CN110272987A - A kind of site mthfr gene C677T rapid typing detection reagent box and method - Google Patents
A kind of site mthfr gene C677T rapid typing detection reagent box and method Download PDFInfo
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Abstract
The present invention provides a kind of site mthfr gene C677T rapid typing detection reagent box and methods.Specifically, the present invention is used through simply dealt blood sample, is then directly detected to MTHFR C677T loci polymorphism using classical fluorescent PCR.This detection method is not necessarily to carry out nucleic acid extraction or purifying to sample, only need a small amount of sample can to the accurate parting in the site MTHFR C677T, overcome normal PCR reaction to the dependence of nucleic acid extraction, simplify testing process, greatly shorten the SNP parting time, reduce research cost and technical complexity.
Description
Technical field
The invention belongs to biotechnologys and detection field, specifically, the present invention relates to a kind of mthfr gene C677T
Point rapid typing detection reagent box and method.
Background technique
Methylenetetrahydrofolate reductase (MTHFR) is the key enzyme of folic acid transhipment and metabolism.It is sub- that MTHFR is catalyzed 5,10-
Methyl tetrahydrofolate is reduced to 5-methyltetrahydrofolate, and one kind participates in nucleotide synthesis and repairs；One kind participates in together with MTRR
Methyl transmission path maintains the normal level of blood homocysteine, and the synthesis for DNA and protein provides methyl.MTHFR
Gene mutation can lead to the reduction of its enzymatic activity, cause folate metabolism disorder, and homocysteine level rises in blood, cause pregnant
Woman's gestation hypertension, spontaneous abortion, the diseases such as newborn's neural tube defect, congenital heart disease, harelip, Down syndrome
The onset risk of disease improves.
Currently, the common detection method in the site mthfr gene 677C > T has the allele-specific of based on PCR method
PCR method (AS-PCR), fluorescent PCR method and PCR sequencing PCR, these detection methods are required to be detected by template of nucleic acid, commonly
Method for extracting nucleic acid has magnetic bead nucleic acid extraction method and column to propose nucleic acid extraction method.
Common method for detecting single nucleotide polymorphism includes:
(1) Allele-specific polymerase chain reaction (allele specific PGR, AS-PCR) is a kind of more simple
The strategy of single detection SNP genotype.The basic principle is that according to the base type design primer of SNP site, this primer packet
Include shared upstream primer (or downstream primer) chain, specific downstream primer (or upstream primer) chain.General primer
Chain is designed according to a conventional method, and specific primer chain need to be mutated direction and type to the end 3' of special chain according to SNP site
The last one base is configured.Wild type and mutation type-special primer chain are different at two from shared primer strand respectively
It is reacted in PCR pipe, then the generation of purpose PCR product is detected whether by agarose gel electrophoresis, to reach
The purpose of SNP Genotyping.
(2) a specific fluorescence probe is added when fluorescent PCR method is PCR amplification while pair of primers is added,
The probe is an oligonucleotides, both ends one reporter fluorescence group of label and a quenching fluorescence group respectively.When probe is complete,
The fluorescence signal of reporter group transmitting is quenched group absorptions；Just start when, probe be incorporated in DNA any one it is single-stranded on；PCR
When amplification ,~3 ' the end 5 prime excision enzyme activity of 5 ' end of Taq enzyme degrades probe digestion, makes reporter fluorescence group and quenching fluorescence group
Separation, so that fluorescence monitoring system can receive fluorescence signal, as soon as that is, every amplification DNA chain, has a fluorescent molecule to be formed,
Realize the accumulation of fluorescence signal and PCR product formed it is fully synchronized.
Specific amplification products are detected using " allele specific pcr " technology combination fluorescence probe.Only when equipotential base
Because 3 ' terminal bases of specific primer match clock synchronization with corresponding allelotype template complete complementary, pcr amplification reaction can be just
It often carries out obtaining specific product, and generates fluorescence signal, conversely, then without specific PCR amplified reaction, thus do not observe glimmering
Optical signal or its Ct value are very high.
(3) CE PCR sequencing PCR is suitble to the SNP screening to short gene segment, and the inspection of SNP detection is carried out by direct Sequencing method
Extracting rate accuracy is close to 100%；Its principle are as follows: will likely SNP site carry out PCR amplification, find SNP in conjunction with DNA sequencing
It puts and determines that base replaces type.But a disadvantage is that the period is long, it is expensive, and it is easy to happen cross contamination.
Common method for extracting nucleic acid includes:
(1) magnetic bead nucleic acid extraction method can be dissolved rapidly according to strong protein denaturant is contained in magnetic bead combination liquid
Protein dissociates nucleic acid and comes；In the presence of its, the nucleic acid compositions released can be combined on magnetic bead, with
The effect for passing through magnetic bead cleaning solution afterwards removes protein, inorganic ion and many organic magazines.Then eluent will be pure
Nucleic Acid Elution get off.
(2) it is using efficiently cracking buffer system that column, which proposes nucleic acid extraction method, and binding silica gel film adsorbs column technology rapidly and efficiently
Extract sample in nucleic acid.Contain strong protein denaturant in lysate, the rapid solubilising protein of energy makes protein solution
It separates out and；In the presence of lysate and ethyl alcohol, the nucleic acid compositions released can be in conjunction on pellosil；Then pass through inhibition
Object removes liquid, the effect of deionization liquid, and protein, inorganic ion and many organic impurities are removed.Then eluent will be pure
Nucleic Acid Elution get off.
The detection of mononucleotide polymorphic (single nucleotide polymorphism SNP) mostly uses periphery at present
The genomic DNA that blood sample is extracted since this method is unsuitable for execute-in-place, and is extracted effect by Sample preservation condition and DNA
The limitation of fruit is not easy to promote in large-scale crowd SNP screening.
Therefore, there is an urgent need in the art to develop high specific, high sensitivity, detection mthfr gene C677T easy to operate
The technology of single nucleotide polymorphisms.
Summary of the invention
The purpose of the present invention is to provide a kind of site mthfr gene C677T rapid typing detection reagent box and methods.
In the first aspect of the present invention, a kind of primer sets for detecting mthfr gene polymorphism, the primer sets packet are provided
It includes:
The upstream primer in the site mthfr gene C677T: 5 '-GAGGCCAGCCTCTCCTGAC-3 '；
The site mthfr gene C677T wild type downstream primer: 5 '-CTGCGTGATGATGAAATATG-3 '；With
Mthfr gene C677T site mutation type downstream primer: 5 '-CTGCGTGATGATGAAATATA-3 '.
In another preferred example, the primer sets further include:
Internal control upstream primer: 5 '-GTGGCATAGTGGGGTGGTGA-3 '；With
Internal control downstream primer: 5 '-GCTCCTCCCACACCAGCTTTG-3 '.
The second aspect of the present invention provides a kind of probe groups for detecting mthfr gene polymorphism, the probe groups packet
It includes:
The site mthfr gene C677T probe: 5 '-GCCACCCCGAAGCAGGGAGCT-3 '.
In another preferred example, the probe groups further include:
Internal control probe: 5 '-ATTCGCCCTCTTAATGGGGAGG-3 '.
In another preferred example, 5 ' ends of the site mthfr gene C677T probe include fluorescent reporter group；With/
Or, 3 ' ends of the probe include fluorescent quenching group.
In another preferred example, 5 ' ends of the internal control probe include to visit different from the site the mthfr gene C677T
The fluorescent reporter group of needle；And/or 3 ' ends of the probe include fluorescent quenching group.
The third aspect of the present invention provides a kind of for detecting the kit of mthfr gene polymorphism, the kit
Including primer sets described in first aspect present invention.
In another preferred example, the kit further includes probe groups described in second aspect of the present invention.
In another preferred example, the kit includes that the first primer probe mixed liquor independently dispensed and the second primer are visited
Needle mixed liquor,
Wherein, the first primer probe mixed liquor includes:
The upstream primer in the site mthfr gene C677T: 5 '-GAGGCCAGCCTCTCCTGAC-3 '；
Mthfr gene C677T site mutation type downstream primer: 5 '-CTGCGTGATGATGAAATATA-3 '；With
The site mthfr gene C677T probe: 5 '-GCCACCCCGAAGCAGGGAGCT-3 '.
The second primed probe mixed liquor includes:
The upstream primer in the site mthfr gene C677T: 5 '-GAGGCCAGCCTCTCCTGAC-3 '；
The site mthfr gene C677T wild type downstream primer: 5 '-CTGCGTGATGATGAAATATG-3 '；With
The site mthfr gene C677T probe: 5 '-GCCACCCCGAAGCAGGGAGCT-3 '.
In another preferred example, the first primer probe mixed liquor and/or the second primed probe mixed liquor also wrap
It includes:
Internal control upstream primer: 5 '-GTGGCATAGTGGGGTGGTGA-3 '；
Internal control downstream primer: 5 '-GCTCCTCCCACACCAGCTTTG-3 '；With
Internal control probe: 5 '-ATTCGCCCTCTTAATGGGGAGG-3 '.
In another preferred example, the kit further includes the positive control and/or negative control independently dispensed.
In another preferred example, the kit further includes Fluorescence PCR enzyme；And/or fluorescent PCR buffer.
The fourth aspect of the present invention provides a kind of method for detecting mthfr gene polymorphism, and the method includes steps
It is rapid:
(1) DNA sample of object to be detected is provided；
(2) it prepares PCR reaction system and carries out PCR detection:
Wherein, the PCR reaction system include step (1) provide the DNA sample, described in first aspect present invention
Probe groups described in primer sets and second aspect of the present invention.
In another preferred example, the DNA sample comes from peripheral blood.
In another preferred example, the method is the detection method of non-diagnostic purpose.
In another preferred example, in step (1) comprising steps of
Periphery whole blood sample is drawn, cell pyrolysis liquid is added, shakes vigorously and mix well rear brief centrifugation with oscillator, 95 DEG C-
100 DEG C are handled 5~15 minutes；Supernatant is centrifugated up to the DNA sample (whole blood supernatant).
In another preferred example, the volume ratio of periphery whole blood sample and cell pyrolysis liquid is 2:4~5；Preferably 2:4.5.
In another preferred example, in the step (2), the PCR reaction system includes DNA described in 0.75 μ of μ L~1.25 L
Sample.
In another preferred example, in the step (2), the first PCR reaction system and the 2nd PCR reaction system are prepared respectively
And PCR reaction is carried out respectively, wherein include the first primer probe mixed liquor in the first PCR reaction system, described the
It include the second primed probe mixed liquor in two PCR reaction systems.
In another preferred example, the PCR reaction system further includes PCR reaction enzymes；And/or PCR buffer.
The fifth aspect of the present invention provides primer sets described in first aspect present invention, and/or second aspect of the present invention
The purposes of the probe groups, is used to prepare PCR detection kit, and the PCR detection kit is more for detecting mthfr gene
State property.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Detailed description of the invention
The PCR result of Fig. 1 present invention detection site mthfr gene 677C > T wild type PCR reaction tube negative control.
The PCR result of Fig. 2 present invention detection mthfr gene 677C > T site mutation type PCR reaction tube negative control.
The PCR result of Fig. 3 present invention detection site mthfr gene 677C > T wild type PCR reaction tube positive control.
The PCR result of Fig. 4 present invention detection mthfr gene 677C > T site mutation type PCR reaction tube positive control.
Fig. 5 compares primer sets 1 and detects mthfr gene 677C > T site mutation type PCR reaction result.
Specific embodiment
The present inventor develops a kind of side for detecting MTHFR C677T loci polymorphism by extensive and in-depth research
Method and kit, kit of the invention can carry out Direct PCR amplification with whole blood sample, eliminate nucleic acid extraction step, because
This is more convenient to use, has the characteristics that quick detection, high accuracy, high sensitivity and low cost.
Specifically, the present invention is used through simply dealt blood sample, then directly right using classical fluorescent PCR
MTHFRC677T loci polymorphism is detected.This detection method is not necessarily to carry out nucleic acid extraction or purifying to sample, it is only necessary to a small amount of
Sample can to the accurate parting in the site MTHFR C677T, overcome normal PCR reaction to the dependence of nucleic acid extraction, simplify inspection
Flow gauge greatly shortens the SNP parting time, reduces research cost and technical complexity.
The detection of mononucleotide polymorphic (single nucleotide polymorphism, SNP) mostly uses periphery at present
The genomic DNA that blood sample is extracted, since this method is unsuitable for execute-in-place, and by Sample preservation condition and Nucleic acid quality
Limitation, is not easy to promote in large-scale crowd SNP screening.The whole blood after simple process can be directly used as PCR in the present invention
The template of reaction carries out the detection of MTHFR C677T loci polymorphism using Standard PCR Buffer.This detection method overcomes biography
PCR reaction unite dependent on nucleic acid extraction and to the demanding disadvantage of Nucleic acid quality, simplifies operating process, reduces testing cost
And technical complexity, shorten detection time.
The present invention provides one kind to be based on real-time fluorescence PCR platform, using outside the fast qualitative detection people for exempting from nucleic acid extraction
The method of methylenetetrahydrofolate reductase (MTHFR) gene C 677T loci polymorphism in all whole blood samples, blood sample are fast
Speed processing primer, probe and the kit including the primed probe mixed liquor, with operating process is simple, detection time is short, inspection
Survey the advantages that at low cost.
Technical scheme is as follows:
One kind being based on real-time fluorescence PCR platform, exempts from the Asia in fast qualitative detection people's periphery whole blood sample of nucleic acid extraction
The method of methyl tetrahydrofolate reductase (MTHFR) gene C 677T loci polymorphism, blood sample quickly handle primer, probe
And the kit including the primed probe mixed liquor.It provides for spy needed for detecting mthfr gene C677T loci polymorphism
Specific primer and probe.
One, the invention discloses a kind of simple blood sample processing method, supernatant can directly carry out PCR amplification inspection
It surveys:
The blood sample processing method is to draw the periphery whole blood sample of 20 μ L, 45 μ L cell pyrolysis liquids is added, with vibration
It swings device to shake vigorously and mix well 15 seconds, the brief centrifugation several seconds.100 DEG C of constant temperature are handled 5~15 minutes；12,000rpm centrifugations 5 minutes,
It is spare to separate supernatant.Supernatant can be directly used for subsequent experimental or be placed in -80 DEG C saving backup.
The cell pyrolysis liquid is that routine DNA extracts reagent, such as the second of Da'an Gene Company, Zhongshan University
DNA extracting solution I (kit article No.: DA0030) in Hepatitis virus nucleic acid detection kit.
Two, the invention discloses the methylenetetrahydrofolate reductases in a kind of fast qualitative detection people periphery whole blood sample
(MTHFR) primer of gene C 677T loci polymorphism, probe:
The upstream primer nucleotide sequence in the detection site mthfr gene C677T is described as shown in SEQ ID NO:1
The downstream primer nucleotide sequence of the site mthfr gene C677T wild type is detected as shown in SEQ ID NO:2, the detection
The downstream primer nucleotide sequence of mthfr gene C677T site mutation type is as shown in SEQ ID NO:3, the detection internal control base
The upstream primer nucleotide sequence of cause is as shown in SEQ ID NO:4, the downstream primer nucleotide sequence of the detection internal control gene
As shown in SEQ ID NO:5；
The probe includes the probe and internal control gene probe for detecting mthfr gene C677T loci polymorphism；It is described
The probe nucleotide sequence in the site mthfr gene C677T such as SEQ ID NO:6, the probe nucleotide sequence of internal control such as SEQ ID
Shown in NO:7.
Further, 5 ' ends of the SEQ ID NO:6 nucleotide sequence are marked with FAM, 3 ' ends are marked with BHQ1, described
5 ' ends of SEQ ID NO:7 nucleotide sequence are marked with VIC, 3 ' ends are marked with BHQ1.
Preferably, the final concentration of 0.8 μm of ol/L of the mthfr gene C677T site upstream primer in the reaction system,
The final concentration of 0.8 μm of ol/L of the downstream primer of the site mthfr gene C677T wild type in the reaction system, it is described
The final concentration of 0.8 μm of ol/L of the downstream primer of mthfr gene C677T site mutation type in the reaction system, the MTHFR base
Because of the final concentration of 0.4 μm of ol/L of the site C677T probe in the reaction system；The internal control upstream primer is in the reaction system
Final concentration of 1 μm of ol/L, the final concentration of 1 μm of ol/L of the internal control downstream primer in the reaction system, the internal control probe exist
Final concentration of 0.3 μm of ol/L in reaction system.
The primer probe sequence of the site the mthfr gene C677T and internal control such as following table,
The primed probe nucleotide sequence information in the detection of table 1 site mthfr gene C677T
Above-mentioned PCR primer and the probe for indicating different type fluorescence can be respectively used to polymorphic to the site mthfr gene C677T
Property detected, according to the fluorescence signal that wild reaction tube and jump reaction pipe result are presented, whether there is or not MTHFR bases in judgement sample
Because of the site C677T type；Above-mentioned primer and probe is according to section where the site people mthfr gene C677T and internal control gene DNA sequence
Column design and synthesize, and can detecte the type in the site mthfr gene C677T；
The specific primer and probe can accurately detect the type in the site mthfr gene C677T.
Three, the invention also discloses a kind of methylenetetrahydrofolate reductase (MTHFR) gene C 677T loci polymorphism is fast
Fast detection kit, including the primed probe mixed liquor, cell pyrolysis liquid, 5 × DNAbuffer, heat for preparing PCR reaction solution
Start Taq enzyme, check sample and purified water, wherein primed probe mixed liquor includes following ingredient, such as table 2.
2 primed probe mixed liquor of table
Wherein, the primer for detecting the site mthfr gene C677T and internal control gene is distinguished as follows with probe:
The upstream primer nucleotide sequence in the detection site mthfr gene C677T is described as shown in SEQ ID NO:1
The downstream primer nucleotide sequence of the site mthfr gene C677T wild type is detected as shown in SEQ ID NO:2, the detection
The downstream primer nucleotide sequence of mthfr gene C677T site mutation type is as shown in SEQ ID NO:3, the detection internal control base
The upstream primer nucleotide sequence of cause is as shown in SEQ ID NO:4, the downstream primer nucleotide sequence of the detection internal control gene
As shown in SEQ ID NO:5；
The probe includes the probe and internal control probe for detecting the site mthfr gene C677T；The mthfr gene C677T
For the probe nucleotide sequence in site as shown in SEQ ID NO:6,5 ' ends are marked with FAM fluorescent reporter group；In the GAPDH
For the probe nucleotide sequence of control as shown in SEQ ID NO:7,5 ' ends are marked with VIC fluorescent reporter group；All probes
3 ' end be marked with BHQ1 fluorescent quenching group.
When detecting mthfr gene C677T loci polymorphism, probe and the upstream primer difference in the site mthfr gene C677T
With wild type and saltant type downstream primer composition wild type reaction tube and saltant type reaction tube, respectively with the target fragment that is expanded
Segment where the upper site C677T combines, and discharges FAM fluorescence signal respectively；The internal control primer and probe are according to house keeper people
Gene conservative fragments design and synthesize, the monitoring for sample and experimentation；
The cell pyrolysis liquid of the PCR reaction solution includes following ingredient, such as table 3,
3 cell pyrolysis liquid of table
| Number | Component | Main component in component |
| 1 | Cell pyrolysis liquid | Tris-HCl、EDTA、NP-40 |
5 × DNA buffer of the PCR reaction solution includes following ingredient, such as table 4,
45 × DNA of table buffer
| Number | Component | Main component in component |
| 1 | 5×DNA buffer | (NH4)2SO4、KCl、Tris-HCl、MgCl2、DTT |
The reference substance includes following ingredient, such as table 5,
5 check sample of table
| Number | Component | Main component in component |
| 1 | Negative control | Purified water |
| 2 | Positive control | Periphery whole blood |
The positive control is the site mthfr gene C677T heterozygous mutant periphery whole blood sample mixed liquor, negative right
This is purified water in the same old way.
It is people's periphery whole blood that kit of the present invention, which is applicable in sample,.
Kit of the present invention be used for determines detect validity standard are as follows: every time detection be respectively provided with negative control group with
Positive controls show that experimental result has when the positive controls of testing result are the positive, and negative control group is feminine gender
Effect.
Four, the invention also discloses a kind of method that mthfr gene C677T loci polymorphism quickly detects, specific steps
Include:
1. handling sample to be tested；Preferably, sample to be tested is people's periphery whole blood；
People's periphery whole blood sample of 20 μ L is taken, 45 μ L cell pyrolysis liquids are added, shake vigorously and mix well 15 seconds with oscillator, wink
When be centrifuged the several seconds.100 DEG C of constant temperature are handled 5~15 minutes；12,000rpm centrifugations 5 minutes, take supernatant spare.
2. PCR reaction system is prepared, by specific primer and probe, 5 × DNA buffer, hot start Taq polymerase, purified water
Mixing is added treated 1 μ L of sample to be tested supernatant, wild type PCR reaction solution and saltant type PCR reaction solution is prepared；
PCR reaction solution constitutes such as table 6 and table 7 respectively,
6 wild type PCR reaction solution of table
7 saltant type PCR reaction solution of table
3. PCR reaction tube is put into instrument sample slot, and record placement order.
4. fluorescence channel selects:
1) FAM Air conduct measurement mthfr gene 677C > T site wild type and saltant type are selected；
2) VIC Air conduct measurement internal standard channel is selected；
3) select reference fluorescent (Passive Reference) for None (ABI7500 is needed).
5. cycling condition such as following table is arranged, reaction system volume is set as 25 μ L.
6. after being provided with, saving file, program is run.
7. the PCR testing principle that the application uses is as follows: real-time fluorescent PCR technology is mainly applied, to human peripheral's whole blood
Mthfr gene 677C > T SNP site design specific primer and probe, expand to distinguish its type in genome.Design is special
Property primer and probe, be equipped with thermal starting archaeal dna polymerase etc. at being grouped as nucleic acid amplification agents, fluorescent PCR instrument used to carry out PCR expansion
Increase, and detect fluorescence signal, being boiled that treated, periphery whole blood supernatant is added in PCR reaction tube, instrument software system from
It is dynamic to draw out real-time amplification curve, the qualitative detection to unknown sample is realized according to threshold cycle values (Ct value).
The present invention proposes that a kind of method of the site mthfr gene 677C > T fast qualitative detection, blood sample are quickly handled
Method, primer, probe and kit, the kit are based on real-time fluorescence PCR platform, allow detection architecture directly to simple
Treated, and blood sample is detected.Specific implementation step is as follows,
Step 1: sample to be tested is handled
Extract human peripheral 2mL be placed in EDTA or citron receive anti-coagulants heparin tube in, marking ensures label information
After errorless, 4 DEG C of preservations.The periphery whole blood sample of 20 μ L and the yin and yang attribute quality-control product of 20 μ L are taken, 45 μ L cell crackings are separately added into
Liquid is shaken vigorously and mix well 15 seconds, the brief centrifugation several seconds with oscillator.100 DEG C of constant temperature are handled 5~15 minutes；12,000rpm centrifugation
5 minutes, separation supernatant was spare.Supernatant can be directly used for subsequent experimental or be placed in -80 DEG C saving backup.
Step 2: the preparation of PCR system
PCR system prepares before preparing: taking out the specific primer in kit and probe, 5 × DNA buffer, thermal starting
Taq enzyme, purified water etc., room temperature are melted, and the concussion that is vortexed is centrifuged 10 seconds after mixing, and prepare PCR system；PCR system constitutes such as 8 He of table
Table 9:
8 wild type PCR system of table
9 saltant type PCR system of table
The nucleotide sequence information difference of the primed probe is as follows:
Detect the primed probe nucleotide sequence information of mthfr gene C677T loci polymorphism:
Step 3: sample-adding
People's periphery whole blood sample and yin and yang attribute quality-control product prepared by step 1 are taken treated each 1 μ L of supernatant, is added
In eight connecting legs of the PCR reaction system prepared to step 2, make the 25 μ L of total volume of every pipe PCR reaction solution；Cover tightly eight unions
Pipe lid, mixes well, and high speed centrifugation 10 seconds；The check sample of the kit such as table 10:
The check sample of 10 kit of table
| Number | Component | Main component in component |
| 1 | Negative control | Purified water |
| 2 | Positive control | Periphery whole blood |
The positive control is the site mthfr gene C677T heterozygous mutant periphery whole blood sample, and negative control sample is
Purified water.
Step 4: PCR amplification
PCR reaction tube is put into instrument sample slot, and records placement order.Select FAM Air conduct measurement mthfr gene
The site 677C > T wild type and saltant type；Select VIC Air conduct measurement internal standard channel；Select reference fluorescent
It (PassiveReference) is None (ABI7500 is needed)；Cycling condition such as following table is set, and reaction system volume is set as 25 μ
L；After being provided with, file is saved, runs program.
Step 5: result is read and analysis
It is automatically saved after reaction as a result, delimiting suitable baseline and threshold value according to amplification curve.Baseline
(Baseline) initial value (Start value) general recommendations is between 3~15；Stop value (End value) general recommendations of baseline is 5
Between~20；Threshold value (Threshold value) suggests being the 1/20 of each Air conduct measurement highest fluorescent value.Baseline and threshold value are provided with
Afterwards, the Ct value that analysis (Reanalyse) automatically obtains each channel is clicked, result judgement is carried out according to each channel C t value.
Main advantages of the present invention are:
(1) this kit optimizes specific primer probe, is directed to mthfr gene 677C > T site wild type respectively and dashes forward
Modification is detected, and can directly carry out PCR amplification detection with through simply dealt whole blood supernatant.
(2) treated template that whole blood supernatant reacts as PCR provided by the invention quickly is used, this reagent is equipped with
Box specific primer and probe are, it can be achieved that quick detection to mthfr gene 677C > T loci polymorphism.
(3) detection method of the invention overcomes normal PCR reaction dependent on nucleic acid extraction, to sample of nucleic acid quality requirement
High disadvantage, greatly simplifies operating process, reduces research cost and technical complexity, shortens detection time.
(4) present invention is suitable for detecting mthfr gene 677C > T loci polymorphism of pregnant woman, and mthfr gene is prominent
Change can lead to the reduction of its enzymatic activity, cause folate metabolism disorder；After being identified by type, can appropriate Supplement of folic acid, to prevent
Pregnant woman because in blood homocysteine level rise caused by gestation hypertension, spontaneous abortion, newborn's nerve channel lack
The onset risk of the diseases such as sunken, congenital heart disease, harelip, Down syndrome.
Method of the invention is equally applicable to non-diagnostic purpose, for example, during new drug development, using of the invention
Detection method obtains the gene mutation information for being used as intermediate result, these gene mutation information can be used as public health and manage it
It needs, can be used for research and the targeting new drug development of drug metabolism mechanism.
Combined with specific embodiments below, the further old present invention in detail.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.The experimental method of detailed conditions is not specified in the following example, usually according to conventional strip
Part such as U.S. Sambrook.J etc. writes " Molecular Cloning: A Laboratory room guide " (Huang Peitang etc. is translated, Beijing: Science Press, 2002)
Described in condition, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number be by weight
It calculates.Experimental material used in following embodiment and reagent can obtain unless otherwise instructed from commercially available channel.
The kit of embodiment 1:MTHFR gene C 677T site fast typing detection
The kit forms ingredient, packaging and quantity (48 reactions/box) of the present embodiment, such as table 11:
Constituent, packaging and the quantity of 11 kit of table
Embodiment 2: sample detection range and the sensitivity technique experiment of the application kit
Choose 20 conducts of the site testing result MTHFR C677T wild type EDTA or citrate anticoagulation periphery whole blood sample
Sample is detected, number is A1~A20, chooses the site testing result MTHFR C677T heterozygous mutant EDTA or citric acid periphery
Whole blood sample 20 as detection sample, number is B1~B20, chooses the site testing result MTHFRC677T homozygous mutant
EDTA or 20, citric acid periphery whole blood sample conduct detection samples, number is C1~C20.The periphery whole blood sample of 20 μ L is taken,
45 μ L cell pyrolysis liquids are added, are shaken vigorously and mix well 15 seconds with oscillator, the brief centrifugation several seconds；100 DEG C of constant temperature handle 5~15 points
Clock；12,000rpm centrifugations 5 minutes, separate supernatant.Each 0.25 μ L, 0.5 μ L, 0.75 μ L, 1 μ L, 1.25 μ L, 1.5 μ L, 1.75 μ
L, 2 μ L supernatant sample detection, negative control is purified water, is loaded in eight connecting legs of the PCR reaction system prepared to step 2,
Make every 25 μ L of pipe PCR reaction solution total volume；Eight connecting leg pipe lids are covered tightly, are mixed well, high speed centrifugation 10 seconds.
PCR reaction tube is put into instrument sample slot, and records placement order.Select FAM Air conduct measurement mthfr gene
The site 677C > T wild type and saltant type；Select VIC Air conduct measurement internal standard channel；Select reference fluorescent
It (PassiveReference) is None (ABI7500 is needed)；Cycling condition such as following table is set, and reaction system volume is set as 25 μ
L；After being provided with, file is saved, runs program.
Sample detection range of the invention is measured with PCR system, actually measured result is as shown in table 12,
12 sample detection range experimental result of table
The lowest detection of this kit is limited to 0.75 μ L, and highest detection is limited to 1.25 μ L, by the site MTHFR C677T
Other 0.75 μ L of different shaped, 1 μ L and 1.25 μ L sample supernatants are repeated 20 times are detected respectively, and sensitivity technique is good；Cause
This, sample detection range of the invention is 0.75 μ of μ L~1.25 L.Recommendation applied sample amount is 1 μ L.
Embodiment 3: the accuracy detection of the application kit
It is prominent that kit detects 677C containing the MTHFR > site T wild-type positive reference material P1, MTHFR 677C > T site heterozygosis
The site the MTHFR 677C > T homozygous mutant positive reference product P3 of modification positive reference product P2 sum, each 20 repetitions are negative right
According to for purified water, each positive reference product of 20 μ L are taken respectively, are separately added into 45 μ L cell pyrolysis liquids, acutely vibrated with oscillator mixed
Even 15 seconds, the brief centrifugation several seconds；100 DEG C of constant temperature are handled 5~15 minutes；12,000rpm centrifugations 5 minutes, separate supernatant.Respectively
It takes 1 μ L supernatant to be loaded in eight unions of the PCR reaction system prepared to step 2, makes every 25 μ of pipe PCR reaction solution total volume
L；Eight union pipe lids are covered tightly, are mixed well, high speed centrifugation 10 seconds.
PCR reaction tube is put into instrument sample slot, and records placement order.Select FAM Air conduct measurement mthfr gene
The site 677C > T wild type and saltant type；Select VIC Air conduct measurement internal standard channel；Select reference fluorescent (Passive
It Reference is) None (ABI7500 is needed)；Cycling condition such as following table is set, and reaction system volume is set as 25 μ L；It sets up
Cheng Hou saves file, runs program.
It is detected with accuracy of the PCR system to kit of the present invention, obtains result such as table 13,
13 accuracy testing result of table
As a result, the testing result positive rate of each quality-control product accuracy is 100% according to upper table, show reagent of the present invention
The accuracy detection of box meets the requirements.
Embodiment 4: clinical application experiment
Extract 20 each 2mL of people's periphery whole blood be placed in EDTA or citron receive anti-coagulants heparin tube in, which has led to
After crossing nucleic acid extraction, sequencing is detected, and is clearly a kind of the site mthfr gene 677C > T type, carries out sample labeling
And ensure that label information is errorless, 4 DEG C of preservations.The periphery whole blood sample of 20 μ L is taken, 45 μ L cell pyrolysis liquids are added, with oscillator play
Strong oscillation mixes 15 seconds, the brief centrifugation several seconds；100 DEG C of constant temperature are handled 10 ± 5 minutes；12,000rpm centrifugations 5 minutes, in separation
Clear liquid obtains whole blood supernatant.Supernatant can be directly used for subsequent experimental or be placed in -80 DEG C saving backup, and avoid multigelation.
Each 1 μ L of check sample in the 1 μ L of supernatant and kit of each sample is taken, it is anti-to be loaded the PCR prepared to step 2
It answers in eight unions of system, makes the 25 μ L of total volume of every pipe PCR reaction solution；Pipe lid is covered tightly, is mixed well, high speed centrifugation 10
Second.
PCR reaction tube is put into instrument sample slot, and records placement order.Select FAM Air conduct measurement mthfr gene
The site 677C > T wild type and saltant type；Select VIC Air conduct measurement internal standard channel；Select reference fluorescent (Passive
It Reference is) None (ABI7500 is needed)；Cycling condition such as following table is set, and reaction system volume is set as 25 μ L；It sets up
Cheng Hou saves file, runs program.
20 human peripheral samples are detected with kit of the present invention, obtain result such as table 14,
14 20 human peripheral pattern detection results of table
Testing result are as follows: in 20 samples, 6 samples are in the site mthfr gene 677C > T wild type, and 9 samples are in
The site mthfr gene 677C > T heterozygous mutant, 5 samples are in the site mthfr gene 677C > T homozygous mutant, examined result
It is 100% with sequencing assay result consistency.
Comparative example 1
The present inventor has screened tens of groups of PCR primers and spy in the course of the research, for the site mthfr gene 677C > T
Needle shows that low specificity, poor sensitivity, accuracy are low wherein the overwhelming majority is not used to the detection of whole blood supernatant samples
The problems such as, it is unable to satisfy application demand.
Part of test results enumerates as follows, and detection method is the same as (the site the MTHFR C677T heterozygous mutant pattern of embodiment 2
This):
Compare primer sets 1:
The upstream primer in the site mthfr gene C677T: 5 '-CTGTTGGAAGGTGCAAGAT-3 ' (SEQ ID NO:8)
The site mthfr gene C677T wild type downstream primer: 5 '-CCGATTTCACCATCACGCAGCTT-3 ' (SEQ ID
NO:9)
Mthfr gene C677T site mutation type downstream primer: 5 '-TCGATTTCACCATCACGCAGCTT-3 ' (SEQ ID
NO:10)
The site mthfr gene C677T probe: 5 '-ACTCTCTCTGCCCAGTCCCTGTGGT-3 ' (SEQ ID NO:11)
Testing result is as shown in Figure 5, the results showed that, control primer sets 1 can not be suitable for whole blood supernatant samples completely
Detection.
Compare primer sets 2:
The upstream primer in the site mthfr gene C677T: 5 '-GAGCCCCCAAAGCAGAGGA-3 ' (SEQ ID NO:12)
The site mthfr gene C677T wild type downstream primer: 5 '-CCGTCTTCATCATCACGCAGC-3 ' (SEQ ID
NO:13)
Mthfr gene C677T site mutation type downstream primer: 5 '-TCGTCTTCATCATCACGCAGC-3 ' (SEQ ID
NO:14)
The site mthfr gene C677T probe: 5 '-CTCTTCATCCCTCGCCTTGA-3 ' (SEQ ID NO:15)
Testing result shows to detect whole blood supernatant samples using control primer sets 2, after detection limit optimization, 20
The highest coincidence rate of the site MTHFRC677T heterozygous mutant sample is only 45% (9/20).
Compare primer sets 3:
The upstream primer in the site mthfr gene C677T: 5 '-CGCCTTGAACAGGTGGAG-3 ' (SEQ ID NO:16)
The site mthfr gene C677T wild type downstream primer: 5 '-CCAATTTCATCATCACGCA-3 ' (SEQ ID NO:
17)
Mthfr gene C677T site mutation type downstream primer: 5 '-TCACTTTCATCATCACGCA-3 ' (SEQ ID NO:
18)
The site mthfr gene C677T probe: 5 '-AGGCCACCCCGAAGCAGGGAG-3 ' (SEQ ID NO:19)
Testing result shows to detect whole blood supernatant samples using control primer sets 3, after detection limit optimization, 20
The highest coincidence rate of the site MTHFRC677T heterozygous mutant sample can reach 80% (16/20).
Accuracy detection is carried out using the method for embodiment 3, test sample includes 677C containing the MTHFR > site T wild type
Positive reference product P1, MTHFR 677C > site T heterozygous mutant positive reference product P2 sum MTHFR 677C > T site homozygosis are prominent
Modification positive reference product P3, each 20 repetitions.Control primer sets 3 for the Detection accuracy of P1, P2, P3 are respectively 50%,
60%, 90%, it is unable to satisfy actual clinical demand.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Sequence table
<110>Da'an Gene Company, Zhongshan University
<120>a kind of site mthfr gene C677T rapid typing detection reagent box and method
<130> 000027
<160> 19
<170> PatentIn version 3.5
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<213>artificial sequence (Artificial sequence)
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gaggccagcc tctcctgac 19
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<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 2
ctgcgtgatg atgaaatatg 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 3
ctgcgtgatg atgaaatata 20
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Artificial sequence)
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gtggcatagt ggggtggtga 20
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<211> 21
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gctcctccca caccagcttt g 21
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<213>artificial sequence (Artificial sequence)
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gccaccccga agcagggagc t 21
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<213>artificial sequence (Artificial sequence)
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attcgccctc ttaatgggga gg 22
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tcgatttcac catcacgcag ctt 23
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cgccttgaac aggtggag 18
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ccaatttcat catcacgca 19
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Claims (10)
1. a kind of primer sets for detecting mthfr gene polymorphism, which is characterized in that the primer sets include:
The upstream primer in the site mthfr gene C677T: 5 '-GAGGCCAGCCTCTCCTGAC-3 '；
The site mthfr gene C677T wild type downstream primer: 5 '-CTGCGTGATGATGAAATATG-3 '；With
Mthfr gene C677T site mutation type downstream primer: 5 '-CTGCGTGATGATGAAATATA-3 '.
2. primer sets as described in claim 1, which is characterized in that the primer sets further include:
Internal control upstream primer: 5 '-GTGGCATAGTGGGGTGGTGA-3 '；With
Internal control downstream primer: 5 '-GCTCCTCCCACACCAGCTTTG-3 '.
3. a kind of probe groups for detecting mthfr gene polymorphism, which is characterized in that the probe groups include:
The site mthfr gene C677T probe: 5 '-GCCACCCCGAAGCAGGGAGCT-3 '.
4. probe groups as claimed in claim 3, which is characterized in that the probe groups further include:
Internal control probe: 5 '-ATTCGCCCTCTTAATGGGGAGG-3 '.
5. a kind of for detecting the kit of mthfr gene polymorphism, which is characterized in that the kit includes claim 1
The primer sets.
6. kit as claimed in claim 5, which is characterized in that the kit further includes probe as claimed in claim 3
Group.
7. kit as claimed in claim 5, which is characterized in that the kit includes the first primer probe independently dispensed
Mixed liquor and the second primed probe mixed liquor,
Wherein, the first primer probe mixed liquor includes:
The upstream primer in the site mthfr gene C677T: 5 '-GAGGCCAGCCTCTCCTGAC-3 '；
Mthfr gene C677T site mutation type downstream primer: 5 '-CTGCGTGATGATGAAATATA-3 '；With
The site mthfr gene C677T probe: 5 '-GCCACCCCGAAGCAGGGAGCT-3 '；And/or
The second primed probe mixed liquor includes:
The upstream primer in the site mthfr gene C677T: 5 '-GAGGCCAGCCTCTCCTGAC-3 '；
The site mthfr gene C677T wild type downstream primer: 5 '-CTGCGTGATGATGAAATATG-3 '；With
The site mthfr gene C677T probe: 5 '-GCCACCCCGAAGCAGGGAGCT-3 '.
8. kit as claimed in claim 5, which is characterized in that the first primer probe mixed liquor and/or described second
Primed probe mixed liquor further include:
Internal control upstream primer: 5 '-GTGGCATAGTGGGGTGGTGA-3 '；
Internal control downstream primer: 5 '-GCTCCTCCCACACCAGCTTTG-3 '；With
Internal control probe: 5 '-ATTCGCCCTCTTAATGGGGAGG-3 '.
9. a kind of method for detecting mthfr gene polymorphism, which is characterized in that the method includes the steps:
(1) DNA sample of object to be detected is provided；
(2) it prepares PCR reaction system and carries out PCR detection:
Wherein, the PCR reaction system include step (1) provide the DNA sample, primer sets described in claim 1 and
Probe groups as claimed in claim 2.
10. the purposes of primer sets described in claim 1, and/or probe groups as claimed in claim 3 is used to prepare PCR detection
Kit, the PCR detection kit is for detecting mthfr gene polymorphism.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2021128659A1 (en) * | 2019-12-24 | 2021-07-01 | 陕西佰美基因股份有限公司 | Specific primer probe combination and applicatoin thereof suitable for test of folate metabolic capability gene by direct blood amplification combined with fluorescent pcr method |
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Address after: 510665 No. 19 incense Hill Road, hi tech Industrial Development Zone, Guangdong, Guangzhou Applicant after: Guangzhou Da'an gene Co.,Ltd. Address before: 510665 No. 19 incense Hill Road, hi tech Industrial Development Zone, Guangdong, Guangzhou Applicant before: DAAN GENE CO., LTD. OF SUN YAT-SEN University |
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