene (MTHFR) gene pleiomorphism, the kit includes specificity ARMS detection primers, internal control primer and PCR reaction solution, by designing ARMS primers and Scorpions probes being changed to SYBR dyestuffs, so that testing cost substantially reduces, it is more suitable for the detection of Chinese patients mthfr gene polymorphism, it is quick with detection, high sensitivity, high specificity, method is simple, as a result accurate advantage, it is suitable for popularization and application.
Description
Technical field
The invention belongs to technical field of gene detection, are related to a kind of primer combination for detecting folic acid metabolism related gene
Object and kit.
Background technology
Folic acid is also referred to as Vitamin B9, belongs to vitamin B complex, is a kind of water soluble vitamin, because being from spinach earliest
It is extracted in leaf, so being widely known as folic acid.But actually there is more than in greens, fruit and eggs for folic acid
Content is also very considerable.Folic acid plays the role of promoting juvenile cell maturation in marrow, is object necessary to body cell growth and breeding
The necessary material of matter and human body when using sugar and amino acid, the mankind can cause macrocytic anemia as lacked folic acid
And leukopenia, it is even more important to pregnant woman.
Human body cannot directly synthesize folic acid, and being absorbed into internal folic acid by diet cannot directly be utilized by body, need
Becoming by a series of bioconversions has the folic acid of physiological activity.Internal folic acid metabolism balance also receives the shadow of inherent cause
It rings, the mutation of some genes can cause folic acid metabolism disorderly.Genetic epidemiology has found base related with folic acid metabolism at present
Because having MTHFR, MTHFD, RFC, MTR, MTRR, TYMS etc..
Two functional sites rs1801131 and rs1801133 on MTHFR (methylenetetrahydrofolate reductase) gene
Mutation can make MTHFR encode protein active reduce, so that the efficiency of human body trans-utilization folic acid is reduced, lead to internal homotype
Cysteine (Hyc) is accumulated, and DNA, protein, lipid undermethylation, chromosome instability is fixed, influences body anabolism, is increased
Add the risk of the diseases such as Down syndrome, nerve channel disease, angiocardiopathy.
Detection method of gene mutation has very much, and scholars have carried out this numerous studies, it has been reported that method packet
Include direct sequencing, dhplc analysis (DHPLC), PCR-SSCP/RFLP, Scorpions ARMS, TaqMan
PCR, ME-PCR etc..These methods respectively have advantage and disadvantage, in clinical and scientific research more common method be direct sequencing and
ARMS (Amplification refractory mutation system, amplification refractory mutation system) method.
Wherein, direct sequencing is it can be found that some new unknown mutations, but detectability is limited, and detection sensitivity is about
20% or so, and step is complicated, and entire detection process is related to a series of steps such as the deciphering of PCR- electrophoresis-sequencing-sequencing result
Suddenly, time-consuming and laborious.
ARMS methods are to be combined to create with the ARMS primers of specificity by molecular beacon (probe), ARMS primers
3 ' end designs are in mutational site, the last one base is matched with mutating alkali yl, using the Taq DNA without 3 ' → 5 ' 5 prime excision enzyme activities
Polymerase specifically identifies that 3 ' ends of primer, only 3 ' end of primer match clock synchronization, could normally expand, work as primer completely
When mispairing occurs for 3 ' ends, cannot effectively it expand.After primer is combined with mutagenesis template and extends corresponding product, probe
The fluorophor and quenching group at both ends detach and generate fluorescence.However, similar reagents box currently on the market is expensive, answer
With being restricted.
Invention content
It is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide one kind for detecting folic acid
The Primer composition and kit of metabolism related gene.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of Primer composition for detecting folic acid metabolism related gene, which includes MTHFR primer sets,
The MTHFR primer sets include following primer：
P1-FW：5’-AGGAGCTGACCAGTGAATA-3’；
P1-FS：5’-GAGGAGCTGACCAGTGAACC-3’；
P1-R：5’-CCTTCCAGGTGGAGGTCTC-3’；
P2-FW：5’-GAGAAGGTGTCTGCGGGATC-3’；
P2-FS：5’-GAGAAGGTGTCTGCGGGACT-3’；
P2-R：5’-AGCGAACTCAGCACTCCACC-3’.
Further, which further includes internal control primer sets, which includes following primer：
CF：5’-AGCAAGCAGGAGTATGACG-3’；
CR：5’-GAAAGGGTGTAACGCAACT-3’.
A kind of Primer composition answering in the genetic polymorphism detection reagent for preparing folic acid metabolism related gene
With.
It is a kind of for detecting the kit of folic acid metabolism related gene, which includes the Primer composition.
Further, which further includes PCR reaction solution, the PCR reaction solution include PCR buffer solutions, Taq enzyme,
MgCl2, dNTPs and SYBR Green I dyestuffs.The PCR buffer solutions are Tris-HCl buffer solutions, Tris- acetic acid (TAE)
Buffer solution or Tris- boric acid (TBE) buffer solution.
The PCR reaction solution includes 3 ' → 5 ' 5 prime excision enzyme activity high-fidelity Taq enzyme (enzyme activities as a preferred technical solution,
For 2.5U), 1.0-5.0mM MgCl2, 4.0-20.0mM dNTPs (including each 1.0-5.0mM of dATP, dUTP, dGTP, dCTP)
And SYBR Green I dyestuffs.
Further, which further includes positive control solution and negative controls.
The positive control solution is to be corresponded to containing detectable 2 sites in kit as a preferred technical solution,
Plasmid mixed liquor, contain 2 kinds of different ALDH2 allele with mutational site to be detected, plasmid in plasmid mixed liquor
Plasmid concentration in mixed liquor is 1000copies/ μ l；The negative controls are Tris-HCL buffer solutions, the Tris-HCL
A concentration of 7-13mM of buffer solution, pH 7.5-8.5.
The gene pleiomorphism detecting method of folic acid metabolism related gene (MTHFR), includes the following steps：
1) extraction of the processing of sample to be tested and template；
2) quantitative fluorescent PCR reaction system is prepared using the Primer composition；
3) ARMS methods are used, divide wild type and mutated genes sequence using each guiding region, are contaminated by SYBR Green I
The hybridization of material and amplified production, detects the SYBR fluorescence intensities of reaction system to judge testing result.SYBR is detection signal, with
SYBR signals reach the cycle-index Ct values needed for the threshold value of setting as criterion, and Ct values are less than 32 for the positive, and Ct values are big
It is feminine gender in 35, Ct values are weakly positive between 32 to 35.
The sample to be tested described in step 1) includes whole blood, blood plasma, serum or pleural effusion as a preferred technical solution,.
As a preferred technical solution, in the PCR reaction systems described in step 2), the content of each component is as follows：
Wherein, the concentration of each primer is 300nM in Primer composition.
The genetic polymorphism detection kit of folic acid metabolism related gene of the present invention uses SYBR dye methods, establishes and is directed to
The Multiplex real-time PCR detection method of the gene pleiomorphism (as shown in table 1) of mankind's related gene (MTHFR).The present invention is through excessive
Amount experiment, research and analysis, which successfully filter out, can be used in primer quick, sensitive, that mthfr gene polymorphism is effectively detected
Composition, and go out the method and kit for detecting folic acid metabolism related gene polymorphism using these primer developments.
The gene pleiomorphism form of 1 folic acid metabolism related gene of table
The specific primer of folic acid metabolism related gene see the table below 2, table 3, and table 4 is the detection primer of internal reference.
The detection primer of table 2 MTHFR (16685) gene pleiomorphism
The detection primer of table 3 MTHFR (14783) gene pleiomorphism
The detection primer of 4 internal reference of table
In the present invention, according to the base mutation form of each gene, using ARMS-PCR methods, design and wild type and prominent
The compatible primer of modification genetic fragment, and these primers are synthesized using custom primer synthetic method, for related gene
Gene pleiomorphism be detected.The basic principle of ARMS-PCR methods is：If 3 ' the end bases and template base of primer are not
Complementation can not then be extended with general hot resistant DNA polymerase, therefore design corresponding primer, 3 ' end alkali according to known point mutation
Base respectively with mutation and normal template base complementrity, so that the template for having certain point mutation and normal template be distinguished.
SYBR Green I are a kind of dyes with green excitation wavelength being incorporated into all dsDNA minor grooves region
Material.Under free state, SYBR Green I send out faint fluorescence, but once combined with double-stranded DNA, fluorescence increases greatly
By force.Therefore, the fluorescence signal intensity of SYBR Green I and the quantity of double-stranded DNA are related, can be detected according to fluorescence signal
Double-stranded DNA quantity existing for PCR system.The maximum absorption wavelength of SYBR Green I is about 497nm, and launch wavelength is about
520nm。
Compared with prior art, the invention has the characteristics that：
1) use the method that is combined with SYBR dyestuffs of ARMS technologies, obtain one kind can quickly, sensitivity and easily
Detect folic acid metabolism related gene (MTHFR) gene pleiomorphism kit, the kit include specificity ARMS detection primers,
Internal control primer and PCR reaction solution, by designing ARMS primers and Scorpions probes being changed to SYBR dyestuffs, so that detection
Cost substantially reduces, and is more suitable for the detection of Chinese patients mthfr gene polymorphism, has and detects quick, high sensitivity, specificity
By force, method is simple, the accurate advantage of result, is suitable for popularization and application；
2) kit can accurately detect 1% mutant DNA under 50ng wild type gene group DNA backgrounds, and specific aim is set
The specific primer for counting different mutational sites, is detected using quantitative fluorescent PCR, and detection process is stopped pipe reaction, significantly
Reduce pollution.
Description of the drawings
Fig. 1 is the amplification curve diagram that genetic polymorphism detection kit of the present invention detects MTHFR (16685) sample；
Fig. 2 is the amplification curve diagram that genetic polymorphism detection kit of the present invention detects MTHFR (14783) sample.
Specific implementation mode
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.The present embodiment is with technical solution of the present invention
Premised on implemented, give detailed embodiment and specific operating process.Those skilled in the art can be by this explanation
Content disclosed by book understands other advantages and effect of the present invention easily.The present invention can also be by addition different specific
Embodiment is embodied or practiced, and the various details in this specification can also be based on different viewpoints and application, not carry on the back
Various modifications or alterations are carried out under spirit from the present invention.
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, the protection domain being not intended to be limiting of the invention；In description of the invention and claims, unless in text
In addition explicitly point out, singulative "one", " one " and " this " include plural form.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range
Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, in the present invention all technologies for using and
Scientific terminology is identical as the normally understood meaning of those skilled in the art of the present technique.Except used in embodiment specific method, equipment,
Outside material, the record according to those skilled in the art to the grasp of the prior art and the present invention can also use and this
Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real
The existing present invention.
Embodiment 1：
1, sample process and nucleic acid extraction：
Sample is handled using the DNA extraction kit of commercialization, concrete operations are needed referring to kit specification, carried DNA
With ultraviolet specrophotometer measured concentration and purity, DNA OD260/OD280Value should 1.8~2.0, concentration should 5~
Between 50ng/ μ L, sample DNA person off quality must not be used to detect, and re-start nucleic acid extraction less than 5ng/ μ L persons, be higher than
50ng/ μ L persons are suitably diluted to defined concentration range, and the DNA extracted should be detected immediately, otherwise in -20 DEG C with
Lower preservation, holding time do not exceed 6 months.Kit quality-control product is melted using preceding room temperature, vortex oscillation 10 seconds, 2000rpm
Centrifuge 15 seconds for use.
2, reagent configures：
2.1 configuration instruction：Detection reaction setting internal control detection, negative quality-control product detection and positive quality control product detection.
2.2 configuration process：
Reagent was taken out in 30 minutes in advance, room temperature is melted, vortex oscillation 10 seconds, and 2000rpm centrifuges 15 seconds for use.It determines every
A detection site stoichiometric number N and internal control testing number M, N=sample numbers (n) to be checked+quality-control product number (2)+1；M=sample numbers to be checked
(n)+1.The amount for being added to each reagent in reaction mixture is calculated, such as the following table 5 is calculated：
5 PCR reaction system allocation lists of table
| Reagent | Detection site | PCR reaction solution P | Primer mixed liquor | Purified water |
| Reaction solution 1 (μ L) | MTHFR(16685)-A | 12.5*N | 2.0*N | 8.5*N |
| Reaction solution 2 (μ L) | MTHFR(16685)-C | 12.5*N | 2.0*N | 8.5*N |
| Reaction solution 3 (μ L) | MTHFR(14783)-C | 12.5*N | 2.0*N | 8.5*N |
| Reaction solution 4 (μ L) | MTHFR(14783)-T | 12.5*N | 2.0*N | 8.5*N |
| Reaction solution 5 (μ L) | Internal control | 12.5*M | 2.0*N | 8.5*N |
5 sterile centrifugation tubes are taken to configure above-mentioned 5 reaction systems, vortex oscillation 10 seconds, 2000rpm after reagent is all added
Centrifugation 15 seconds.Then 23 μ L/ pipes of above-mentioned mixed liquor are dispensed into PCR reaction tubes (sterile and RNase-Free).
3, it is loaded：
6 sample of table and quality-control product loading table
| Title | Ingredient | Sample-adding amount |
| Sample to be checked | Sample DNA | 2μL |
| STD1 | Positive quality control product 1 | 2μL |
| STD2 | Positive quality control product 2 | 2μL |
| NTC | Negative quality-control product | 2μL |
Note：STD represents positive quality control product；NTC represents negative quality-control product.
According to the loading ratio that table 6 prompts, reaction tube is added in processed sample, positive quality control product and negative quality-control product
In, internal control primer need to only detect sample DNA, cover tightly pipe lid (bubble is avoided to generate), 2000rpm is centrifuged 15 seconds will be on tube wall
Liquid all get rid of to tube bottom, carry out pcr amplification reaction immediately after.
4, PCR amplification program is arranged：
The setting of 7 PCR response procedures of table
5, the deciphering of inspection result：
Divide wild type and mutated genes sequence using the guiding regions ARMS, passes through SYBR Green dyestuffs and amplified production
Hybridization, detects the SYBR fluorescence intensities of reaction system to judge testing result.Detect the amplification curve diagram institute as shown in Figure 1, Figure 2 of sample
Show.SYBR is detection signal, reaches the cycle-index Ct values needed for the threshold value of setting as criterion, Ct values using SYBR signals
It is the positive less than 32, Ct values are more than 35 for feminine gender, and Ct values are weakly positive between 32 to 35.
Embodiment 2：
A kind of Primer composition for detecting folic acid metabolism related gene, which includes MTHFR primer sets,
The MTHFR primer sets include following primer：
P1-FW：5’-AGGAGCTGACCAGTGAATA-3’；
P1-FS：5’-GAGGAGCTGACCAGTGAACC-3’；
P1-R：5’-CCTTCCAGGTGGAGGTCTC-3’；
P2-FW：5’-GAGAAGGTGTCTGCGGGATC-3’；
P2-FS：5’-GAGAAGGTGTCTGCGGGACT-3’；
P2-R：5’-AGCGAACTCAGCACTCCACC-3’.
Application of the above-mentioned Primer composition in the genetic polymorphism detection reagent for preparing folic acid metabolism related gene.
Kit for detecting folic acid metabolism related gene includes above-mentioned Primer composition and PCR reaction solution, and the PCR is anti-
It includes PCR buffer solutions, Taq enzyme, MgCl to answer liquid2, dNTPs and SYBR Green I dyestuffs.
Embodiment 3：
A kind of Primer composition for detecting folic acid metabolism related gene, which includes MTHFR primer sets,
The MTHFR primer sets include following primer：
P1-FW：5’-AGGAGCTGACCAGTGAATA-3’；
P1-FS：5’-GAGGAGCTGACCAGTGAACC-3’；
P1-R：5’-CCTTCCAGGTGGAGGTCTC-3’；
P2-FW：5’-GAGAAGGTGTCTGCGGGATC-3’；
P2-FS：5’-GAGAAGGTGTCTGCGGGACT-3’；
P2-R：5’-AGCGAACTCAGCACTCCACC-3’.
The Primer composition further includes internal control primer sets, which includes following primer：
CF：5’-AGCAAGCAGGAGTATGACG-3’；
CR：5’-GAAAGGGTGTAACGCAACT-3’.
Application of the above-mentioned Primer composition in the genetic polymorphism detection reagent for preparing folic acid metabolism related gene.
Kit for detecting folic acid metabolism related gene includes above-mentioned Primer composition and PCR reaction solution, and the PCR is anti-
It includes PCR buffer solutions, Taq enzyme, MgCl to answer liquid2, dNTPs and SYBR Green I dyestuffs.
Kit further includes positive control solution and negative controls.
The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention.
Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general
Principle is applied in other embodiment without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability
Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be the present invention's
Within protection domain.
Claims (6)
1. a kind of for detecting the Primer composition of folic acid metabolism related gene, which is characterized in that the Primer composition includes
MTHFR primer sets, the MTHFR primer sets include following primer：
P1-FW：5’-AGGAGCTGACCAGTGAATA-3’；
P1-FS：5’-GAGGAGCTGACCAGTGAACC-3’；
P1-R：5’-CCTTCCAGGTGGAGGTCTC-3’；
P2-FW：5’-GAGAAGGTGTCTGCGGGATC-3’；
P2-FS：5’-GAGAAGGTGTCTGCGGGACT-3’；
P2-R：5’-AGCGAACTCAGCACTCCACC-3’.
2. a kind of Primer composition for detecting folic acid metabolism related gene according to claim 1, which is characterized in that
The Primer composition further includes internal control primer sets, which includes following primer：
CF：5’-AGCAAGCAGGAGTATGACG-3’；
CR：5’-GAAAGGGTGTAACGCAACT-3’.
3. a kind of Primer composition as claimed in claim 1 or 2 is in the gene pleiomorphism inspection for preparing folic acid metabolism related gene
Application in test agent.
4. a kind of kit for detecting folic acid metabolism related gene, which is characterized in that the kit includes such as claim 1
Or the Primer composition described in 2.
5. a kind of kit for detecting folic acid metabolism related gene according to claim 4, which is characterized in that the examination
Agent box further includes PCR reaction solution, which includes PCR buffer solutions, Taq enzyme, MgCl2, dNTPs and SYBR Green I
Dyestuff.
6. a kind of kit for detecting folic acid metabolism related gene according to claim 4, which is characterized in that the examination
Agent box further includes positive control solution and negative controls.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667435A (en) * | 2012-09-25 | 2014-03-26 | 浙江爱易生物医学科技有限公司 | Kit for detecting individual folate metabolism disorder |
| CN105463122A (en) * | 2016-01-27 | 2016-04-06 | 宁波海尔施基因科技有限公司 | MTHFR, MTRR and RFC1 gene polymorphism detection primer combination and kit and application of MTHFR, MTRR and RFC1 gene polymorphism detection kit |
-
2018
- 2018-04-27 CN CN201810393250.4A patent/CN108315409A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667435A (en) * | 2012-09-25 | 2014-03-26 | 浙江爱易生物医学科技有限公司 | Kit for detecting individual folate metabolism disorder |
| CN105463122A (en) * | 2016-01-27 | 2016-04-06 | 宁波海尔施基因科技有限公司 | MTHFR, MTRR and RFC1 gene polymorphism detection primer combination and kit and application of MTHFR, MTRR and RFC1 gene polymorphism detection kit |
Non-Patent Citations (5)
| Title |
|---|
| 1000GENOMES: "ss107994602", 《DBSNP DATABASE》 * |
| APPLERA_GI: "ss48422848", 《DBSNP DATABASE》 * |
| RAZIEH MOGHTADERI NASAB等: "three coagulation related mutations and increased risk of myoma inwomen of fars province", 《ZAHEDAN JOURNAL OF RESEARCH IN MEDICAL SCIENCES》 * |
| 林宁等: "孕前女性人群中MTHFR基因多态性与叶酸、同型半胱氨酸的相关性", 《南京医科大学学报（自然科学版）》 * |
| 黄璐琦: "《分子生药学》", 30 September 2006, 北京大学医学出版社 * |
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