CN109182537A - A kind of amplimer, kit and detection method for evaluating folic acid metabolism ability - Google Patents
A kind of amplimer, kit and detection method for evaluating folic acid metabolism ability Download PDFInfo
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- CN109182537A CN109182537A CN201811119417.4A CN201811119417A CN109182537A CN 109182537 A CN109182537 A CN 109182537A CN 201811119417 A CN201811119417 A CN 201811119417A CN 109182537 A CN109182537 A CN 109182537A
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- 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
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Abstract
The invention discloses a kind of amplimer, kit and detection methods for evaluating folic acid metabolism ability, belong to field of biotechnology.The amplimer of the evaluation folic acid metabolism ability, including the first primer for detecting rs1801133 to, the second primer pair for detecting rs1801131, the third primer pair for detecting rs1801394 and the 4th primer pair for detecting rs1805087.The invention also discloses a kind of kit for evaluating folic acid metabolism ability and a kind of methods for evaluating folic acid metabolism ability.The amplimer of evaluation folic acid metabolism ability of the invention, has many advantages, such as high sensitivity, high specific, high accurancy and precision, easy to operate, it is able to achieve the genetic test of 4 polymorphic sites relevant to folic acid metabolism, screening goes out mutational site, personalized supplement folic acid is realized, to reduce Newborn Birth-defects risk.
Description
Technical field
The present invention relates to a kind of amplimer, kit and detection methods for evaluating folic acid metabolism ability, belong to biological skill
Art field.
Background technique
Folic acid, also known as Vitamin B9, the indispensable substance during fetal growth, is one of vitamin B complex nutrient,
Have the characteristics that water-soluble, therefore can not gather together in vivo.This nutrient is found from the leaf of spinach at the beginning,
So can just be called " folic acid ".Folic acid plays the synthesis of the merisis and nucleic acid of cell, amino acid, protein important
Effect.Human body lacks the exception that folic acid can lead to red blood cell, the increase of immature cell, and anaemia and white blood cell are reduced.Folic acid
It is also the indispensable nutrient of embryo growth and development.Pregnant woman lack folic acid be likely to result in occurring when fetal birth under-weight,
Harelip, heart defect etc..If lacking folic acid in pregnancy head 3 months, fetal neural tube developmental defect can be caused, and caused
Deformity.
China since 2016, it is specified that Supplement of folic acid pregnancy period first trimester, (most often to prevent newborn's neural tube malformation
What is seen is spina bifida and anencephalus), recommended dose is daily 0.4 milligram.But research in recent years and it has been observed that for certain
For standby pregnant women, this dosage be not necessarily it is enough because some to folic acid absorb it is insensitive, generate folic acid metabolism barrier
Hinder.
Folate metabolism disorder be as caused by gene defect, cannot normally assimilate food in folic acid be converted into human body institute
The tetrahydrofolic acid needed, if it is pregnant woman, the shortage of folic acid may result in the developmental deformity of fetus.The difference of folic acid metabolism gene
It is different, determine the difference of folic acid Utilization ability.
Mononucleotide polymorphic (SNP), belongs to one kind of DNA polymorphism, refers to list of the occurrence frequency greater than 1% in DNA sequence dna
The variation such as nucleotide variation, including single base conversion, transversion, insertion or missing.SNP, may as a kind of common hereditary variation
The difference and Different Individual for leading to individual phenotype to the neurological susceptibility of disease, especially complex disease, and to environmental factor and
The difference of drug and therapeutic response.Studies have shown that the especially needed Supplement of folic acid of the Pregnant women of MTHFR and MTRR gene defect,
Therefore, MTHFR the and MTRR gene SNP site of Pregnant women, especially MTHFR C677T rs1801133, MTRR are detected
A1298C rs1801131, the acid site methionine synthetase reductase MTRR A66G rs1801394, can be used as Supplement of folic acid
Personalized diet suggestion foundation.
Currently, the method on the market in relation to the detection of folic acid related gene is mainly PCR sequencing PCR.Polymerase chain reaction
(Polymerase Chain Reaction), i.e. round pcr are a kind of rapid amplifying specific gene or DNA sequence dnas in vitro
Method, therefore the amplification in vitro method of also known as gene.Round pcr has become in molecular biology research using most, widest hand
One of section, and design of primers is a vital ring in round pcr, is easy to cause experiment to lose using inappropriate PCR primer
It loses.
The prior art is about the generally existing detection poor accuracy of detection method of folic acid metabolism related gene, the period is long, at
This height, defect complicated for operation.Therefore, it is necessary to provide a kind of new detection method, so as to solve the deficiencies in the prior art.
Summary of the invention
An object of the present invention is to provide a kind of amplimer for evaluating folic acid metabolism ability.Evaluation leaf of the invention
The amplimer of acid metabolic ability has many advantages, such as high sensitivity, high specific, high accurancy and precision, easy to operate, is able to achieve to leaf
The genetic test of relevant 4 polymorphic sites of acid metabolic, screening go out mutational site, personalized supplement folic acid are realized, to reduce
Newborn Birth-defects risk.
The technical scheme to solve the above technical problems is that a kind of amplimer for evaluating folic acid metabolism ability,
Including the first primer for detecting rs1801133 to, the second primer pair for detecting rs1801131, for detecting
The third primer pair of rs1801394 and the 4th primer pair for detecting rs1805087, wherein the first primer is to including
Nucleotide sequence shown in SEQ ID NO.1 and SEQ ID NO.2, second primer pair include SEQ ID NO.3 and SEQ
Nucleotide sequence shown in ID NO.4, the third primer pair include nucleosides shown in SEQ ID NO.5 and SEQ ID NO.6
Acid sequence, the 4th primer pair include nucleotide sequence shown in SEQ ID NO.7 and SEQ ID NO.8.
The principle of the present invention explanation:
It come what is indicated, is being for letter with reference to the position on cDNA that above-mentioned SNP site, which marks the site according to HGVS nomenclature,
Just refer to some specific SNP site, a SNP site can also have other representations, such as it is following will appear opened with " rs "
The SNP site representation of head, which is the naming method of GenBank snp database, with " rs " plus 7 Arabic numerals
To indicate a SNP.Believe according to being determined in database such as snp database and obtaining the position in specific SNP site gene order
While breath, its context, distribution frequency etc. can be also obtained.
The present invention utilizes 4 SNP sites, it is determined that 4 pairs of primers in 4 sites obtain 4 with PCR amplification Sanger sequencing
The genotype in a site.Rs1801133 and mthfr gene C677T indicates the same site, rs1801131 and mthfr gene
A1298C indicates the same site, and rs1801394 and MTRR Gene A 66G indicate the same site, rs1805087 and MTR gene
A2756G indicates the same site.
Wherein, mthfr gene is methylenetetrahydrofolate reductase, is one of key enzyme of homocysteine metabolism.
There are Genotyping in 3, i.e. wild type CC, heterozygous mutant CT and homozygous mutant TT in 677 sites of mthfr gene.There is research
It was found that mthfr gene C677T mutation can be such that enzymatic activity is decreased obviously, so that the generation of methyl donor is insufficient, intracorporal homotype half
Cystine (Hcy) metabolic disorder, so as to cause the raising of homocysteine (Hcy) concentration.
MTR gene is 5-methyltetrahydrofolate-homocysteine methyl transferase, be primarily involved in folic acid metabolism access with
Homocysteine metabolism access.5-methyltetrahydrofolate is under the catalysis of methionine synthetase and its cobamamide
It provides a methyl to be allowed to transform into methionine to homocysteine, and itself is converted to tetrahydrofolic acid, methionine
Then S-adenosylmethionine (SAM) is converted into the case where ATP is energized.S-adenosylmethionine is the first of internal biochemical reaction
Base lays in substance, can provide methyl for the methylation of protein and the methylation of DNA.MTR is in this metabolic cycles process
In play a crucial role, if MTR gene defect or enzymatic activity decline, conversion of the homocysteine to methionine will be sent out
Raw obstacle, causes a series of pathological changes in succession.
Design of primers of the invention follows design of primers principle, and guarantees the specificity and homogeneity of amplified band, adopts
Determine the genotype in site with generation sequencing technologies, thus the PCR product length of design of primers of the invention guarantee 700bp with
Interior (it is 700bp or so that it is online that primary first-order equation sequencing length maximum is sequenced in a generation), and mutational site is greater than apart from upstream primer
50bp (base sequencing result is often inaccurate in generation sequencing starting 50bp).
The sequence table of the amplimer of evaluation folic acid metabolism ability of the invention, as shown in table 1.
1 sequence table of table
The second object of the present invention is to provide a kind of kit for evaluating folic acid metabolism ability.Evaluation folic acid of the invention
The kit of metabolic capability can accurately detect one in rs1801133, rs1801131, rs1801394, rs1805087
Kind, two kinds, three kinds or four kinds of site mutations, to judge the magnitude of recruitment of folic acid.
The technical scheme to solve the above technical problems is that a kind of kit for evaluating folic acid metabolism ability, institute
State the amplimer that kit includes above-mentioned evaluation folic acid metabolism ability.
Based on the above technical solution, the present invention can also be improved as follows.
Further, the kit also includes PCR buffer, dNTPs, Mg2+, 2XTaq enzyme and for extracting sample to be tested
The reagent of Oral Mucosal Cells DNA.
Further, the reagent for extracting sample to be tested Oral Mucosal Cells DNA is purchased from as Tiangeng biochemical technology
(Beijing) Co., Ltd, model buccal swab genome DNA extracting reagent kit (DP322).
The third object of the present invention is to provide a kind of method for evaluating folic acid metabolism ability.The present invention evaluates folic acid metabolism
The method of ability establishes stable reaction system using above-mentioned primer, establishes ideal reaction condition, can be accurate, fast
It is prominent that one of rs1801133, rs1801131, rs1801394, rs1805087, two kinds, three kinds or four kinds of sites are detected fastly
Become, easy to operate, low in cost, as a result accurately, wide market is suitble to scale application.
The technical scheme to solve the above technical problems is that a kind of method for evaluating folic acid metabolism ability, including
Following steps:
Step 1: extracting the Oral Mucosal Cells DNA of sample to be tested；
Step 2: the nucleotide sequence amplification mthfr gene shown in SEQ ID NO.1 and SEQ ID NO.2 respectively
C677T, the nucleotide sequence amplification mthfr gene A1298C shown in SEQ ID NO.3 and SEQ ID NO.4, with SEQ ID
Nucleotide sequence amplification MTRR Gene A 66G shown in NO.5 and SEQ ID NO.6, with SEQ ID NO.7 and SEQ ID NO.8
Shown in nucleotide sequence amplification MTR Gene A 2756G, using step 1 extract Oral Mucosal Cells DNA as template, carry out respectively
PCR reaction, respectively takes 20uL system, respectively include:
SEQ ID NO.2, the 10uL of SEQ ID NO.1 of 50ng/uL Oral Mucosal Cells DNA profiling 1uL, 1uL, 1uL
2X Taq Mix and 7uL ddH2O；
SEQ ID NO.4, the 10uL of SEQ ID NO.3 of 50ng/uL Oral Mucosal Cells DNA profiling 1uL, 1uL, 1uL
2X Taq Mix and 7uL ddH2O；
SEQ ID NO.5 of 50ng/uL Oral Mucosal Cells DNA profiling 1uL1uL, SEQ ID NO.6 of 1uL, 10uL
The ddH of 2X Taq Mix and 7uL2O；
SEQ ID NO.8, the 10uL of SEQ ID NO.7 of 50ng/uL Oral Mucosal Cells DNA profiling 1uL, 1uL, 1uL
2X Taq Mix and 7uL ddH2O；
Step 3: carrying out PCR amplification respectively, amplified production is detected through agarose gel electrophoresis；
Step 4: amplified reaction being purified, PCR sequencing is carried out；
Step 5: with the PCR sequencing result of Chromas software analytical procedure 4.
Based on the above technical solution, the present invention can also be improved as follows.
Further, in step 1, the Oral Mucosal Cells DNA for extracting sample to be tested, using buccal swab genome
DNA extraction kit carries out.
Above-mentioned buccal swab genome DNA extracting reagent kit, being purchased from is TIANGEN Biotech (Beijing) Co., Ltd., model
For buccal swab genome DNA extracting reagent kit (DP322).
Further, in step 3, the response procedures of the PCR amplification are as follows: 94 DEG C of initial denaturation 5min；Subsequently enter circulation, 94
DEG C denaturation 30s, 60 DEG C of annealing 20s, 72 DEG C of extension 30s, totally 34 recycle；Later, continue 72 DEG C of extension 5min；4 DEG C of preservations.
Further, in step 3, the mass percent of the Ago-Gel is 1%.
Further, in step 4, the response procedures of the PCR sequencing are as follows: 96 DEG C of initial denaturation 1min；Subsequently enter circulation, 96
DEG C denaturation 10s, 50 DEG C of annealing 5s, 60 DEG C of annealing 4min, totally 25 recycle；4 DEG C of preservations.
The beneficial effects of the present invention are:
1. the amplimer of evaluation folic acid metabolism ability of the invention, have high sensitivity, high specific, high accurancy and precision,
The advantages that easy to operate, is able to achieve the genetic test of 4 polymorphic sites relevant to folic acid metabolism, and screening goes out mutational site,
Personalized supplement folic acid is realized, to reduce Newborn Birth-defects risk.
2. the kit of evaluation folic acid metabolism ability of the invention, can accurately detect rs1801133, rs1801131,
One of rs1801394, rs1805087, two kinds, three kinds or four kinds of site mutations, to judge the magnitude of recruitment of folic acid.
The method that the present invention evaluates folic acid metabolism ability is established stable reaction system, is established using above-mentioned primer
Ideal reaction condition can accurately and fast detect in rs1801133, rs1801131, rs1801394, rs1805087
A kind of, two kinds, three kinds or four kinds of site mutations, easy to operate, low in cost, as a result accurately, wide market is suitble to scale
Change application.
Detailed description of the invention
Fig. 1 is that PCR product of the invention expands schematic diagram.From left to right, respectively Maker, MTHFR C677T, MTHFR
The Ago-Gel result figure of A1298C, MTRR A66G and MTR A2756G amplified production.
Fig. 2 is the sequencing result figure of the pcr amplification product of mthfr gene C677T of the invention.
Fig. 3 is the sequencing result figure of the pcr amplification product of mthfr gene A1298C of the invention.
Fig. 4 is the sequencing result figure of the pcr amplification product of MTRR Gene A 66G of the invention.
Fig. 5 is the sequencing result figure of the pcr amplification product of MTR Gene A 2756G of the invention.
Specific embodiment
The principle and features of the present invention will be described below with reference to the accompanying drawings, and the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the invention.
Embodiment 1
It is a kind of evaluate folic acid metabolism ability amplimer, including the first primer for detecting rs1801133 to, be used for
Detect the second primer pair of rs1801131, the third primer pair for detecting rs1801394 and for detecting rs1805087's
4th primer pair, wherein the first primer is to including nucleotide sequence shown in SEQ ID NO.1 and SEQ ID NO.2, institute
Stating the second primer pair includes nucleotide sequence shown in SEQ ID NO.3 and SEQ ID NO.4, and the third primer pair includes
Nucleotide sequence shown in SEQ ID NO.5 and SEQ ID NO.6, the 4th primer pair include SEQ ID NO.7 and SEQ
Nucleotide sequence shown in ID NO.8.
The sequence table of the amplimer of above-mentioned evaluation folic acid metabolism ability, as shown in table 1.
1 sequence table of table
Embodiment 2
A kind of kit for evaluating folic acid metabolism ability, the kit include the amplification of above-mentioned evaluation folic acid metabolism ability
Primer.
The kit also includes PCR buffer, dNTPs, Mg2+, 2XTaq enzyme and for extracting sample to be tested mucous membrane of mouth
The reagent of cell DNA.
It is that Tiangeng biochemical technology (Beijing) is limited that the reagent for extracting sample to be tested Oral Mucosal Cells DNA, which is purchased from,
Company, model buccal swab genome DNA extracting reagent kit (DP322)
Embodiment 3
A method of evaluation folic acid metabolism ability includes the following steps:
Step 1: extracting the Oral Mucosal Cells DNA of sample to be tested
TIANGEN Biotech (Beijing) Co., Ltd., model buccal swab genome DNA extracting reagent kit (DP322),
DNA is extracted to the Oral Mucosal Cells of tester.
Step 2: the nucleotide sequence amplification mthfr gene shown in SEQ ID NO.1 and SEQ ID NO.2 respectively
C677T, the nucleotide sequence amplification mthfr gene A1298C shown in SEQ ID NO.3 and SEQ ID NO.4, with SEQ ID
Nucleotide sequence amplification MTRR Gene A 66G shown in NO.5 and SEQ ID NO.6, with SEQ ID NO.7 and SEQ ID NO.8
Shown in nucleotide sequence amplification MTR Gene A 2756G, the Oral Mucosal Cells that are obtained using step 1 extract DNA as template, respectively
PCR reaction is carried out, 20uL system is respectively taken, respectively include:
SEQ ID NO.1 of template 1uL, 1uL of 50ng/uL, SEQ ID NO.2 of 1uL, 10uL 2X Taq Mix and
The ddH of 7uL2O；
SEQ ID NO.3 of template 1uL, 1uL of 50ng/uL, SEQ ID NO.4 of 1uL, 10uL 2X Taq Mix and
The ddH of 7uL2O；
SEQ ID NO.5 of template 1uL, 1uL of 50ng/uL, SEQ ID NO.6 of 1uL, 10uL 2X Taq Mix and
The ddH of 7uL2O；
SEQ ID NO.7 of template 1uL, 1uL of 50ng/uL, SEQ ID NO.8 of 1uL, 10uL 2X Taq Mix and
The ddH of 7uL2O；
Step 3: carrying out PCR amplification, the response procedures of the PCR amplification respectively are as follows: 94 DEG C of initial denaturation 5min；It subsequently enters
Circulation, 94 DEG C of denaturation 30s, 60 DEG C of annealing 20s, 72 DEG C of extension 30s, totally 34 recycle；Later, continue 72 DEG C of extension 5min；4℃
It saves；Amplified production is detected through the agarose gel electrophoresis that mass percent is 1%.As a result as shown in Figure 1.
Step 4: amplified reaction being purified, according to exonuclease I (Exo I), the effect of alkaline phosphatase (AIP)
Concentration is added in PCR reaction product, and 37 DEG C of digestion 15mn, 85 DEG C make enzyme inactivate 15min.Purification system is as follows: PCR product
5uL, Exo I 0.5uL, alkaline phosphatase 1uL.
PCR product after purification is subjected to PCR sequencing, the response procedures of the PCR sequencing are as follows: 96 DEG C of initial denaturation 1min；
Circulation is subsequently entered, 96 DEG C of denaturation 10s, 50 DEG C of annealing 5s, 60 DEG C of annealing 4min, totally 25 recycle；4 DEG C of preservations.
Step 5: with the PCR sequencing result of Chromas software analytical procedure 4.The pcr amplification product of mthfr gene C677T
Sequencing result figure, as shown in Figure 2.The sequencing result figure of the pcr amplification product of mthfr gene A1298C, as shown in Figure 3.
The sequencing result figure of the pcr amplification product of MTRR Gene A 66G, as shown in Figure 4.The pcr amplification product of MTR Gene A 2756G
Sequencing result figure, as shown in Figure 5.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
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Claims (8)
1. a kind of amplimer for evaluating folic acid metabolism ability, which is characterized in that draw including first for detecting rs1801133
Object to, the second primer pair for detecting rs1801131, the third primer pair for detecting rs1801394 and for detecting
The 4th primer pair of rs1805087, wherein the first primer is to including core shown in SEQ ID NO.1 and SEQ ID NO.2
Nucleotide sequence, second primer pair include nucleotide sequence shown in SEQ ID NO.3 and SEQ ID NO.4, the third
Primer pair includes nucleotide sequence shown in SEQ ID NO.5 and SEQ ID NO.6, and the 4th primer pair includes SEQ ID
Nucleotide sequence shown in NO.7 and SEQ ID NO.8.
2. a kind of kit for evaluating folic acid metabolism ability, which is characterized in that the kit includes described in claim 1 comments
The amplimer of valence folic acid metabolism ability.
3. a kind of kit for evaluating folic acid metabolism ability according to claim 2, which is characterized in that the kit is also
Include PCR buffer, dNTPs, Mg2+, 2X Taq enzyme and the reagent for extracting sample to be tested Oral Mucosal Cells DNA.
4. a kind of method for evaluating folic acid metabolism ability, which comprises the steps of:
Step 1: extracting the Oral Mucosal Cells DNA of sample to be tested；
Step 2: the nucleotide sequence amplification mthfr gene C677T shown in SEQ ID NO.1 and SEQ ID NO.2 respectively is used
Nucleotide sequence amplification mthfr gene A1298C shown in SEQ ID NO.3 and SEQ ID NO.4, with SEQ ID NO.5 and
Nucleotide sequence amplification MTRR Gene A 66G shown in SEQ ID NO.6, shown in SEQ ID NO.7 and SEQ ID NO.8
It is anti-to carry out PCR as template respectively by nucleotide sequence amplification MTR Gene A 2756G, the Oral Mucosal Cells DNA extracted using step 1
It answers, respectively takes 20uL system, respectively include:
The 2X of SEQ ID NO.1 of 50ng/uL Oral Mucosal Cells DNA profiling 1uL, 1uL, SEQ ID NO.2 of 1uL, 10uL
The ddH of Taq Mix and 7uL2O；
The 2X of SEQ ID NO.3 of 50ng/uL Oral Mucosal Cells DNA profiling 1uL, 1uL, SEQ ID NO.4 of 1uL, 10uL
The ddH of Taq Mix and 7uL2O；
The 2X of SEQ ID NO.5 of 50ng/uL Oral Mucosal Cells DNA profiling 1uL1uL, SEQ ID NO.6 of 1uL, 10uL
The ddH of Taq Mix and 7uL2O；
The 2X of SEQ ID NO.7 of 50ng/uL Oral Mucosal Cells DNA profiling 1uL, 1uL, SEQ ID NO.8 of 1uL, 10uL
The ddH of Taq Mix and 7uL2O；
Step 3: carrying out PCR amplification respectively, amplified production is detected through agarose gel electrophoresis；
Step 4: amplified reaction being purified, PCR sequencing is carried out；
Step 5: with the PCR sequencing result of Chromas software analytical procedure 4.
5. a kind of method for evaluating folic acid metabolism ability according to claim 4, which is characterized in that described to mention in step 1
The Oral Mucosal Cells DNA for taking sample to be tested is carried out using buccal swab genome DNA extracting reagent kit.
6. a kind of method for evaluating folic acid metabolism ability according to claim 4, which is characterized in that described in step 3
The response procedures of PCR amplification are as follows: 94 DEG C of initial denaturation 5min；Subsequently enter circulation, 94 DEG C of denaturation 30s, 60 DEG C of annealing 20s, 72 DEG C
Extend 30s, totally 34 circulations；Later, continue 72 DEG C of extension 5min；4 DEG C of preservations.
7. a kind of method for evaluating folic acid metabolism ability according to claim 4, which is characterized in that in step 3, the fine jade
The mass percent of sepharose is 1%.
8. a kind of method for evaluating folic acid metabolism ability according to claim 4, which is characterized in that described in step 4
The response procedures of PCR sequencing are as follows: 96 DEG C of initial denaturation 1min；Circulation is subsequently entered, 96 DEG C of denaturation 10s, 50 DEG C of annealing 5s, 60 DEG C are moved back
Fiery 4min, totally 25 recycle；4 DEG C of preservations.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110241195A (en) * | 2019-04-17 | 2019-09-17 | 青海大学 | A kind of gene tester of individuation folic acid supplement amount |
| CN110257507A (en) * | 2019-07-11 | 2019-09-20 | 上海联吉医学检验所有限公司 | A kind of pregnancy period folic acid metabolism, calcium metabolism and H-type hypertension associated detecting method |
| CN111187828A (en) * | 2020-02-11 | 2020-05-22 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting polymorphism of human folate metabolism gene |
| CN111394449A (en) * | 2020-03-24 | 2020-07-10 | 海南主健细胞分子遗传医学检验中心有限公司 | Kit for detecting folate metabolism pathway related genes |
| CN111500709A (en) * | 2020-05-12 | 2020-08-07 | 珠海横琴润孚创新科技有限公司 | Metabolic disease gene detection and clinical depth data analysis method |
| CN111876482A (en) * | 2020-09-21 | 2020-11-03 | 郑州安图生物工程股份有限公司 | Kit for simultaneously detecting human MTHFR and MTRR genes |
| CN112094899A (en) * | 2020-09-24 | 2020-12-18 | 江苏先声医疗器械有限公司 | Detection method of folic acid metabolism capability based on MassArray nucleic acid mass spectrum and application thereof |
| CN113637735A (en) * | 2021-08-09 | 2021-11-12 | 优葆优保健康科技(宁波)有限公司 | Kit for detecting children nutrition genome and application |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015081052A1 (en) * | 2013-11-27 | 2015-06-04 | Yasko Amy | Use of polymorphisms for identifying individuals at risk of developing autism |
| CN107446994A (en) * | 2017-06-16 | 2017-12-08 | 安徽安龙基因医学检验所有限公司 | A kind of detection method of alcohol metabolism related gene |
| 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 |
| CN108048563A (en) * | 2018-02-08 | 2018-05-18 | 中国疾病预防控制中心营养与健康所 | A kind of method for lacking risk assessment to people's nutrient based on SNP site |
-
2018
- 2018-09-25 CN CN201811119417.4A patent/CN109182537A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015081052A1 (en) * | 2013-11-27 | 2015-06-04 | Yasko Amy | Use of polymorphisms for identifying individuals at risk of developing autism |
| CN107446994A (en) * | 2017-06-16 | 2017-12-08 | 安徽安龙基因医学检验所有限公司 | A kind of detection method of alcohol metabolism related gene |
| 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 |
| CN108048563A (en) * | 2018-02-08 | 2018-05-18 | 中国疾病预防控制中心营养与健康所 | A kind of method for lacking risk assessment to people's nutrient based on SNP site |
Non-Patent Citations (1)
| Title |
|---|
| 谢晓媛 等: "叶酸代谢酶MTHFR、MTRR基因多态性与原因不明复发性流产的关系 ", 《天津医药》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110241195A (en) * | 2019-04-17 | 2019-09-17 | 青海大学 | A kind of gene tester of individuation folic acid supplement amount |
| CN110257507A (en) * | 2019-07-11 | 2019-09-20 | 上海联吉医学检验所有限公司 | A kind of pregnancy period folic acid metabolism, calcium metabolism and H-type hypertension associated detecting method |
| CN110257507B (en) * | 2019-07-11 | 2023-11-03 | 上海联吉医学检验所有限公司 | Pregnancy folic acid metabolism, calcium metabolism and H-type hypertension combined detection method |
| CN111187828A (en) * | 2020-02-11 | 2020-05-22 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting polymorphism of human folate metabolism gene |
| CN111187828B (en) * | 2020-02-11 | 2023-09-15 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting polymorphism of human folic acid metabolism gene |
| CN111394449A (en) * | 2020-03-24 | 2020-07-10 | 海南主健细胞分子遗传医学检验中心有限公司 | Kit for detecting folate metabolism pathway related genes |
| CN111500709A (en) * | 2020-05-12 | 2020-08-07 | 珠海横琴润孚创新科技有限公司 | Metabolic disease gene detection and clinical depth data analysis method |
| CN111876482A (en) * | 2020-09-21 | 2020-11-03 | 郑州安图生物工程股份有限公司 | Kit for simultaneously detecting human MTHFR and MTRR genes |
| CN112094899A (en) * | 2020-09-24 | 2020-12-18 | 江苏先声医疗器械有限公司 | Detection method of folic acid metabolism capability based on MassArray nucleic acid mass spectrum and application thereof |
| CN112094899B (en) * | 2020-09-24 | 2021-06-25 | 江苏先声医疗器械有限公司 | Detection method of folic acid metabolism capability based on MassArray nucleic acid mass spectrum and application thereof |
| CN113637735A (en) * | 2021-08-09 | 2021-11-12 | 优葆优保健康科技(宁波)有限公司 | Kit for detecting children nutrition genome and application |
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