CN105695618A - SNP detecting kit for methionine-folic acid metabolism key enzymes MTHFR and use method thereof - Google Patents
SNP detecting kit for methionine-folic acid metabolism key enzymes MTHFR and use method thereof Download PDFInfo
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Abstract
The invention discloses an SNP detecting kit for methionine-folic acid metabolism key enzymes MTHFR and a use method thereof. Corresponding specific primers and fluorescence probes for detecting MTHFR C677T polymorphism and MTHFR A1298C polymorphism are added, a real-time fluorescence quantification PCR system is formed, and through a real-time PCR, technicists in the field determine and detect the genotype of a corresponding MTHFR gene locus through data collected by a fluorogenic quantitative PCR amplifier. The advantages of being fast and sensitive, facilitating judgment and the like are achieved, and the SNP detecting kit and the use method thereof are suitable for clinical detection and popularization.
Description
Technical field
The present invention relates to medical treatment detection field, more particularly to SNP detection kit and the using method thereof of a kind of methionine-folic acid metabolism key enzyme MTHFR。
Background technology
MTHFR full name: Methylene tetrahydrofolate reductase, it is the key enzyme in methionine-folic acid metabolism, it can make 5,10-methylene tetrahydrofolate is reduced to 5-methyltetrahydrofolate, thus participating in internal purine, the synthesis of pyrimidine and DNA, RNA, the methylating of protein as the indirect donor of methyl, maintain internal normal homocysteine level simultaneously。
Big quantity research all confirms that mother MTHFRC677T and MTHFRA1298C sudden change is the genetic risk factors of fertility neural tube defect infant。MTHFRC677T and MTHFRA1298C causes that the enzyme activity of MTHFR reduces, and causes blood plasma hyperhomocysteinemiainjury。It is hypercoagulability that trimester of pregnancy hyperhomocysteinemiainjury may result in blood, increases the thrombotic danger of Placenta Hominis, causes Placenta Hominis thromboembolism, causes female foetal circulation not enough, thus may result in miscarriage, FGR and placental abruption；On the other hand, hyperhomocysteinemiainjury can generate peroxide and oxygen-derived free radicals under metal ion mediates, and the 26S Proteasome Structure and Function of vascular endothelial cell injury also further results in patient vessel and relaxes contracting Balance disorders, thus causing pregnancy induced hypertension syndrome。Lack additionally, due to MTHFR vigor in placenta tissue, cause the 5-methyltetrahydrofolate dyspoiesis as the indirect donor of methyl, affect metabolism and the DNA methylation of purine, pyrimidine and nucleic acid further, make the undermethylation of the required biotic component such as DNA of fetal development and protein, be also cause one of miscarriage or the reason making FGR。This kind of people requires supplementation with more folic acid and can be only achieved the prevention pathogenetic effect of disease。
Detection method most common process currently for gene pleiomorphism is sequencing, and the method is suitable for the detection in the many sites of high flux, but operates length consuming time and sensitivity is low, is not suitable for rapid clinical detection；High-resolution solubility curve method is more special to equipment requirements, it is necessary to have installation high-resolution software, temperature could use than more sensitive machine, and its clinical expansion there be difficulties involved when。
Summary of the invention
For the deficiency that prior art exists, it is an object of the invention to provide a kind of SNP detection kit and using method thereof being capable of quickly detecting methionine-folic acid metabolism key enzyme MTHFR。
For achieving the above object, the technical scheme is that
A kind of SNP detection kit of methionine-folic acid metabolism key enzyme MTHFR, the composition including following parts by volume is constituted:
Described primer is MTHFRC677T primer and MTHFRA1298C primer,
Described MTHFRC677T primer includes:
Forward primer: MTHFRC677T-F:5 '-GCACTTGAAGGAGAAGGTGTCT-3 '；
Reverse primer: MTHFRC677T-R:5 '-TGTGTCAGCCTCAAAGAAAAGCT-3 '；
Described MTHFRA1298C primer includes:
Forward primer: MTHFRA1298C-F:5 '-GGAGGAGCTGCTGAAGATGTG-3 '；
Reverse primer: MTHFRA1298C-R:5 '-CCCGAGAGGTAAAGAACAAAGACTT-3 '；
Described probe is MTHFRC677T probe and MTHFRA1298C probe
MTHFRC677T probe includes:
MTHFRC677T wild-type probe: 5 '-fluorophor-ATGAAATCGGCTCCCGC-MGB-3 '；
MTHFRC677T saltant type probe: 5 '-fluorophor-ATGAAATCGACTCCCGC-MGB-3 '；
Described MTHFRA1298C probe includes:
MTHFRA1298C wild-type probe: 5 '-fluorophor-CAAAGACACTTTCTTC-MGB-3 '；
MTHFRA1298C saltant type probe: 5 '-fluorophor-AGACACTTGCTTCACT-MGB-3 '。
As a further improvement on the present invention, described pre-composition is 2 × PCR enzyme reaction premixed liquid, and described 2 × PCR enzyme reaction premixed liquid contains the MgCl of the TaqDNAPolymerase (recombinant) of 0.1units/ μ l, 4mmol/L2, 0.5mmol/L dNTPs。
As a further improvement on the present invention, described solvent is distilled water。
As a further improvement on the present invention, described primer and probe volume ratio are 1: 1。
As a further improvement on the present invention, described forward primer is identical with reverse primer consumption。
As a further improvement on the present invention, described MTHFRC677T wild-type probe is identical with MTHFRC677T saltant type probe consumption。
As a further improvement on the present invention, described MTHFRA1298C wild-type probe is identical with MTHFRA1298C saltant type probe consumption。
The using method of test kit, comprises the following steps:
1) genomic DNA of people to be measured is extracted；
2) each PCR system is comprise parts by volume: premixed liquid 10～25 parts, DNA profiling 1 part, primer 1～2 part, probe 1～2 part；
3) PCR system configured is put in quantitative real time PCR Instrument, carry out Real-timePCR detection；Reaction condition is:
95 DEG C of 10 minutes denaturations；Cycle stage, 95 DEG C 15 seconds and at this gather fluorescence signal, 60 DEG C 1 minute, 40-60 circulation；
4) genotype of detection site is determined by data collected by quantitative real time PCR Instrument。
As the most preferably scheme of the present invention,
1) genomic DNA of people to be measured is extracted；
2), in each PCR system, 2 × PCR enzyme reaction premixed liquid 15 μ L, DNA profiling 1 μ L, each 1 μ L of Direct/Reverse primer, wild type and saltant type fluorescent probe each 1 μ L, distilled water 10 μ L are comprised；Wherein 2 × PCR enzyme reaction premixed liquid composition and content: TaqDNAPolymerase (recombinant): 0.1units/ μ l；MgCl2:4mmol/L；DNTPs (dATP, dCTP, dGTP, dTTP): 0.5mmol/L；Wherein DNA content 10-100ng in genomic DNA template, Direct/Reverse primer concentration 100-1000nM, wild type and saltant type fluorescent probe concentration 100-1000nM；
3) PCR system configured is put in quantitative real time PCR Instrument, carry out Real-timePCR detection；Reaction condition is:
95 DEG C of 10 minutes denaturations；Cycle stage, 95 DEG C 15 seconds and at this gather fluorescence signal, 60 DEG C 1 minute, 40-60 circulation；
4) genotype of detection site is determined by data collected by quantitative real time PCR Instrument。
With human gene group DNA for template, it is separately added into detection MTHFRC677T type, the corresponding special primer of MTHFRA1298C type polymorphism and fluorescent probe, constitute real-time fluorescence quantitative PCR system, react through Real-timePCR, determined the genotype of MTHFR by the data collected by quantitative real time PCR Instrument。
Beneficial effects of the present invention, the present invention is compared with direct sequencing of the prior art, it is not necessary to complicated equipment, inherently a sequence in human genome is detected。Directly the people DNA extracted is detected by fluorescent PCR method, there is higher resolution, it is not necessary to order-checking instrument, it is possible to realize the detection for methionine-folic acid metabolism key enzyme related gene。Meanwhile, substantially reduce the detection time, also very directly perceived on judging, from the final datagram derived, just can immediately arrive at conclusion, be more prone on judging。
The application scheme have quick, sensitive, the advantage such as easily determine, be suitable for clinical detection and popularization。
Accompanying drawing explanation
Fig. 1 is MTHFRC677T site wild-type samples quantitative fluorescent PCR curve chart。
Fig. 2 is MTHFRC677T site heterozygous mutant fluorescent quantitative PCR curve chart。
Fig. 3 is MTHFRC677T site homozygous mutant fluorescent quantitative PCR curve chart。
Fig. 4 is MTHFRA1298C site wild-type samples quantitative fluorescent PCR curve chart。
Fig. 5 is MTHFRA1298C site heterozygous mutant fluorescent quantitative PCR curve chart。
Fig. 6 is MTHFRA1298C site homozygous mutant fluorescent quantitative PCR curve chart。
Detailed description of the invention
Below in conjunction with the embodiment given by accompanying drawing, the present invention is described in further detail。
Shown in reference Fig. 1, the SNP detection kit of a kind of methionine of the present embodiment-folic acid metabolism key enzyme MTHFR, the composition including following parts by volume is constituted:
Described primer is MTHFRC677T primer and MTHFRA1298C primer,
Described MTHFRC677T primer includes:
Forward primer: MTHFRC677T-F:5 '-GCACTTGAAGGAGAAGGTGTCT-3 '；
Reverse primer: MTHFRC677T-R:5 '-TGTGTCAGCCTCAAAGAAAAGCT-3 '；
Described MTHFRA1298C primer includes:
Forward primer: MTHFRA1298C-F:5 '-GGAGGAGCTGCTGAAGATGTG-3 '；
Reverse primer: MTHFRA1298C-R:5 '-CCCGAGAGGTAAAGAACAAAGACTT-3 '；
Described probe is MTHFRC677T probe and MTHFRA1298C probe
MTHFRC677T probe includes:
MTHFRC677T wild-type probe: 5 '-fluorophor-ATGAAATCGGCTCCCGC-MGB-3 '；
MTHFRC677T saltant type probe: 5 '-fluorophor-ATGAAATCGACTCCCGC-MGB-3 '；
Described MTHFRA1298C probe includes:
MTHFRA1298C wild-type probe: 5 '-fluorophor-CAAAGACACTTTCTTC-MGB-3 '；
MTHFRA1298C saltant type probe: 5 '-fluorophor-AGACACTTGCTTCACT-MGB-3 '。
As a kind of detailed description of the invention improved, described solvent is distilled water。
As a kind of detailed description of the invention improved, described pre-composition is 2 × PCR enzyme reaction premixed liquid, and described 2 × PCR enzyme reaction premixed liquid contains the MgCl of the TaqDNAPolymerase (recombinant) of 0.1units/ μ l, 4mmol/L2, 0.5mmol/L dNTPs。
As a kind of detailed description of the invention improved, described primer and probe volume ratio is for 1:1。
As a kind of detailed description of the invention improved, described forward primer is identical with reverse primer consumption。
As a kind of detailed description of the invention improved, described MTHFRC677T wild-type probe is identical with MTHFRC677T saltant type probe consumption。
As a kind of detailed description of the invention improved, described MTHFRA1298C wild-type probe is identical with MTHFRA1298C saltant type probe consumption。
The using method of test kit, comprises the following steps:
1) genomic DNA of people to be measured is extracted；
2) each PCR system is comprise parts by volume: premixed liquid 10～25 parts, DNA profiling 1 part, primer 1～2 part, probe 1～2 part；
3) PCR system configured is put in quantitative real time PCR Instrument, carry out Real-timePCR detection；Reaction condition is:
95 DEG C of 10 minutes denaturations；Cycle stage, 95 DEG C 15 seconds and at this gather fluorescence signal, 60 DEG C 1 minute, 40-60 circulation；
4) genotype of detection site is determined by data collected by quantitative real time PCR Instrument。
As the most preferably scheme of the present invention,
1) genomic DNA of people to be measured is extracted；
2), in each PCR system, 2 × PCR enzyme reaction premixed liquid 15 μ L, DNA profiling 1 μ L, each 1 μ L of Direct/Reverse primer, wild type and saltant type fluorescent probe each 1 μ L, distilled water 10 μ L are comprised；Wherein 2 × PCR enzyme reaction premixed liquid composition and content: TaqDNAPolymerase (recombinant): 0.1units/ μ l；MgCl2: 4mmol/L；DNTPs (dATP, dCTP, dGTP, dTTP): 0.5mmol/L；Wherein DNA content 10-100ng in genomic DNA template, Direct/Reverse primer concentration 100-1000nM, wild type and saltant type fluorescent probe concentration 100-1000nM；
3) PCR system configured is put in quantitative real time PCR Instrument, carry out Real-timePCR detection；Reaction condition is:
95 DEG C of 10 minutes denaturations；Cycle stage, 95 DEG C 15 seconds and at this gather fluorescence signal, 60 DEG C 1 minute, 40-60 circulation；
4) genotype of detection site is determined by data collected by quantitative real time PCR Instrument。
Detecting with optimal case, choosing sample is that MTHFRC677T site wild-type samples, MTHFRC677T site heterozygous mutant sample, MTHFRC677T site homozygous mutant sample, MTHFRA1298C site wild-type samples, MTHFRA1298C site heterozygous mutant sample, MTHFRA1298C site homozygous mutant sample detect。Testing result is as shown in Figures 1 to 6。
Shown by the data of Fig. 1 to Fig. 6, the test kit of the present invention when detecting methionine-folic acid metabolism key enzyme related gene, have quick, sensitive, the advantage such as easily determine, be suitable for clinical detection and popularization。
The above is only the preferred embodiment of the present invention, and protection scope of the present invention is not limited merely to above-described embodiment, and all technical schemes belonged under thinking of the present invention belong to protection scope of the present invention。It should be pointed out that, for those skilled in the art, some improvements and modifications without departing from the principles of the present invention, these improvements and modifications also should be regarded as protection scope of the present invention。
Claims (8)
1. the SNP detection kit of methionine-folic acid metabolism key enzyme MTHFR, it is characterised in that: the composition including following parts by volume is constituted:
Described primer is MTHFRC677T primer and MTHFRA1298C primer,
Described MTHFRC677T primer includes:
Forward primer: MTHFRC677T-F:5 '-GCACTTGAAGGAGAAGGTGTCT-3 '；
Reverse primer: MTHFRC677T-R:5 '-TGTGTCAGCCTCAAAGAAAAGCT-3 '；
Described MTHFRA1298C primer includes:
Forward primer: MTHFRA1298C-F:5 '-GGAGGAGCTGCTGAAGATGTG-3 '；
Reverse primer: MTHFRA1298C-R:5 '-CCCGAGAGGTAAAGAACAAAGACTT-3 '；
Described probe is MTHFRC677T probe and MTHFRA1298C probe
MTHFRC677T probe includes:
MTHFRC677T wild-type probe: 5 '-fluorophor-ATGAAATCGGCTCCCGC-MGB-3 '；
MTHFRC677T saltant type probe: 5 '-fluorophor-ATGAAATCGACTCCCGC-MGB-3 '；
Described MTHFRA1298C probe includes:
MTHFRA1298C wild-type probe: 5 '-fluorophor-CAAAGACACTTTCTTC-MGB-3 '；
MTHFRA1298C saltant type probe: 5 '-fluorophor-AGACACTTGCTTCACT-MGB-3 '。
2. the SNP detection kit of methionine according to claim 1-folic acid metabolism key enzyme MTHFR, it is characterized in that: described pre-composition is 2 × PCR enzyme reaction premixed liquid, described 2 × PCR enzyme reaction premixed liquid contains the MgCl of the TaqDNAPolymerase (recombinant) of 0.1units/ μ l, 4mmol/L2, 0.5mmol/L dNTPs。
3. the SNP detection kit of the methionine according to claim 1 and 2-folic acid metabolism key enzyme MTHFR, it is characterised in that: described solvent is distilled water。
4. the SNP detection kit of methionine according to claim 3-folic acid metabolism key enzyme MTHFR, it is characterised in that: described primer and probe volume ratio are 1: 1。
5. the SNP detection kit of methionine according to claim 1-folic acid metabolism key enzyme MTHFR, it is characterised in that: described forward primer is identical with reverse primer consumption。
6. the SNP detection kit of methionine according to claim 1-folic acid metabolism key enzyme MTHFR, it is characterised in that: described wild-type probe is identical with saltant type probe consumption。
7. the using method of the test kit as described in claim 1 to 6, it is characterised in that: comprise the following steps:
1) genomic DNA of people to be measured is extracted；
2) each PCR system is comprise parts by volume: premixed liquid 10～25 parts, DNA profiling 1 part, primer 1～2 part, probe 1～2 part；
3) PCR system configured is put in quantitative real time PCR Instrument, carry out Real-timePCR detection；Reaction condition is:
95 DEG C of 10 minutes denaturations；Cycle stage, 95 DEG C 15 seconds and at this gather fluorescence signal, 60 DEG C 1 minute, 40-60 circulation；
4) genotype of detection site is determined by data collected by quantitative real time PCR Instrument。
8. the using method of test kit as claimed in claim 7, it is characterised in that: comprise the following steps:
1) genomic DNA of people to be measured is extracted；
2) each PCR system, comprises 2 × PCR enzyme reaction premixed liquid 15 μ L, DNA profiling 1 μ L, each 1 μ L of Direct/Reverse primer, wild type and saltant type fluorescent probe each 1 μ L, distilled water 10 μ L；
Wherein 2 × PCR enzyme reaction premixed liquid composition and content: TaqDNAPolymerase (recombinant): 0.1units/ μ l；MgCl2: 4mmol/L；DNTPs (dATP, dCTP, dGTP, dTTP): 0.5mmol/L；
Wherein DNA content 10-100ng in genomic DNA template, Direct/Reverse primer concentration 100-1000nM, wild type and saltant type fluorescent probe concentration 100-1000nM；
3) PCR system configured is put in quantitative real time PCR Instrument, carry out Real-timePCR detection；Reaction condition is:
95 DEG C of 10 minutes denaturations；Cycle stage, 95 DEG C 15 seconds and at this gather fluorescence signal, 60 DEG C 1 minute, 40-60 circulation；
4) genotype of detection site is determined by data collected by quantitative real time PCR Instrument。
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN107267645A (en) * | 2017-07-31 | 2017-10-20 | 广州德臻生物技术有限公司 | Primer pair, probe and kit for detecting mthfr gene polymorphism |
| 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 |
| CN110408685A (en) * | 2019-08-29 | 2019-11-05 | 无锡市申瑞生物制品有限公司 | For detecting primer combination of probe object, kit and the detection method of people's mthfr gene parting |
| CN112210591A (en) * | 2020-11-16 | 2021-01-12 | 合肥中科支众医学检验有限公司 | Method for detecting polymorphism of C677T locus of human MTHFR gene |
-
2016
- 2016-04-22 CN CN201610257598.1A patent/CN105695618A/en active Pending
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| Title |
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| LOÏC LE MARCHANDDENG等: "MTHFR polymorphisms, diet, HRT, and breast cancer risk：the multiethnic cohort study", 《CANCER EPIDEMIOL BIOMARKERS PREV》 * |
| MATTIAS JOHANSSON等: "The MTHFR 677C→T polymorphism and risk of prostate cancer： results from the CAPS study", 《CANCER CAUSES CONTROL》 * |
| NABIL MTIRAOUI等: "MTHFR C677T and A1298C gene polymorphisms and hyperhomocysteinemia as risk factors of diabetic nephropathy in type 2 diabetes patients", 《DIABETES RESEARCH AND CLINICAL PRACTICE》 * |
| WU ZHANG等: "Association of Methylenetetrahydrofolate Reductase Gene Polymorphisms and Sex-Specific Survival in Patients With Metastatic Colon Cance", 《JOURNAL OF CLINICAL ONCOLOGY》 * |
| 周胜利等: "MTHFR基因C677T和A1298C多态性与精神分裂症关联的家系研究", 《精神医学杂志》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN107267645A (en) * | 2017-07-31 | 2017-10-20 | 广州德臻生物技术有限公司 | Primer pair, probe and kit for detecting mthfr gene polymorphism |
| CN107267645B (en) * | 2017-07-31 | 2021-10-08 | 广州德臻生物技术有限公司 | Primer pair, probe and kit for detecting MTHFR gene polymorphism |
| 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 |
| CN110408685A (en) * | 2019-08-29 | 2019-11-05 | 无锡市申瑞生物制品有限公司 | For detecting primer combination of probe object, kit and the detection method of people's mthfr gene parting |
| CN112210591A (en) * | 2020-11-16 | 2021-01-12 | 合肥中科支众医学检验有限公司 | Method for detecting polymorphism of C677T locus of human MTHFR gene |
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