CN104109714A - Primer for detecting folic acid metabolism related gene SNP, fluorescent probe and application - Google Patents
Primer for detecting folic acid metabolism related gene SNP, fluorescent probe and application Download PDFInfo
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Abstract
The invention discloses primers for detecting folic acid metabolism related gene SNP, fluorescent probes and applications. The provided primers have four groups. Each group of the primers has a corresponding fluorescent probe group for carrying out rapid gene typing on rs1801133, rs11018628 and rs1047891. Detected results obtained by the primers and the fluorescent probe are accurate and reliable. The primers have low cost and relatively good practicality.
Description
Technical field
The present invention relates to biological technical field, refer to particularly a kind of primer for detection of folic acid metabolism genes involved SNP, fluorescent probe and application.
Background technology
Folic acid (Folic acid) is one of vitamin B complex, being equivalent to VitB11 (pteroylglutamic acid, PGA), is Mitchell (H.K.Mitchell, 1941) from the leaf of spinach, extract purifying, so called after folic acid.Folic acid is the required important nutrient of body, rich content in green vegetable, fruit and animal livers.Folic acid form with tetrahydrofolic acid (THFA) in human body works, and tetrahydrofolic acid (THFA) is the vehicle of one carbon unit, can deliver the one carbon units such as methyl, methene, carboxaldehyde radicals, participates in the synthetic of vivo acid, purine, pyrimidine.Thereby folic acid is all indispensable nutrient substance to cell fission propagation, tissue repair and body development etc.
Folic acid be confirmed as far back as 1948 to the important trophism of human body, and the mankind (or other animals) can cause megaloblastic anemia and leukopenia as lacked folic acid.In addition, research is also found, folic acid is even more important to pregnant woman, folic acid deficiency can cause two kinds of results: the first, and make homocysteine transform and occur obstacle to methionine(Met), and then cause hyperhomocysteinemiainjury, the latter can cause blood vessel endothelium injury, promote vascular smooth muscle cell curing, causing blood coagulation and Fibrinolytic System imbalance, make blood in hypercoagulative state, is also the important factor that causes pregnancy induced hypertension syndrome; The second, cause that S-adenosylmethionine/AdoHcy ratio reduces, suppress DNA methyl and turn solemn enzymic activity and DNA methylation, cause karyomit(e) nondisjunction, cause multiple fetal anomaly.After deliberation, in inborn defect TOP V, have four relevant with folic acid deficiency, i.e. congenital heart disease, harelip, spina bifida and anencephalus.
Gestation 4 weeks is the important period of fetal neural tube differentiation and formation, and this phase folic acid deficiency can increase the risk of Fetal neurotubules malformation and premature labor; More studies show that, women took folic acid after 4 weeks, and the anacid state of body internal lobe just can be significantly improved.Therefore, the women of child-bearing age at least should start at pregnant first 3 months, suitably eat the food and the Supplement of folic acid that are rich in folic acid more, to guarantee that embryo has a good folic acid nutrition state in early days, and the generation of prevention fetal neural tube and other organ deformities.
When the women of child-bearing age take folic acid supplementing agent, due to individual genetic constitution difference, have the difference of folic acid metabolism ability, the required folic acid dosage of different crowd cannot treat different things as the same.As the actual demand of taking dose deficiency, do not reach the effect of Supplement of folic acid; And blindness excessive use high density folic acid can disturb pregnant woman's nutritive element metabolism, can affect equally fetation, excess is taken folic acid also can cause adverse drug reaction, can cause serious adverse consequences to pregnant woman and fetus on the contrary.So the women of child-bearing age of different physical appearances and quality must be according to s own situation, prepare with a definite target in view and supplementary needed folic acid.
Therefore, choose with Pre-pregnancy women/pregnancy period and the required folic acid metabolism of fetation closely related and study some clear and definite important gene sites, comprehensive utilization modern scientific research achievement, develop a folic acid supplementing agent based on Molecular and genetic basis and take quantitative guidance product, the folic acid supplement amount that can propose to be applicable to himself demand for different genetic constitution crowds instructs, and has very strong social effect and wide market outlook.
The simplest method that detects at present folic acid metabolism genes involved genetic polymorphism is PCR-RFLP, and its cardinal principle is by application specific digestion with restriction enzyme pcr amplification product, and whether disappears to judge that according to digestion site whether its variation exists.But the method complicated operation, easily causes the crossed contamination of PCR product and easily occurs that enzyme cuts insufficient or enzyme and cut excessively and occur false negative or false positive results when sample size is many, reliability is low.Although multiple PCR method specificity increases, this side's ratio juris remains the principle based on regular-PCR, and these factors such as primer specificity and Lo-Fi Taq enzyme all can cause the impact on result.Although DNA sequencing method is the gold standard that carries out at present gene diagnosis, complex steps, process complexity, reagent is expensive, easily occurs the crossed contamination between sample and causes checking order unsuccessfully; The equipment of sequenator has also exceeded the tolerance range of general Clinical Test Lab in addition.High resolving power melting curve method is fast a kind of, easy, economy, and practical classifying method, but gene type depends on the accuracy of instrument temperature control, and false positive is high.The method of Taqman probe adopts specific fluorescently-labeled probe, and high specificity is highly sensitive and easy to operate, fast.The method is considered to the gold standard of SNP somatotype equally, at the prospect Worth Expecting of clinical application.
Summary of the invention
The object of the present invention is to provide a kind of primer for detection of folic acid metabolism genes involved SNP, fluorescent probe and application.
For achieving the above object, first group of primer for detection of folic acid metabolism genes involved SNP provided by the invention (hereinafter to be referred as MTHFR primer), described primer is following primer pair:
(1) forward primer MTHFR-F, its nucleotide sequence is as shown in SEQ ID NO.1;
Reverse primer MTHFR-R, its nucleotide sequence is as shown in SEQ ID NO.2;
(2) primer being obtained through increase, disappearance or replacement single core thuja acid by the nucleotide sequence of forward primer MTHFR-F or reverse primer MTHFR-R;
The corresponding goal gene of above-mentioned primer is Methylene tetrahydrofolate reductase gene, comprises rs1801133SNP site taking this gene as template amplification gained DNA fragmentation.
The present invention also provides one group of fluorescent probe being used in conjunction with MTHFR primer (hereinafter to be referred as MTHFR probe), and described fluorescent probe comprises following two:
MTHFR-T probe, its nucleotide sequence is as shown in SEQ ID NO.3;
MTHFR-C probe, its nucleotide sequence is as shown in SEQ ID NO.4;
5 ' end of described MTHFR-T probe and MTHFR-C probe is marked with reporter group, and 3 ' end is marked with fluorescent quenching group, and 5 ' end reporter group of MTHFR-T probe is different with 5 ' end reporter group of MTHFR-C probe.
Wherein, the reporter group of 5 ' end mark can be selected from: FAM, HEX, TET, JOE, VIC, ROX, Cy3, Cy5, MAR, JUP, SAT, PLU or NEP, and be not limited to above-mentioned group; The fluorescent quenching group of 3 ' end mark can be selected from: TAMRA, Eclipse, BHQ1, BHQ2, BHQ3, DABCYL, MGB, and be not limited to above-mentioned group.
Preferably, the reporter group of 5 ' end mark is FAM or VIC, and the fluorescent quenching group of 3 ' end mark is MGB.
The present invention also provides MT reconnaissance HFR primer and/or the application of MTHFR probe in the test kit of preparation detection Methylene tetrahydrofolate reductase gene rs1801133SNP loci polymorphism.
Second group of primer for detection of folic acid metabolism genes involved SNP provided by the invention (hereinafter to be referred as NOX primer), described primer is following primer pair:
(1) forward primer NOX-F, its nucleotide sequence is as shown in SEQ ID NO.5;
Reverse primer NOX-R, its nucleotide sequence is as shown in SEQ ID NO.6;
(2) primer being obtained through increase, disappearance or replacement single core thuja acid by the nucleotide sequence of forward primer NOX-F or reverse primer NOX-R;
The corresponding goal gene of above-mentioned primer is nadph oxidase gene, comprises rs11018628SNP site taking this gene as template amplification gained DNA fragmentation.
The present invention also provides one group of fluorescent probe being used in conjunction with NOX primer (hereinafter to be referred as NOX probe), and described fluorescent probe comprises following two:
NOX-T probe, its nucleotide sequence is as shown in SEQ ID NO.7;
NOX-C probe, its nucleotide sequence is as shown in SEQ ID NO.8;
5 ' end of described NOX-T probe and NOX-C probe is marked with reporter group, and 3 ' end is marked with fluorescent quenching group, and 5 ' end reporter group of NOX-T probe is different with 5 ' end reporter group of NOX-C probe.
Wherein, the reporter group of 5 ' end mark can be selected from: FAM, HEX, TET, JOE, VIC, ROX, Cy3, Cy5, MAR, JUP, SAT, PLU or NEP, and be not limited to above-mentioned group; The fluorescent quenching group of 3 ' end mark can be selected from: TAMRA, Eclipse, BHQ1, BHQ2, BHQ3, DABCYL, MGB, and be not limited to above-mentioned group.
Preferably, the reporter group of 5 ' end mark is FAM or VIC, and the fluorescent quenching group of 3 ' end mark is MGB.
The present invention also provides above-mentioned NOX primer and/or the application of NOX probe in the test kit of preparation detection nadph oxidase gene rs11018628SNP loci polymorphism.
The 3rd group of primer for detection of folic acid metabolism genes involved SNP provided by the invention (hereinafter to be referred as CPS primer), described primer is following primer pair:
(1) forward primer CPS-F, its nucleotide sequence is as shown in SEQ ID NO.9;
Reverse primer CPS-R, its nucleotide sequence is as shown in SEQ ID NO.10;
(2) primer being obtained through increase, disappearance or replacement single core thuja acid by the nucleotide sequence of forward primer CPS-F or reverse primer CPS-R;
The corresponding goal gene of above-mentioned primer is carbamyl phosphate synthase gene, comprises rs1047891SNP site taking this gene as template amplification gained DNA fragmentation.
The present invention also provides one group of fluorescent probe being used in conjunction with CPS primer (hereinafter to be referred as CPS probe), and described fluorescent probe comprises following two:
CPS-G probe, its nucleotide sequence is as shown in SEQ ID NO.11;
CPS-T probe, its nucleotide sequence is as shown in SEQ ID NO.12;
5 ' end of described CPS-G probe and CPS-T probe is marked with reporter group, and 3 ' end is marked with fluorescent quenching group, and 5 ' end reporter group of CPS-T probe is different with 5 ' end reporter group of CPS-C probe.
Wherein, the reporter group of 5 ' end mark can be selected from: FAM, HEX, TET, JOE, VIC, ROX, Cy3, Cy5, MAR, JUP, SAT, PLU or NEP, and be not limited to above-mentioned group; The fluorescent quenching group of 3 ' end mark can be selected from: TAMRA, Eclipse, BHQ1, BHQ2, BHQ3, DABCYL, MGB, and be not limited to above-mentioned group.
Preferably, the reporter group of 5 ' end mark is FAM or VIC, and the fluorescent quenching group of 3 ' end mark is MGB.
The present invention also provides above-mentioned CPS primer and/or the application of CPS probe in the detection kit of rs1047891SNP loci polymorphism of preparing carbamyl phosphate synthase gene.
The 4th group of primer sets for detection of folic acid metabolism genes involved SNP provided by the invention (hereinafter to be referred as the 4th group of primer), described primer sets comprises MT reconnaissance HFR primer, NOX primer and CPS primer:
1) forward primer MTHFR-F, its nucleotide sequence is as shown in SEQ ID NO.1;
Reverse primer MTHFR-R, its nucleotide sequence is as shown in SEQ ID NO.2;
2) forward primer NOX-F, its nucleotide sequence is as shown in SEQ ID NO.5;
Reverse primer NOX-R, its nucleotide sequence is as shown in SEQ ID NO.6;
3) forward primer CPS-F, its nucleotide sequence is as shown in SEQ ID NO.9;
Reverse primer CPS-R, its nucleotide sequence is as shown in SEQ ID NO.10.
The present invention also provides one group of fluorescent probe group being used in conjunction with above-mentioned the 4th group of primer (hereinafter to be referred as the 4th group of fluorescent probe), and described fluorescent probe group comprises following probe:
MTHFR-T probe, its nucleotide sequence is as shown in SEQ ID NO.3;
MTHFR-C probe, its nucleotide sequence is as shown in SEQ ID NO.4;
NOX-T probe, its nucleotide sequence is as shown in SEQ ID NO.7;
NOX-C probe, its nucleotide sequence is as shown in SEQ ID NO.8;
CPS-G probe, its nucleotide sequence is as shown in SEQ ID NO.11;
CPS-T probe, its nucleotide sequence is as shown in SEQ ID NO.12;
5 ' end of described MTHFR-T probe and MTHFR-C probe is marked with reporter group, and 3 ' end is marked with fluorescent quenching group, and 5 ' end reporter group of MTHFR-T probe is different with 5 ' end reporter group of MTHFR-C probe;
5 ' end of described NOX-T probe and NOX-C probe is marked with reporter group, and 3 ' end is marked with fluorescent quenching group, and 5 ' end reporter group of NOX-T probe is different with 5 ' end reporter group of NOX-C probe;
5 ' end of described CPS-G probe and CPS-T probe is marked with reporter group, and 3 ' end is marked with fluorescent quenching group, and 5 ' end reporter group of CPS-T probe is different with 5 ' end reporter group of CPS-C probe.
Wherein, the reporter group of 5 ' end mark can be selected from: FAM, HEX, TET, JOE, VIC, ROX, Cy3, Cy5, MAR, JUP, SAT, PLU or NEP, and be not limited to above-mentioned group; The fluorescent quenching group of 3 ' end mark can be selected from: TAMRA, Eclipse, BHQ1, BHQ2, BHQ3, DABCYL, MGB, and be not limited to above-mentioned group.
Preferably, the reporter group of 5 ' end mark is FAM or VIC, and the fluorescent quenching group of 3 ' end mark is MGB.
The present invention also provides a kind of PCR kit for fluorescence quantitative for detection of folic acid metabolism genes involved SNP, comprises Taq enzyme, dNTPs, Mgcl
2, 10 × PCR buffer, ROX reference fluorescence, ultrapure water and the 4th group of primer, the 4th group of fluorescent probe.It should be noted that, about the consumption of above-mentioned each reagent, primer, fluorescent probe, and the time of PCR, temperature parameter setting is common technology means, is that those skilled in the art can adjust according to practical situation, not in order to limit the present invention.PCR buffer is commercially available prod.
In addition; although the present invention only provides the 4th group of primer that comprises MTHFR primer, NOX primer, CPS primer; and the combination of the 4th group of fluorescent probe; but combination and the application of any two kinds in MTHFR primer, NOX primer, CPS primer, or also should be considered as protection scope of the present invention with the coupling of corresponding fluorescent probe.
The present invention also provides a kind of method that detects folic acid metabolism genes involved SNP polymorphism, the method is to utilize the 4th group of primer, the 4th group of fluorescent probe, carry out quantitative fluorescent PCR reaction for human gene group DNA, the fluorescent signal producing according to reaction process is distinguished the polymorphism in corresponding SNP site.
Above-mentioned primer, fluorescent probe can be applied in the correlation detection of warfarin personalized medicine.
Principle of the present invention:
Study discovery through the present inventor, the gene relevant to folic acid metabolism comprises following three kinds:
1) MTHFR: coding Methylene tetrahydrofolate reductase, can catalysis form 5-methyltetrahydrofolate, the latter participates in homocysteine metabolism, mthfr gene sudden change can cause folic acid utilization ratio low, homocysteine accumulates in vivo, cause hyperhomocysteinemiainjury, can affect the early stage cardiovascular growth of embryo.
2) NOX4: coding nadph oxidase, content is more in kidney.In the genome health research of participating in 140,000 women, find that NOX4 gene is relevant to homotype semicystinol concentration investigating in blood, NOX4 transgenation meeting reduces homotype semicystinol concentration investigating.
3) CPS1: encoding carbamoyl phosphate synthase, catalysis forms carbamyl phosphate, participates in urea synthesis, pyrimidine etc.CPS transgenation meeting affects homotype semicystinol concentration investigating in women's blood, and appropriate folic acid supplements can metabolism homocysteine, prevention hyperhomocysteinemiainjury.
So the present invention has used rs1801133 site, the rs11018628 site of NOX4 gene and the rs1047891 site of CPS1 gene of the TaqMan probe technique of generally admitting in the world at present to mthfr gene to carry out gene type, its principle is mainly, in the PCR of TaqMan probe method reaction system, comprise one couple of PCR primers and a pair of probe.Probe only with template specificity combination, its binding site is between two primers.5 ' end of probe is marked with reporter gene, 3 ' end is marked with fluorescent quenching group, in the time that probe is complete, the fluorescent energy that reporter gene is launched is quenched group and absorbs, and instrument can't detect signal, along with the carrying out of PCR, Taq enzyme runs into the probe of being combined with template in chain extension process, and its 3 '-5 ' exonuclease activity will cut off probe, and reporter group is away from quenching group, its energy can not be absorbed, and produces fluorescent signal.With two kinds of different fluorescence dyes (as FAM, HEX, VIC) this pair of probe of mark respectively, for the different genotype of two equipotential SNP, just can in a PCR reaction, complete the genotype in single SNP site is judged.Primer length in the method, probe length, primer specificity, specificity and the susceptibility of probe specificity to detected result is extremely important, so needs rational design primer and repeatedly scientific validation just can obtain good primer and probe.
Beneficial effect of the present invention: a kind of somatotype reagent and application of the related SNP of folic acid metabolism are efficiently provided, and the detected result that application the present invention obtains is accurately reliable, and cost is lower, has good practicality.
Brief description of the drawings
Fig. 1 is the genotypic PCR fluorometric analysis of SNP somatotype result rs1801133TT figure in the embodiment of the present invention;
Fig. 2 is the genotypic PCR fluorometric analysis of SNP somatotype result rs1801133TC figure in the embodiment of the present invention;
Fig. 3 is the genotypic PCR fluorometric analysis of SNP somatotype result rs1801133CC figure in the embodiment of the present invention;
Fig. 4 is the genotypic PCR fluorometric analysis of SNP somatotype result rs11018628TT figure in the embodiment of the present invention;
Fig. 5 is the genotypic PCR fluorometric analysis of SNP somatotype result rs11018628TC figure in the embodiment of the present invention;
Fig. 6 is the genotypic PCR fluorometric analysis of SNP somatotype result rs11018628CC figure in the embodiment of the present invention;
Fig. 7 is the genotypic PCR fluorometric analysis of SNP somatotype result rs1047891GG figure in the embodiment of the present invention;
Fig. 8 is the genotypic PCR fluorometric analysis of SNP somatotype result rs1047891GT figure in the embodiment of the present invention;
Fig. 9 is the genotypic PCR fluorometric analysis of SNP somatotype result rs1047891TT figure in the embodiment of the present invention;
Figure 10 is the type map of sequencing result in the embodiment of the present invention.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
Embodiment
1, prepare following primer and probe:
(1) for primer and the fluorescent probe in Methylene tetrahydrofolate reductase gene rs1801133SNP site:
Forward primer MTHFR-F:5 '-TACCCCAAAGGCCACCCCG-3 '
Reverse primer MTHFR-R:5 '-CATGCCTTCACAAAGCGGA-3 '
MTHFR-T probe:
FAM-GTGTCTGCGGGAG
TCGATTTCATCATCACGC-MGB
MTHFR-C probe:
VIC-GTGTCTGCGGGAG
CCGATTTCATCATCACGC-MGB
(2) for primer and the fluorescent probe in NOX oxidase gene rs11018628SNP site:
Forward primer NOX-F:5 '-TGCAGAGAGCATGGTTGACTAG-3 '
Reverse primer NOX-R:5 '-TAAAGAGGAATGAGGCAACAT-3 '
NOX-T probe:
FAM-TGATTGTTTTTGATG
TGGAGTAGATGAGAG-MGB
NOX-C probe:
VIC-TGATTGTTTTTGATG
CGGAGTAGATGAGAG-MGB
(3) for primer and the fluorescent probe in the rs1047891SNP site of carbamyl phosphate synthase gene:
Forward primer CPS-F:5 '-TAAATTATTTCAGAAAACAG-3 '
Reverse primer CPS-R:5 '-AAAAATAAGAAATCACTGTGA-3 '
CPS-G probe:
FAM-ATGCCACTGGG
GTGGCAGGGACATTG-MGB
CPS-T probe:
VIC-ATGCCACTGGG
TTGGCAGGGACATTG-MGB
Above-mentioned 3 groups of primers are prepared into mixed solution with corresponding probe respectively, and in mixed solution, primer concentration is 18 μ mol/L, and fluorescent probe concentration is 5 μ mol/L.
2, prepare pcr amplification system:
96 examinates' of preparation DNA sample, and add respectively the mixed solution of 3 groups of primers and probe to do 3 PCR reactions to each DNA, coming respectively each SNP to be carried out to somatotype, required reagent comprises Taqman Genotyping Master Mix5 μ l, ddH
2o3.5 μ l, fluorescent probe and primer mixed solution 0.5 μ l, all the other are examinate's DNA sample 1 μ l of 20ng/ μ l, cumulative volume 10 μ l.
3, pcr amplification program:
PCR reacts the first step: 60 DEG C, 30s; PCR reacts second step: 95 DEG C, 10min; PCR reacts the 3rd step: 92 DEG C, 15s; PCR reacts the 4th step: 60 DEG C, 90s; PCR reacts the 5th step: circulation the 3rd step the 4th step 50 times, then 60 DEG C, 30s.Adopt ABI Stepone quantitative real time PCR Instrument (Applied Biosystems, Applied Biotechnology company limited of the U.S.).
4, experimental result:
After PCR reaction finishes, application Stepone software V2.1 (Applied Biosystems, Applied Biotechnology company limited of the U.S.) analyze, the 96 routine results that obtain are classified, its result type, as shown in Fig. 1～9, is divided into and discerns 9 kinds of genotype according to probe of the present invention: SNP rs1801133 tri-kinds of genotype TT, TC and CC; SNP rs11018628 tri-kinds of genotype TT, TC and CC; SNP rs1047891 tri-kinds of genotype GG, GT and TT.
5, the analysis of the accuracy of test kit
For the accuracy that checking test kit detects, 96 routine samples are carried out to sequence verification.SNP rs1801133T/T genotype has 10 examples, and T/C genotype has 41 examples, and C/C genotype has 45 examples; SNP rs11018628C/C genotype has 7 examples, and T/C genotype has 39 examples, and T/T genotype has 50 examples; SNP rs1047891T/T genotype has 9 examples, and G/T genotype has 40 examples, and G/G genotype has 47 examples.The accuracy that test kit detects has reached 100%.The type of sequencing result as shown in figure 10.
Claims (13)
1. for detection of a primer of folic acid metabolism genes involved SNP, described primer is following primer pair:
(1) forward primer MTHFR-F, its nucleotide sequence is as shown in SEQ ID NO.1;
Reverse primer MTHFR-R, its nucleotide sequence is as shown in SEQ ID NO.2;
(2) primer being obtained through increase, disappearance or replacement single core thuja acid by the nucleotide sequence of forward primer MTHFR-F or reverse primer MTHFR-R;
The corresponding goal gene of above-mentioned primer is Methylene tetrahydrofolate reductase gene, comprises rs1801133SNP site taking this gene as template amplification gained DNA fragmentation.
2. with the fluorescent probe being used in conjunction with for detection of the primer of folic acid metabolism genes involved SNP described in claim 1, described fluorescent probe comprises following two:
MTHFR-T probe, its nucleotide sequence is as shown in SEQ ID NO.3;
MTHFR-C probe, its nucleotide sequence is as shown in SEQ ID NO.4;
5 ' end of described MTHFR-T probe and MTHFR-C probe is marked with reporter group, and 3 ' end is marked with fluorescent quenching group, and 5 ' end reporter group of MTHFR-T probe is different with 5 ' end reporter group of MTHFR-C probe.
3. the application of fluorescent probe in the detection kit of the rs1801133SNP loci polymorphism of preparation Methylene tetrahydrofolate reductase gene described in primer and/or claim 2 described in claim 1.
4. for detection of a primer of folic acid metabolism genes involved SNP, described primer is following primer pair:
(1) forward primer NOX-F, its nucleotide sequence is as shown in SEQ ID NO.5;
Reverse primer NOX-R, its nucleotide sequence is as shown in SEQ ID NO.6;
(2) primer being obtained through increase, disappearance or replacement single core thuja acid by the nucleotide sequence of forward primer NOX-F or reverse primer NOX-R;
The corresponding goal gene of above-mentioned primer is nadph oxidase gene, comprises rs11018628SNP site taking this gene as template amplification gained DNA fragmentation.
5. with the fluorescent probe being used in conjunction with for detection of the primer of folic acid metabolism genes involved SNP described in claim 4, described fluorescent probe comprises following two:
NOX-T probe, its nucleotide sequence is as shown in SEQ ID NO.7;
NOX-C probe, its nucleotide sequence is as shown in SEQ ID NO.8;
5 ' end of described NOX-T probe and NOX-C probe is marked with reporter group, and 3 ' end is marked with fluorescent quenching group, and 5 ' end reporter group of NOX-T probe is different with 5 ' end reporter group of NOX-C probe.
6. the application of fluorescent probe in the detection kit of rs11018628SNP loci polymorphism of preparing nadph oxidase gene described in primer and/or claim 5 described in claim 4.
7. for detection of a primer of folic acid metabolism genes involved SNP, described primer is following primer pair:
(1) forward primer CPS-F, its nucleotide sequence is as shown in SEQ ID NO.9;
Reverse primer CPS-R, its nucleotide sequence is as shown in SEQ ID NO.10;
(2) primer being obtained through increase, disappearance or replacement single core thuja acid by the nucleotide sequence of forward primer CPS-F or reverse primer CPS-R;
The corresponding goal gene of above-mentioned primer is carbamyl phosphate synthase gene, comprises rs1047891SNP site taking this gene as template amplification gained DNA fragmentation.
8. with the fluorescent probe being used in conjunction with for detection of the primer of folic acid metabolism genes involved SNP described in claim 1, described fluorescent probe comprises following two:
CPS-G probe, its nucleotide sequence is as shown in SEQ ID NO.11;
CPS-T probe, its nucleotide sequence is as shown in SEQ ID NO.12;
5 ' end of described CPS-G probe and CPS-T probe is marked with reporter group, and 3 ' end is marked with fluorescent quenching group, and 5 ' end reporter group of CPS-T probe is different with 5 ' end reporter group of CPS-C probe.
9. the application of fluorescent probe in the detection kit of rs1047891SNP loci polymorphism of preparing carbamyl phosphate synthase gene described in primer and/or claim 8 described in claim 7.
10. for detection of a primer sets of folic acid metabolism genes involved SNP, described primer sets comprises following primer pair:
1) forward primer MTHFR-F, its nucleotide sequence is as shown in SEQ ID NO.1;
Reverse primer MTHFR-R, its nucleotide sequence is as shown in SEQ ID NO.2;
2) forward primer NOX-F, its nucleotide sequence is as shown in SEQ ID NO.5;
Reverse primer NOX-R, its nucleotide sequence is as shown in SEQ ID NO.6;
3) forward primer CPS-F, its nucleotide sequence is as shown in SEQ ID NO.9;
Reverse primer CPS-R, its nucleotide sequence is as shown in SEQ ID NO.10.
11. 1 kinds with the fluorescent probe group being used in conjunction with for detection of the primer of folic acid metabolism genes involved SNP described in claim 10, described fluorescent probe group comprises following probe:
MTHFR-T probe, its nucleotide sequence is as shown in SEQ ID NO.3;
MTHFR-C probe, its nucleotide sequence is as shown in SEQ ID NO.4;
NOX-T probe, its nucleotide sequence is as shown in SEQ ID NO.7;
NOX-C probe, its nucleotide sequence is as shown in SEQ ID NO.8;
CPS-G probe, its nucleotide sequence is as shown in SEQ ID NO.11;
CPS-T probe, its nucleotide sequence is as shown in SEQ ID NO.12;
5 ' end of described MTHFR-T probe and MTHFR-C probe is marked with reporter group, and 3 ' end is marked with fluorescent quenching group, and 5 ' end reporter group of MTHFR-T probe is different with 5 ' end reporter group of MTHFR-C probe;
5 ' end of described NOX-T probe and NOX-C probe is marked with reporter group, and 3 ' end is marked with fluorescent quenching group, and 5 ' end reporter group of NOX-T probe is different with 5 ' end reporter group of NOX-C probe;
5 ' end of described CPS-G probe and CPS-T probe is marked with reporter group, and 3 ' end is marked with fluorescent quenching group, and 5 ' end reporter group of CPS-T probe is different with 5 ' end reporter group of CPS-C probe.
12. 1 kinds of PCR kit for fluorescence quantitative for detection of folic acid metabolism genes involved SNP, is characterized in that: comprise Taq enzyme, dNTPs, Mgcl
2, fluorescent probe group described in 10 × PCR buffer, ROX reference fluorescence, ultrapure water and primer sets claimed in claim 10, claim 11.
13. 1 kinds are detected the method for folic acid metabolism genes involved SNP polymorphism, it is characterized in that: the method comprises that the primer sets of utilizing described in claim 10, the fluorescent probe group described in claim 11 carry out quantitative fluorescent PCR reaction for human gene group DNA, and the fluorescent signal producing according to reaction process is distinguished the polymorphism in corresponding SNP site.
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