CN104561296A - Primer, primer set, kit and detection method for detecting polymorphism of folate metabolism related genes with blood direct-PCR (polymerase chain reaction) method - Google Patents
Primer, primer set, kit and detection method for detecting polymorphism of folate metabolism related genes with blood direct-PCR (polymerase chain reaction) method Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention relates to molecular biological detection, in particular to a primer, a primer set, a kit and a detection method for detecting polymorphism of folate metabolism related genes with a blood direct-PCR (polymerase chain reaction) method. The primer set comprises the following primer pairs: (1), a forward primer MTRR-PF and a reverse primer MTRR-PR, the nucleotide sequence of the forward primer MTRR-PF is represented as SEQ ID NO.1: atgccttgaagtgatgag, and the nucleotide sequence of the reverse primer MTRR-PR is represented as SEQ ID NO.2: actgtaacggctctaacc; (2), a forward primer MTHFR-677PF and a reverse primer MTHFR-677PR; and (3), a forward primer MTHFR-1298PF and a reverse primer MTHFR-1298PR. The gene polymorphism can be determined and detected without extracting human genomic DNA (deoxyribose nucleic acid).
Description
Technical field
The present invention relates to molecular Biological Detection, particularly relate to the primer of a kind of blood direct PCR method detection folic acid metabolism related gene polymorphism, primer sets, test kit and detection method.
Background technology
Folic acid belongs to vitamin B group, is the necessary element of nucleic acid, is the necessary material of Growth of Cells and tissue repair, indispensable nutrient substance in embryo development procedure especially.Large quantity research confirms in recent years, and folic acid deficiency is the major cause causing inborn defect.The clinical function of folic acid, except prevention Foetus neural tube defect, can also reduce the sickness rate such as pregnant woman's gestational hypertension, spontaneous abortion and fetal intrauterine growth retardation, premature labor and low birth weight.
Scientific research finds, folic acid Utilization ability is by genetic construction (gene) impact, (i.e. genes involved dysfunction) if on the low side with folic acid metabolism involved enzyme activity, this crowd is as pressed constant (400 micro-grams/day) Supplement of folic acid, and body folate level also can be not enough.The difference of genetic constitution result in the difference of body to folic acid Utilization ability, and therefore, folic acid supplementary behavior should vary with each individual, and augments too much or to be very fewly all unfavorable for foetus health and pregnant woman health.
Scientific research is verified, is MTHFR and MTRR with the closely-related gene of methyl biostearin (folic acid and vitamin B12) metabolism.MTHFR proteins encoded 5,10-CH2-THFA reductase enzyme, MTRR proteins encoded methionine synthetase reductase enzyme.MTHFR and MTRR genovariation can cause corresponding enzymic activity to reduce, and prevents homocysteine to be converted into methionine(Met), causes low folic acid mass formed by blood stasis and hyperhomocysteinemiainjury, thus increases Newborn Birth-defects risk or spontaneous abortion equivalent risk.
MTHFR full name 5,10-methylenetetrahydrofolatereductase(5,10-Methylene tetrahydrofolate reductase), be positioned at a karyomit(e) lp36.3 position.MTHFE total length 19.3kb, mRNA total length 7105bp, common exon 12, coding has the albumen of 657 amino acid coding.Methylene tetrahydrofolate reductase catalysis 5,10-CH2-THFA converts 5-methyltetrahydrofolate salt to, makes it as homocysteine provides methylmethionine.This enzyme is the very important enzyme of in human body folic acid metabolism.
MTRR full name 5-methyltetrahydrofolate-homocysteinemethyltransferase reductase, encodes methionine synthetic enzyme reductase enzyme, is positioned at 5p15.3-p15.2.MTRR full length gene 32021 kb, mRNA total length 3274bp, have 15 exons, and coding has 726 amino acid whose protein.Methionine(Met) is the necessary amino acid of protein synthesis and one carbon metabolism, its synthesis by methionine synthases (MTR coding) catalysis, and methionine synthases because cofactor vitamin B12 is oxidized final inactivation.The methionine synthetase reductase enzyme of MTRR coding can regenerate the methionine synthases with functionally active by reduced form methylation.Methionine synthetase reductase enzyme is the Triphosphopyridine nucleotide photoreductase-NADP(+ with transfer transport effect) reductase enzyme (ferredoxin-NADP(+) reductase, FNR) family member.MTRR sudden change causes the avitaminotic Etiological of folic acid/methyl.
CN103834733A (2014-6-4) discloses test kit and the measuring method thereof in a kind of single tube simultaneous determination aldehyde dehydrogenase 2 gene and Methylene tetrahydrofolate reductase gene mutational site, and described test kit comprises: Whole Blood Genomic DNA extracts reagent, multiplexed PCR amplification primer, multiplexed PCR amplification reaction reagent, single stranded DNA abstraction and purification reagent, Pyrosequencing primer, Manganic pyrophosphate complex initiation reagent and box body.Described method: human whole blood extracting genome DNA; Multiplexed PCR amplification reacts; Single stranded DNA sample separation and purifying; Manganic pyrophosphate complex initiation and interpretation of result.Purposes: wish to understand the individual physical examination of healthy population to ethanol and folic acid metabolism capabilities might; Prediction is individual to pannonit metabolic capacity and curative effect, instructs the heart to obstruct patient's choose reasonable and vasorelaxation first aid medicine for subsequent use; The individual metabolic capacity to folic acid of prediction, instructs pregnant and lying-in women and children to supplement the folic acid supplement etc. of applicable dosage.But the method needs extracting genomic dna.
Detect 5 on the market at present, the method of 3 SNP loci polymorphisms on 10-Methylene tetrahydrofolate reductase gene (MTHFR), methionine synthetase reductase gene (MTRR) is all need extracting human gene group DNA from people's whole blood or oral epithelium tissue, then on quantitative real time PCR Instrument, utilize fluorescent probe or fluorescent dye determination to carry out quantitative fluorescent PCR, judge mutation type according to fluorescent signal power or solubility curve.Although these methods can accomplish that the same day goes out result, be not suitable for detecting in the hospital in domestic two or three line cities or healthcare hospital for women & children.Reason is as follows:
1, quantitative real time PCR Instrument is expensive, and domestic equipment price is also more than 300,000 yuan, and not all hospital can be equipped with;
2, test kit on the quantitative real time PCR Instrument of different manufacturers or different model interpretation method or result inconsistent, could detect on a large scale after needing to do preliminary experiment or sequence verification;
3, need extracting genomic dna, extra increase testing staff's workload and testing cost, and easily cause sample contamination.
Summary of the invention
An object of the present invention is to provide a kind of without the need to extracting human gene group DNA, and from whole blood Direct PCR, without the need to the quantitative real time PCR Instrument of costliness, the blood direct PCR method detecting rs1801133 SNP site on 5,10-Methylene tetrahydrofolate reductase gene detects the primer of folic acid metabolism related gene polymorphism.
Two of object of the present invention is to provide a kind of without the need to extracting human gene group DNA, and from whole blood Direct PCR, without the need to the quantitative real time PCR Instrument of costliness, the blood direct PCR method detecting rs1801131 SNP site on 5,10-Methylene tetrahydrofolate reductase gene detects the primer of folic acid metabolism related gene polymorphism.
Three of object of the present invention is to provide a kind of without the need to extracting human gene group DNA, and from whole blood Direct PCR, without the need to the quantitative real time PCR Instrument of costliness, the blood direct PCR method detecting rs1801394 SNP site on methionine synthetase reductase gene detects the primer of folic acid metabolism related gene polymorphism.
Four of object of the present invention is to provide a kind of without the need to extracting human gene group DNA, and from whole blood Direct PCR, without the need to the quantitative real time PCR Instrument of costliness, detect 5 simultaneously, on 10-Methylene tetrahydrofolate reductase gene, on rs1801133 SNP site, 5,10-Methylene tetrahydrofolate reductase gene, on rs1801131 SNP and methionine synthetase reductase gene, the blood direct PCR method in rs1801394 SNP site detects the primer sets of folic acid metabolism related gene polymorphism.
Five of object of the present invention is to provide a kind of without the need to extracting human gene group DNA, and from whole blood Direct PCR, without the need to the quantitative real time PCR Instrument of costliness, detect 5 simultaneously, on 10-Methylene tetrahydrofolate reductase gene on rs1801133 SNP site, 5,10-Methylene tetrahydrofolate reductase gene on rs1801131 SNP and methionine synthetase reductase gene rs1801394 SNP site, the blood direct PCR method that judges of being checked order by PCR primer detects the test kit of folic acid metabolism related gene polymorphism.
Six of object of the present invention is to provide a kind of without the need to extracting human gene group DNA, and from whole blood Direct PCR, without the need to the quantitative real time PCR Instrument of costliness, detect 5 simultaneously, on 10-Methylene tetrahydrofolate reductase gene on rs1801133 SNP site, 5,10-Methylene tetrahydrofolate reductase gene on rs1801131 SNP and methionine synthetase reductase gene rs1801394 SNP site, the blood direct PCR method that judges of being checked order by PCR primer detects the detection method of folic acid metabolism related gene polymorphism.
First technical purpose of the present invention is achieved by the following technical programs:
Blood direct PCR method detects the primer of folic acid metabolism related gene polymorphism, and it comprises following primer pair:
(1) forward primer MTRR-PF, its nucleotide sequence SEQ ID NO.1:atgccttgaagtgatgag;
Reverse primer MTRR-PR, its nucleotide sequence SEQ ID NO.2:actgtaacggctctaacc; And/or
(2) primer obtained through increasing, lacking or replace single core thuja acid by the nucleotide sequence of forward primer MTRR-PF or reverse primer MTRR-PR;
The goal gene that above-mentioned primer pair is answered is 5,10-Methylene tetrahydrofolate reductase gene, with this gene for template amplification gained DNA fragmentation comprises rs1801133 SNP site.
The present invention sets and synthesizes specific described Auele Specific Primer, can detect rs1801133 SNP site on 5,10-Methylene tetrahydrofolate reductase gene.
Second technical purpose of the present invention is achieved by the following technical programs:
Blood direct PCR method detects the primer of folic acid metabolism related gene polymorphism, and it comprises following primer pair:
(1) forward primer MTHFR-677PF, its nucleotide sequence SEQ ID NO.3:AGGACAGTGTGGGAGTTTGG;
Reverse primer MTHFR-677PR, its nucleotide sequence SEQ ID NO.4:GAAAAGCTGCGTGATGATGA; And/or
(2) primer obtained through increasing, lacking or replace single core thuja acid by the nucleotide sequence of forward primer MTHFR-677PF or reverse primer MTHFR-677PR;
The goal gene that above-mentioned primer pair is answered is 5,10-Methylene tetrahydrofolate reductase gene, with this gene for template amplification gained DNA fragmentation comprises rs1801131 SNP site.
The present invention sets and synthesizes specific described Auele Specific Primer, can detect rs1801131 SNP site on 5,10-Methylene tetrahydrofolate reductase gene.
3rd technical purpose of the present invention is achieved by the following technical programs:
Blood direct PCR method detects the primer of folic acid metabolism related gene polymorphism, and it comprises following primer pair:
(1) forward primer MTHFR-1298PF, its nucleotide sequence SEQ ID NO.5:CCAGACCAAAGAGTTACATCTACCG;
Reverse primer MTHFR-1298PR, its nucleotide sequence SEQ ID NO.6:CTTACCCTTCTCCCTTTGCCA; And/or
(2) primer obtained through increasing, lacking or replace single core thuja acid by the nucleotide sequence of forward primer MTHFR-1298PF or reverse primer MTHFR-1298PR;
The goal gene that above-mentioned primer pair is answered is methionine synthetase reductase gene, with this gene for template amplification gained DNA fragmentation comprises rs1801394 SNP site.
The present invention sets and synthesizes specific described Auele Specific Primer, can detect rs1801394 SNP site on methionine synthetase reductase gene.
4th technical purpose of the present invention is achieved by the following technical programs:
Blood direct PCR method detects the primer sets of folic acid metabolism related gene polymorphism, and described primer sets comprises following primer pair:
1) forward primer MTRR-PF, its nucleotide sequence SEQ ID NO.1:atgccttgaagtgatgag;
Reverse primer MTRR-PR, its nucleotide sequence SEQ ID NO.2:actgtaacggctctaacc;
2) forward primer MTHFR-677PF, its nucleotide sequence SEQ ID NO.3:AGGACAGTGTGGGAGTTTGG;
Reverse primer MTHFR-677PR, its nucleotide sequence SEQ ID NO.4:GAAAAGCTGCGTGATGATGA;
3) forward primer MTHFR-1298PF, its nucleotide sequence SEQ ID NO.5:CCAGACCAAAGAGTTACATCTACCG;
Reverse primer MTHFR-1298PR, its nucleotide sequence SEQ ID NO.6:CTTACCCTTCTCCCTTTGCCA.
5, on 10-Methylene tetrahydrofolate reductase gene, on rs1801133 SNP site, 5,10-Methylene tetrahydrofolate reductase gene, on rs1801131 SNP and methionine synthetase reductase gene, rs1801394 SNP site is the transgenation that the meeting of having generally acknowledged at present causes folate metabolism disorder.These three gene polymorphism sites are MTRR (A66G), MTHFR (C677T) respectively, MTHFR (A1298C).The present invention is without the need to extracting human gene group DNA, and from whole blood Direct PCR, without the need to the quantitative real time PCR Instrument of costliness, 5 can be detected by described primer simultaneously, rs1801394 SNP site on rs1801131 SNP and methionine synthetase reductase gene on rs1801133 SNP site, 5,10-Methylene tetrahydrofolate reductase gene on 10-Methylene tetrahydrofolate reductase gene.
Folic acid metabolism related gene polymorphism
5th technical purpose of the present invention is achieved by the following technical programs:
Blood direct PCR method detects the test kit of folic acid metabolism related gene polymorphism, and test kit comprises 2 × Taq Master Mix, primer sets according to claim 4 and deionized water ddH
2o.
As preferably, the formula of described 2 × Taq Master Mix is: 0.04-0.06U/ml HaemoTaq enzyme, 0.4-0.6mM (mmole) dNTP, 50-70mM Tricine(N-tri-(methylol) methylglycine), 4-6mM (NH
4)
2sO
4, 2.5-4.5mM MgCl
2, 4-7% glycerine, pH 8-9(25 DEG C), 90-110ng/ml T4 32 gene(T4 phage gene 32 proteins encoded).Wherein, ml refers to microlitre, 10
-6rise.
HaemoTaq enzyme, this enzyme is the Taq archaeal dna polymerase after brachymemma, and it has lacked 5'-3' 5 prime excision enzyme activity, can purchased from Shanghai Jinan Technology Co., Ltd..
As preferably, the formula of described 2 × Taq Master Mix is: 0.05U/ml HaemoTaq enzyme, 0.5mM dNTP, 60mM Tricine, 5mM (NH
4)
2sO
4, 3.5mM MgCl
2, 6% glycerine, pH 8.7(25 DEG C), 100ng/ml T4 32 gene.
6th technical purpose of the present invention is achieved by the following technical programs:
Blood direct PCR method detects the detection method of folic acid metabolism related gene polymorphism, and method described in it comprises and utilizes test kit of the present invention to carry out PCR reaction, the PCR reaction product of gained is checked order, sequencer map analyzes mutational site.
As preferably, described method comprises preparation PCR reaction system, and each sample amounts to 3 reactions, is specially:
MTRR (A66G) suddenlys change:
MTHFR (C677T) suddenlys change:
MTHFR (A1298C) suddenlys change:
。
As preferably, described method comprises the setting that PCR reacts specific procedure, is specially:
Hangzhou lattice KF960 PCR instrument can be used to react.
Detection kit of the present invention has the following advantages:
1, blood ingredient is complicated, and the factor of a lot of suppression PCR reaction is contained in the inside, and blood plasma, oxyphorase and lactoferrin etc. can suppress Taq enzyme active, and amplification efficiency declines, and also has some to become branch that primer is degraded, thus causes the failure of whole blood Direct PCR.We devise Specific PCR primers, use the PCR damping fluid of supressor impact in the Taq enzyme and minimizing blood of transformation, have prepared the reaction system of applicable whole blood PCR, made it PCR inhibitor in tolerance whole blood.
2, PCR condition is wide in range, without the need to preliminary experiment, is applicable to most regular-PCR instrument.
3, pcr template is whole blood, no matter is EDTA anti-freezing, and anticoagulant heparin or Trisodium Citrate anti-freezing are all applicable.Without the need to extracting genomic dna, so do not need additionally to buy genome DNA extraction test kit, cost-saving, decreasing pollution chance simultaneously, is applicable to the detection of extensive sample.
4, required whole blood sample is few, and minimum needs 2ul, and to the blood sample in 4 degree Refrigerator stores 7 days, detected result is stablized, and is conducive to check, or can utilize blood samples of other detections of patient, takes a blood sample specially, alleviate patient's misery without the need to patient.
5, PCR primer directly sends the order-checking of commercialization company outside, without the need to Purified in electrophoresis, reduces workload.Meanwhile, sequencer map analyzes mutational site, very clear, accuracy rate 100%.Simple to operate, with low cost, quick accurately high-throughput, meets the detection of two or three line city general hospitals.
Accompanying drawing explanation
Fig. 1 is PCR detection method Gel electrophoresis results figure of the present invention;
Fig. 2 is the blood PCR Gel electrophoresis results figure in the different anti-freezing source of the embodiment of the present invention one;
Fig. 3 is the embodiment of the present invention two PCR result electrophorogram;
Fig. 4 is that the MTRR (A66G) of the embodiment of the present invention three different annealing temperature suddenlys change PCR electrophorogram;
Fig. 5 is that the MTHFR (C677T) of the embodiment of the present invention three different annealing temperature suddenlys change PCR electrophorogram;
Fig. 6 is that the MTHFR (A1298C) of the embodiment of the present invention three different annealing temperature suddenlys change PCR electrophorogram.
Embodiment
Below in conjunction with accompanying drawing, the present invention is described in further detail.
Folic acid metabolism related gene polymorphism
PCR detection method Gel electrophoresis results figure is shown in Fig. 1,
M:DL2000 molecule Marker, is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom.Wherein 750bp is bright band.
1:MTRR (A66G) suddenlys change
2:MTHFR (C677T) suddenlys change
3:MTHFR (A1298C) suddenlys change
Electrophoresis result shows, PCR primer size is consistent with expection, and band is clear, without assorted band, illustrates that PCR system, reaction conditions and primer specificity are very good.
PCR primer transfers to Mei Ji bio tech ltd, Shanghai to check order.The sequencing result of various mutation type is as follows:.
PCR primer sequence is as follows, and wherein, it is primer combining site that PCR primer sequence draws horizontal line, and italicized item is gene extron, and black overstriking is intron, and two line position is SNP site.
MTRR A66G：
ATGCCTTGAAGTG
ATGAG GAGGTTTCTGTTACTATATGCTACACAGCAGGGACAGGCAAAGGCCATCGCAGAAGAAAT
A/G TGTGAGCAAGCTGTGGTACATGGATTTTCTGCAGATCTTCACTGTATTAGTGAATCCGATAA
G gttagagccgttacagt。
MTHFR C677T：
ggaaggtgcaagatcagagcccccaaagcagaggactctctctgcccagtccctgtggtctcttcatccctcgccttgaacaggtggaggccagcctctcctgactgtcatccctattggcag
GTTACCCCAAAGGCCACCCCGAAGCAGGGAGCTTTGAGGCTGACCTGAAGCACTTGAAGGAGAAGGTGTCTGCGGGAG
C/T
CGATTTCATCATCACGCAGCTTTTCTTTGAGGCTGACACATTCTTCCGCTTTGTGAAGGCATGCACCGACATGGGCATCACTTGCCCCATCGTCCCCGGGATCTTTCCCATCCAG
gtgaggggcccaggagagcccataagctccctccaccccactctcaccgcaccgtcctcgcacaggctgggggctctgggtggagtgctga
gttcgctgagttcttcccag。
MTHFR A1298C：
CCAGACCAAAGAGTTACATCTACCGTACCCAGGAGTGGGACGAGTTCCCTAACGGCCGCTG
gtgagggcctgcagaccttccttgcaaatacatctttgttcttgggagcgggagggcagaagaagtttgcatgcttgtggttgacctgggaggagtcaggggcagaatttacaggaatggcctcctgggcatgtggtggcactgccctctgtcaggagtgtgccctgacctctgggcacccctctgccag
GGGCAATTCCTCTTCCCCTGCCTTTGGGGAGCTGAAGGACTACTACCTCTTCTACCTGAAGAGCAAGTCCCCCAAGGAGGAGCTGCTGAAGATGTGGGGGGAGGAGCTGACCAGTGAAG
A/C AAGTGTCTTTGAAGTCTTCGTTCTTTACCTCTCGGGAGAACCAAACCGGAATGGTCACAAA
gtgagtgatgctggagtggggaccctggttcatcccctgcccctggcctgaccccagctgcaggccaggctgcggggctgtgacttccccatcctgtgccctcccctccatgctgtggaca
tggcaaagggagaagggtaag。
Embodiment one
The fresh blood of the same people of the different anti-freezing different acquisitions of PCR of the blood sample in different anti-freezing source, is collected in EDTA anticoagulant tube respectively, in heparin sodium anticoagulant tube and Trisodium Citrate anticoagulant tube, is respectively numbered A, B, C.
Prepare PCR reaction solution by the following method,
PCR reaction conditions is as follows:
The blood PCR electrophorogram in different anti-freezing source is shown in Fig. 2, wherein
M:DL2000 molecule Marker, is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom.Wherein 750bp is bright band.
The anticoagulant MTRR of 1:EDTA (A66G) suddenlys change PCR;
The anticoagulant MTHFR of 2:EDTA (C677T) suddenlys change PCR;
The anticoagulant MTHFR of 3:EDTA (A1298C) suddenlys change PCR;
4: the anticoagulant MTRR of heparin sodium (A66G) suddenlys change PCR;
5: the anticoagulant MTHFR of heparin sodium (C677T) suddenlys change PCR;
6: the anticoagulant MTHFR of heparin sodium (A1298C) suddenlys change PCR;
7: the anticoagulant MTRR of Trisodium Citrate (A66G) suddenlys change PCR;
8: the anticoagulant MTHFR of Trisodium Citrate (C677T) suddenlys change PCR;
9: the anticoagulant MTHFR of Trisodium Citrate (A1298C) suddenlys change PCR.
Electrophoresis result shows: the blood in different anticoagulant methods source can achieve desired results in this PCR reaction system, and PCR band is clear, without assorted band.Sequencing result verifies that the blood mutational site in these three kinds of anticoagulant methods sources is consistent.
Embodiment two
The blood of fresh extraction is divided into three groups, and first group of poba gene group DNA extraction agent box with Tian Gen company, according to test kit specification sheets extracting poba gene group DNA, detects genomic dna concentration, use ddH
2o is diluted to PCR after 100ng/ul; Second group of direct blood PCR of fresh blood, places 7 days (7D) direct blood PCR afterwards at 4 DEG C of refrigerators for the 3rd group.The anticoagulant PCR of EDTA.PCR method is according to example 1.PCR the results are shown in Figure 3.
In Fig. 3
The blood MTRR (A66G) that 1:4 degree is preserved 7 days suddenlys change PCR;
The blood MTHFR (C677T) that 2:4 degree is preserved 7 days suddenlys change PCR;
The blood MTHFR (A1298C) that 3:4 degree is preserved 7 days suddenlys change PCR;
4: fresh blood MTRR (A66G) suddenlys change PCR;
5: fresh blood MTHFR (C677T) suddenlys change PCR;
6: fresh blood MTHFR (A1298C) suddenlys change PCR;
7: the MTRR (A66G) of blood extracting genomic dna suddenlys change PCR;
8: blood extracting genomic dna MTHFR (C677T) suddenlys change PCR;
9: blood extracting genomic dna MTHFR (A1298C) suddenlys change PCR.
Electrophoresis and sequence verification sudden change result show, the blood sample that test kit of the present invention places 7 days to 4 DEG C of refrigerators is effective equally, consistent with the result that fresh blood extracts PCR again after genomic dna.
Embodiment three
Different PCR response procedures is on the impact of experimental result
Because although experimenter is provided with identical PCR program in PCR instrument, because different PCR instrument warming and cooling rates can be variant, this species diversity can cause the difference of each thermotonus time.It is annealing that the step affecting PCR result key is played, so we adopt lattice grads PCR instrument to verify different PCR reaction conditionss, mainly annealing temperature is on the impact of PCR result.
By the EDTA anticoagulation of fresh collection according to embodiment one method preparation PCR reaction solution.PCR response procedures is as follows:
The MTRR (A66G) of the different annealing temperature PCR electrophorogram that suddenlys change is shown in Fig. 4, wherein,
1: the MTRR (A66G) that annealing temperature is 50 degree suddenlys change PCR;
2: the MTRR (A66G) that annealing temperature is 52 degree suddenlys change PCR;
3: the MTRR (A66G) that annealing temperature is 54 degree suddenlys change PCR;
4: the MTRR (A66G) that annealing temperature is 56 degree suddenlys change PCR;
5: the MTRR (A66G) that annealing temperature is 58 degree suddenlys change PCR;
6: the MTRR (A66G) that annealing temperature is 60 degree suddenlys change PCR.
The MTHFR (C677T) of the different annealing temperature PCR that suddenlys change is shown in Fig. 5, wherein,
1: the MTHFR (C677T) that annealing temperature is 50 degree suddenlys change PCR;
2: the MTHFR (C677T) that annealing temperature is 52 degree suddenlys change PCR;
3: the MTHFR (C677T) that annealing temperature is 54 degree suddenlys change PCR;
4: the MTHFR (C677T) that annealing temperature is 56 degree suddenlys change PCR;
5: the MTHFR (C677T) that annealing temperature is 58 degree suddenlys change PCR;
6: the MTHFR (C677T) that annealing temperature is 60 degree suddenlys change PCR.
The MTHFR (A1298C) of the different annealing temperature PCR that suddenlys change is shown in Fig. 6, wherein,
1: the MTHFR (A1298C) that annealing temperature is 50 degree suddenlys change PCR;
2: the MTHFR (A1298C) that annealing temperature is 52 degree suddenlys change PCR;
3: the MTHFR (A1298C) that annealing temperature is 54 degree suddenlys change PCR;
4: the MTHFR (A1298C) that annealing temperature is 56 degree suddenlys change PCR;
5: the MTHFR (A1298C) that annealing temperature is 58 degree suddenlys change PCR;
6: the MTHFR (A1298C) that annealing temperature is 60 degree suddenlys change PCR.
Result shows: test kit of the present invention is actual can meet different PCR annealing temperatures, has the experiment condition of wider model, is applicable to different PCR instrument.
This specific embodiment is only explanation of the invention; it is not limitation of the present invention; those skilled in the art can make to the present embodiment the amendment not having creative contribution as required after reading this specification sheets, as long as but be all subject to the protection of patent law in right of the present invention.
SEQUENCE LISTING
Company limited of <110> Huzhou ancient cooking vessel brilliant medical test institute
<120> blood direct PCR method detects the primer of folic acid metabolism related gene polymorphism, primer sets, test kit and inspection
Survey method
<130> 1
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213> artificial sequence
<400> 1
atgccttgaa gtgatgag 18
<210> 2
<211> 18
<212> DNA
<213> artificial sequence
<400> 2
actgtaacgg ctctaacc 18
<210> 3
<211> 20
<212> DNA
<213> artificial sequence
<400> 3
aggacagtgt gggagtttgg 20
<210> 4
<211> 20
<212> DNA
<213> artificial sequence
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gaaaagctgc gtgatgatga 20
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<211> 25
<212> DNA
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ccagaccaaa gagttacatc taccg 25
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<211> 21
<212> DNA
<213> artificial sequence
<400> 6
cttacccttc tccctttgcc a 21
Claims (10)
1. blood direct PCR method detects the primer of folic acid metabolism related gene polymorphism, it is characterized in that comprising following primer pair:
(1) forward primer MTRR-PF, its nucleotide sequence SEQ ID NO.1:atgccttgaagtgatgag;
Reverse primer MTRR-PR, its nucleotide sequence SEQ ID NO.2:actgtaacggctctaacc; And/or
(2) primer obtained through increasing, lacking or replace single core thuja acid by the nucleotide sequence of forward primer MTRR-PF or reverse primer MTRR-PR;
The goal gene that above-mentioned primer pair is answered is 5,10-Methylene tetrahydrofolate reductase gene, with this gene for template amplification gained DNA fragmentation comprises rs1801133 SNP site.
2. blood direct PCR method detects the primer of folic acid metabolism related gene polymorphism, it is characterized in that comprising following primer pair:
(1) forward primer MTHFR-677PF, its nucleotide sequence SEQ ID NO.3:AGGACAGTGTGGGAGTTTGG;
Reverse primer MTHFR-677PR, its nucleotide sequence SEQ ID NO.4:GAAAAGCTGCGTGATGATGA; And/or
(2) primer obtained through increasing, lacking or replace single core thuja acid by the nucleotide sequence of forward primer MTHFR-677PF or reverse primer MTHFR-677PR;
The goal gene that above-mentioned primer pair is answered is 5,10-Methylene tetrahydrofolate reductase gene, with this gene for template amplification gained DNA fragmentation comprises rs1801131 SNP site.
3. blood direct PCR method detects the primer of folic acid metabolism related gene polymorphism, it is characterized in that comprising following primer pair:
(1) forward primer MTHFR-1298PF, its nucleotide sequence SEQ ID NO.5:CCAGACCAAAGAGTTACATCTACCG;
Reverse primer MTHFR-1298PR, its nucleotide sequence SEQ ID NO.6:CTTACCCTTCTCCCTTTGCCA; And/or
(2) primer obtained through increasing, lacking or replace single core thuja acid by the nucleotide sequence of forward primer MTHFR-1298PF or reverse primer MTHFR-1298PR;
The goal gene that above-mentioned primer pair is answered is methionine synthetase reductase gene, with this gene for template amplification gained DNA fragmentation comprises rs1801394 SNP site.
4. blood direct PCR method detects the primer sets of folic acid metabolism related gene polymorphism, it is characterized in that: described primer sets comprises following primer pair:
1) forward primer MTRR-PF, its nucleotide sequence SEQ ID NO.1:atgccttgaagtgatgag;
Reverse primer MTRR-PR, its nucleotide sequence SEQ ID NO.2:actgtaacggctctaacc;
2) forward primer MTHFR-677PF, its nucleotide sequence SEQ ID NO.3:AGGACAGTGTGGGAGTTTGG;
Reverse primer MTHFR-677PR, its nucleotide sequence SEQ ID NO.4:GAAAAGCTGCGTGATGATGA;
3) forward primer MTHFR-1298PF, its nucleotide sequence SEQ ID NO.5:CCAGACCAAAGAGTTACATCTACCG;
Reverse primer MTHFR-1298PR, its nucleotide sequence SEQ ID NO.6:CTTACCCTTCTCCCTTTGCCA.
5. blood direct PCR method detects the test kit of folic acid metabolism related gene polymorphism, it is characterized in that: test kit comprises 2 × Taq Master Mix, primer sets according to claim 4 and deionized water ddH
2o.
6. blood direct PCR method according to claim 5 detects the test kit of folic acid metabolism related gene polymorphism, it is characterized in that: the formula of described 2 × Taq Master Mix is: 0.04-0.06U/ml HaemoTaq enzyme, 0.4-0.6mM dNTP, 50-70mM Tricine, 4-6mM (NH
4)
2sO
4, 2.5-4.5mM MgCl
2, 4-7% glycerine, pH 8-9(25 DEG C), 90-110ng/ml T4 32 gene.
7. blood direct PCR method according to claim 5 detects the test kit of folic acid metabolism related gene polymorphism, it is characterized in that: the formula of described 2 × Taq Master Mix is: 0.05U/ml HaemoTaq enzyme, 0.5mM dNTP, 60mM Tricine, 5mM (NH
4)
2sO
4, 3.5mM MgCl
2, 6% glycerine, pH 8.7(25 DEG C), 100ng/ml T4 32 gene.
8. blood direct PCR method detects the detection method of folic acid metabolism related gene polymorphism, it is characterized in that: described method comprises and utilizes test kit described in any one of claim 5-7 to carry out PCR reaction, the PCR reaction product of gained is checked order, sequencer map analyzes mutational site.
9. blood direct PCR method according to claim 8 detects the detection method of folic acid metabolism related gene polymorphism, it is characterized in that: described method comprises preparation PCR reaction system, and each sample amounts to 3 reactions, is specially:
MTRR (A66G) suddenlys change:
MTHFR (C677T) suddenlys change:
MTHFR (A1298C) suddenlys change:
。
10. blood direct PCR method according to claim 8 detects the detection method of folic acid metabolism related gene polymorphism, it is characterized in that: described method comprises the setting that PCR reacts specific procedure, is specially:
。
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| CN106868180A (en) * | 2017-03-30 | 2017-06-20 | 浙江中迪生物科技有限公司 | The method that the direct amplification of nucleic acid of whole blood detects folic acid metabolism related gene polymorphism |
| CN107058530A (en) * | 2017-03-30 | 2017-08-18 | 浙江中迪生物科技有限公司 | The kit and its application method of folic acid medication sensitive gene are directly detected for blood |
| CN108034710A (en) * | 2017-12-19 | 2018-05-15 | 上海派森诺医学检验所有限公司 | For detecting the primer, fluorescence probe and application of folic acid metabolism related gene SNP |
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| CN109811042A (en) * | 2018-12-29 | 2019-05-28 | 广州凯普医药科技有限公司 | A kind of method of Overlap extension PCR combination Sanger sequencing detection folic acid metabolism associated gene mutation |
| CN109811042B (en) * | 2018-12-29 | 2020-02-07 | 广州凯普医药科技有限公司 | Method for detecting folic acid metabolism related gene mutation by combining overlap extension PCR with Sanger sequencing |
| CN117660632A (en) * | 2024-01-31 | 2024-03-08 | 江苏吉诺思美精准医学科技有限公司 | SNP locus primer and probe for guiding folic acid medication and method thereof |
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