CN111394449A - Kit for detecting folate metabolism pathway related genes - Google Patents
Kit for detecting folate metabolism pathway related genes Download PDFInfo
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Abstract
The invention provides a kit for detecting folate metabolism pathway related genes, which comprises a primer composition for detecting MTR gene RS1805087 site, CBS gene RS5742905 site, MTRR gene RS1801394 site, MTHFR gene RS1801131 site and MTHFR gene RS1801133 site related to folate metabolism pathway, wherein the sequence of the primer composition comprises 5 pairs of PCR primers and 5 forward extension compositions which are close to SNP sites, and the primer compositions are shown as SEQ ID Nos. 1-15. The kit can realize disposable multi-gene locus detection, reduces the minimum detection limit to 1ng/ul, has high detection rate, realizes large-flux rapid genotyping of gene loci, and ensures 100% accuracy of sample detection.
Description
Technical Field
The invention relates to the technical field of gene polymorphism detection, in particular to a kit for detecting folate metabolism pathway related genes.
Background
Folic acid is an important nutrient required by organisms, is a water-soluble B vitamin and participates in a series of important biochemical reactions related to one-carbon unit transportation. With the continuous progress of medical science such as medical genetics, molecular biology and the like, people increasingly recognize many new important functions of folic acid, especially folic acid deficiency can cause damage and breakage of chromosomes, the exchange frequency of sister chromosomes is increased, fragile locus expression is realized, and severe patients can mutate or even become cancerous. Folic acid is an essential element for nucleic acid synthesis, is a necessary substance for cell growth and tissue repair, and is an indispensable nutrient in the process of embryonic development. The clinical function of folic acid can prevent the fetal neural tube defect, and reduce the incidence of pregnant woman gestational hypertension, spontaneous abortion, intrauterine fetal development retardation, premature birth, low birth weight of newborn and the like. A number of studies in recent years have demonstrated that folate deficiency is a major cause of birth defects.
Folate is reduced in the human body by the action of folate reductase to physiologically active tetrahydrofolate, which is a carrier of intracellular one-carbon units, involved in purine and pyrimidine synthesis and provides methyl for methylation of DNA, 5-methyltetrahydrofolate, one of the tetrahydrofolate methyl carriers, is produced by methyltetrahydrofolate reductase and serves as a methyl donor, which promotes the remethylation of homocysteine in blood to produce methionine (methionine) under the catalysis of methionine Synthase, using vitamin B12 as a coenzyme, to maintain normal plasma homocysteine levels, and at the same time, methionine is phosphorylated by ATP to form S-adenosylmethionine, which is involved in the methylation of DNA and proteins in the body to regenerate homocysteine, thus 5-methyltetrahydrofolate is an important factor for maintaining homocysteine levels in the blood and is also an important precursor of methylation reaction, genes involved in regulating plasma homocysteine metabolism and affecting the metabolic pathways include Cystathionine- β -synthosphatase (synthosphatine, synthosphatine-synthetase (cbthionine), methionine Synthase (mtmethyltetrahydrofolate reductase), methionine-reductase (cbe-reductase), and methionine-reductase (mte-reductase).
MTHFR-encoded methionine synthase, which catalyzes the final step of methionine biosynthesis, provides methyl from 5-methyltetrahydrofolate entering the cell, catalyzes the re-methylation of HCY to methionine, and maintains the HCY level and methionine cycle in vivo.
The existing detection technology mainly aims at the polymorphism detection of the related genes of the folic acid metabolic pathway only aiming at the polymorphism detection of MTHFR gene and MTRR gene sites, and when the number of detection sites is increased, the existing detection technology is difficult to realize the efficient and accurate synchronous detection of the polymorphism sites of a plurality of folic acid metabolic pathway key genes due to the problems of complex operation, high requirement on sample quality, low detectable rate and low experiment stability and efficiency, thereby limiting the effective application of the detection product in providing reference basis for guiding pregnant women and pregnant women to reasonably take folic acid.
Disclosure of Invention
Therefore, the kit for detecting the related genes of the folate metabolism pathway provided by the invention can be used for simply, intuitively and accurately detecting the related genotyping of the folate metabolism pathway, the minimum detection concentration of a sample is up to lng/ul, the detection efficiency and accuracy are high, the flux is high, and the kit is easy to interpret.
The technical scheme of the invention is realized as follows:
a kit for detecting folate metabolism pathway related genes comprises a primer composition for detecting MTR gene RS1805087 site, CBS gene RS5742905 site, MTRR gene RS1801394 site, MTHFR gene RS1801131 site and MTHFR gene RS1801133 site related to folate metabolism pathway, wherein the sequence of the primer composition is as follows:
forward and reverse PCR primers of MTR gene RS1805087 site: 1-2, forward extension primer: 11 is shown in SEQ ID No;
forward and reverse PCR primers for the CBS gene at the RS5742905 site: 3-4, forward extension primer: 12 is shown in SEQ ID NO;
forward and reverse PCR primers for MTRR gene RS1801394 site: 5-6 of SEQ ID No, forward extension primer: SEQ ID No. 13;
forward and reverse PCR primers of MTHFR gene RS1801131 site: 7-8 of SEQ ID No, forward extension primer: SEQ ID No. 14;
forward and reverse PCR primers of MTHFR gene RS1801133 site: 9-10 of SEQ ID No, forward extension primer: SEQ ID No. 15.
Further, the 5' end of the forward extension primer sequence was modified with different amounts of ploy T; wherein, the number of ploy T modifications on the forward extension primer at the RS1805087 site of the MTR gene is 11; the number of ploy T modifications on the forward extension primer at position RS5742905 of the CBS gene is 19; the number of ployT modifications on the forward extension primer at the RS1801394 site of the MTRR gene is 32; the number of ploy T modifications on the forward extension primer at the RS1801131 site of the MTHFR gene is 14; the number of ploy T modifications on the forward extension primer at the RS1801133 site of MTHFR gene was 26.
Further indicates that the concentration ratio of forward extension primers of the MTR gene RS1805087 site, the CBS gene RS5742905 site, the MTRR gene RS1801394 site, the MTHFR gene RS1801131 site and the MTHFR gene RS1801133 site is 1:2:3:2: 3.
Further, the concentration ratio of each pair of forward and reverse PCR primers of the MTR gene RS1805087 site, the CBS gene RS5742905 site, the MTRR gene RS1801394 site, the MTHFR gene RS1801131 site and the MTHFR gene RS1801133 site is 1:2:1:1: 1.
Further illustratively, the kit further comprises the following reagents:
(1) multiplex PCR amplification reagents: comprises dNTPs, Taq DNA polymerase, PCR reaction buffer solution, 1 XGC-I buffer solution and Mg2 +:
(2) PCR product purification reagents: AP enzyme, Exol1 enzyme, Seq buffer;
(3) multiple single base extension reaction reagents: SNaPshot Multiplex Kit (ABI);
(4) extension product purification reagent: an SAP enzyme;
(5) 120L iz SIZE STANDARD internal STANDARD and Hi-Di formamide as sequencing denaturing reaction reagent.
Further, the volume ratio of the purified Multiplex PCR product, the forward extension primer composition and the SNaPshot Multiplex Kit (ABI) in the Multiplex single base extension reaction reagent is 2:1: 5.
Further, the volume ratio of the purified extension product to 120L iz SIZESTANDARD internal standard and Hi-Di formamide in the sequencing denaturation reagents was 1:1: 18.
Compared with the prior art, the invention has the beneficial effects that: the kit for detecting the folate metabolism pathway related gene provided by the invention not only can realize disposable multi-gene site detection, but also effectively overcomes the defects of high requirement on sample quality, low detection rate and low detection efficiency in the prior art, and the invention designs 5 pairs of PCR primers and 5 forward extension compositions adjacent to the SNP sites by carrying out optimized selection combination on 5 SNP site systems on MTR gene, CBS gene, MTRR gene and MTHFR gene on the whole, combines the characteristics of the SNP site systems, adjusts different lengths of the forward extension primer combinations aiming at different SNP sites, effectively improves the accurate typing of a plurality of SNP sites in a reaction system, efficiently and intuitively determines the SNP sites corresponding to different extension products according to the moving positions of an electrophoresis front, directly judges the types of the doped bases according to the color of the front, greatly reduces the minimum detection limit to 1ng/ul, the detection rate is high, the genotyping of the gene loci is rapidly carried out with large flux, and the accuracy of sample detection is ensured to be 100%.
Drawings
FIG. 1 is an electropherogram of the reaction of experimental sample 2 on MTR gene RS1805087 locus genotyping in the examples of the present invention;
FIG. 2 is a reaction electropherogram of the experimental sample 2 for genotyping at the RS5742905 locus of the CBS gene in the example of the present invention;
FIG. 3 is a reaction electropherogram showing that the CBS gene can not be genotyped at the RS5742905 site in the example of the present invention;
FIG. 4 is a reaction electropherogram of the experimental sample 2 for genotyping the RS1801394 locus of the MTRR gene in the example of the present invention;
FIG. 5 is an electrophoretogram showing the inability of genotyping the RS1801394 locus of the MTRR gene in the examples of the present invention;
FIG. 6 is a reaction electropherogram of the experimental sample 2 for genotyping at rs1801133 locus of MTHFR gene in the example of the present invention;
FIG. 7 is a reaction electropherogram showing that the rs1801133 locus of MTHFR gene cannot be genotyped in the example of the present invention;
FIG. 8 is an electropherogram of the reaction of the experimental sample 2 on genotyping of RS1801131 locus of MTHFR gene in the example of the present invention;
FIG. 9 is an electrophoretogram of a reaction that does not allow genotyping of the RS1801131 locus of the MTHFR gene in the examples of the present invention.
Detailed Description
In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.
The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.
The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.
Example 1 design of primer compositions
The invention carries out optimization selection on 5 SNP site systems on MTR gene, CBS gene, MTRR gene and MTHFR gene, designs 5 pairs of PCR primers for amplifying DNA fragments containing 5 SNP sites, and designs 5 forward extension guiding compositions which are close to the SNP sites; meanwhile, extension primers with different lengths are designed aiming at different SNP sites, namely, the quantity of poly T modified on 5 single-base extension primers and the concentration of the single-base extension primers are respectively adjusted, so that extension products generated according to respective genes have different molecular weights, thus a plurality of SNP sites are accurately typed in a reaction system, the SNP sites corresponding to different extension products are efficiently and intuitively determined according to the moving positions of an electrophoresis front, and the types of the doped bases are directly judged according to the colors of the fronts.
The primer composition for detecting MTR gene RS1805087 site, CBS gene RS5742905 site, MTRR gene RS1801394 site, MTHFR gene RS1801131 site and MTHFR gene RS1801133 site related to folic acid metabolic pathway is as follows:
example 2 preparation and use of the kit
a. The components and contents of the kit comprise:
(1) multiplex PCR amplification reagents: dNTPs, TaqDNA polymerase, PCR reaction buffer, 1 XGC-I buffer, Mg2 +:
(2) PCR product purification reagents: AP enzyme, Exol1 enzyme, Seq buffer;
(3) multiple single base extension reaction reagents: SNaPshot Multiplex Kit (ABI);
(4) extension product purification reagent: an SAP enzyme;
(5) 120L iz SIZE STANDARD internal STANDARD and Hi-Di formamide as sequencing denaturing reaction reagent.
b. Use of the kit
1. Sampling of samples
Taking an oral test-element saliva sample, carrying out DNA extraction to obtain a DNA sample 1 mu l of 1% agarose electrophoresis, carrying out quality inspection and concentration estimation on the sample, dividing the sample into 4 parts by equal amount according to the estimated concentration, and diluting the samples to the experimental samples 1-4 with the concentrations of 0.8 ng/mu l, 1 ng/mu l, 5 ng/mu l and 10 ng/mu l respectively.
2. Multiplex PCR reaction
(1) PCR reaction conditions
Preparing a reaction system comprising 1.2. mu.l dNTP mix, 0.1. mu.l Taq DNA polymerase, 1. mu.l 10X PCRbuffer, 5. mu.l 1X GC-I buffer, 0.8ul Mg2+, 1. mu.l sample DNA and 1. mu.l multiplex PCR primer of example 1; in particular, see the following table:
| dNTP Mixture | 1.2μl |
| TaqDNA polymerase | 0.1μl |
| 10X PCR buffe | 1μl |
| 1x GC-I buffer | 5μl |
| Mg2+ | 0.8ul |
| Sample DNA | 1μl |
| Multiplex PCR primers | 1μl |
Concentration of each pair of primers in multiplex PCR primers: (mu M)
| rs1805087F/R | rs5742905F/R | rs1801394F/R | rs1801131F/R | rs1801133F/R |
| 1 | 2 | 1 | 1 | 1 |
(2) PCR cycling program
3. PCR product purification
Mu.l of AP enzyme, 0.6. mu.l of Exonuclease I enzyme and 0.7ul of seq buffer were added to 10. mu.l of the PCR product, incubated at 37 ℃ for 30 minutes, and then inactivated at 75 ℃ for 15 minutes.
4. Multiple single base extension reactions
(1) Extension reaction conditions
Preparing an extension reaction system comprising 5. mu.l of the SNaPshot Multiplex Kit (ABI), 2. mu.l of the purified Multiplex PCR product, 1. mu.l of the forward extension primer composition of example 1, and 2. mu.l of ultrapure water; in particular, see the following table:
| NaPshot Multiplex Kit(ABI) | 5μl |
| purified multiplex PCR product | 2μl |
| Forward extension primer composition | 1μl |
| Ultrapure water | 2μl |
Concentration of each extension primer in the extension primer composition: (mu M)
| rs1805087F | rs5742905F | rs1801394F | rs1801131F | rs1801133F |
| 1 | 2 | 3 | 2 | 3 |
(2) Extension reaction procedure
5. Purification of extension products
Mu.l of SAP enzyme was added to 10. mu.l of extension product, incubated for 1 hour at 37 ℃ and then inactivated for 15 minutes at 75 ℃.
6. Denaturation and sequencing of extension products
Mu.l of the purified extension product was mixed with 0.5. mu.l of 120L iz SIZE STANDARD internal STANDARD and 9.0. mu.l of Hi-Di formamide, denatured at 95 ℃ for 5 minutes and electrophoresed on ABI3730X L sequencer.
| Purified extension product | 0.5μl |
| 120L iz SIZE STANDARD internal STANDARD | 0.5μl |
| Hi-Di formamide | 9.0μl |
And analyzing the electrophoresis result by using GeneMapper 4.1 software, determining the genotype of each SNP site according to the condition of each peak in an electrophoretogram after reaction, and comparing the genotype with the detection of the gene site by using a 'gold standard' sanger sequencing method, wherein the results are shown in the following table:
| gene locus | Experimental sample 1 | Experimental sample 2 | Experimental sample 3 | Experimental sample 4 | Sanger sequencing |
| rs1805087 | G,A | G,A | G,A | G,A | G,A |
| rs5742905 | C,T | C,T | C,T | C,T | C,T |
| rs1801394 | G,A | G,A | G,A | G,A | G,A |
| rs1801131 | C,A | C,A | C,A | C,A | C,A |
| rs1801133 | C,T | C,T | C,T | C,T | C,T |
According to the table, 5 pairs of PCR primers and 5 single-base extension primers are designed aiming at the MTR gene RS1805087 site, the CBS gene RS5742905 site, the MTRR gene RS1801394 site, the MTHFR gene RS1801131 site and the MTHFR gene RS1801133 site, and maps of five gene sites are accurately presented according to the SNaPshot reaction electrophoresis: RS1805087, RS5742905, RS1801394, RS1801131 and RS1801133, and according to the typing results of different samples and the detection results of the above gene loci by using the conventional "gold standard" sanger sequencing method, it can be seen that the gene mutation conditions of all gene loci can be detected even at a concentration of 1ng/ul, so as to achieve accurate and rapid genotyping, and the detection results are consistent with the results of the "gold standard" sanger sequencing, and the consistency is 100%, but when the concentration is lower than 0.8ng/ul, the genotyping of which cannot be synchronized at some gene loci occurs, and the genotyping cannot be effectively performed on the RS5742905 locus of the CBS gene, the RS1801394 1394 locus of the MTRR gene, the RS 1801801803 locus of the MTHFR gene and the RS1801131 locus of the MTHFR gene, as shown in fig. 1-9. Therefore, the detection limit of the kit for the folate metabolism pathway related genes can be as low as 1ng/ul, which is far lower than the traditional minimum detection limit of 10ng/ul, and the accuracy of sample detection is 100%. The kit has the advantages of simple and clear detection result, quick judgment of the experimental result and high accuracy.
According to the kit, different numbers of the Poly T modified by each single-base extension primer and the concentration ratio of each extension primer composition are optimized, so that different peaks are located at different positions, the ddNTPs are further utilized for fluorescence labeling, one base after the primer extension carries fluorescein with different weights, the different peaks at the same position cannot be overlapped, the separation uniformity of the peaks is improved, the electrophoresis pattern result of the reaction is judged more accurately and clearly, the detection efficiency, the accuracy, the flux and the easiness in interpretation of the kit are further improved, and the situation that the gene department sites cannot effectively perform genotyping is avoided. Wherein, the 5 'ends in the forward extension primer sequences of the 5 SNP sites in the MTR gene, the CBS gene, the MTRR gene and the MTHFR gene are respectively modified by adopting different amounts of ploy T and the concentration ratio among extension primer compositions is adjusted, so that the optimal 5' ends in the forward extension primer sequences of the 5 SNP sites in the MTR gene, the CBS gene, the MTRR gene and the MTHFR gene are respectively modified by 11, 19, 32, 14 and 26 amounts of ploy T, and when the concentration ratio of the forward extension primers is 1:2:3:2:3, the distance among peaks in a reaction electrophoresis map is uniform and stable, and the typing of each gene site is clear, accurate and stable and easy to judge.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
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Claims (7)
1. A kit for detecting folate metabolism pathway related genes is characterized in that: the primer composition comprises a primer composition for detecting MTR gene RS1805087 site, CBS gene RS5742905 site, MTRR gene RS1801394 site, MTHFR gene RS1801131 site and MTHFR gene RS1801133 site related to folate metabolism pathway, and the sequence of the primer composition is as follows:
forward and reverse PCR primers of MTR gene RS1805087 site: 1-2, forward extension primer: 11 is shown in SEQ ID No;
forward and reverse PCR primers for the CBS gene at the RS5742905 site: 3-4, forward extension primer: 12 in SEQ ID No;
forward and reverse PCR primers for MTRR gene RS1801394 site: 5-6 of SEQ ID No, forward extension primer: 13, SEQ ID No;
forward and reverse PCR primers of MTHFR gene RS1801131 site: 7-8 of SEQ ID No, forward extension primer: 14, SEQ ID No;
forward and reverse PCR primers of MTHFR gene RS1801133 site: 9-10 of SEQ ID No, forward extension primer: 15, SEQ ID No.
2. The kit for detecting a folate metabolism pathway-related gene according to claim 1, wherein: modifying the 5' end in the forward extension primer sequence by adopting different amounts of ploy T;
wherein, the number of ploy T modifications on the forward extension primer at the RS1805087 site of the MTR gene is 11; the number of ploy T modifications on the forward extension primer at position RS5742905 of the CBS gene is 19; the number of ploy T modifications on the forward extension primer at the RS1801394 site of the MTRR gene was 32; the number of ploy T modifications on the forward extension primer at the RS1801131 site of the MTHFR gene is 14; the number of ploy T modifications on the forward extension primer at the RS1801133 site of MTHFR gene was 26.
3. The kit for detecting a folate metabolism pathway-related gene according to claim 2, wherein: the concentration ratio of forward extension primers of the MTR gene RS1805087 site, the CBS gene RS5742905 site, the MTRR gene RS1801394 site, the MTHFR gene RS1801131 site and the MTHFR gene RS1801133 site is 1:2:3:2: 3.
4. The kit for detecting a folate metabolism pathway-related gene according to claim 2, wherein: the concentration ratio of each pair of forward and reverse PCR primers of the MTR gene RS1805087 site, the CBS gene RS5742905 site, the MTRR gene RS1801394 site, the MTHFR gene RS1801131 site and the MTHFR gene RS1801133 site is 1:2:1:1: 1.
5. The kit for detecting a folate metabolism pathway-related gene according to claim 2, wherein: the kit further comprises the following reagents:
(1) multiplex PCR amplification reagents: comprises dNTPs, Taq DNA polymerase, PCR reaction buffer solution, 1 XGC-I buffer solution and Mg2 +:
(2) PCR product purification reagents: AP enzyme, Exol1 enzyme, Seq buffer;
(3) multiple single base extension reaction reagents: SNaPshot Multiplex Kit (ABI);
(4) extension product purification reagent: an SAP enzyme;
(5) 120L izSIZE STANDARD internal STANDARD and Hi-Di formamide as sequencing denaturing reaction reagent.
6. The kit for detecting a folate metabolism pathway-related gene according to claim 5, wherein: the volume ratio of the purified multiplex PCR product, the forward extension primer composition and the SNaPshot multiplex kit (ABI) in the multiplex single-base extension reaction reagent is 2:1: 5.
7. The kit for detecting folate metabolic pathway related genes as in claim 5, wherein the volume ratio of the purified extension products to 120L iz SIZE STANDARD internal STANDARD and Hi-Di formamide in the sequencing denaturation reagents is 1:1: 18.
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| CN112522375A (en) * | 2020-12-11 | 2021-03-19 | 武汉博杰生物医学科技有限公司 | Detection kit and detection method for gene mutation of folate metabolism related molecular marker |
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| CN109182537A (en) * | 2018-09-25 | 2019-01-11 | 北京呈诺医学科技有限公司 | A kind of amplimer, kit and detection method for evaluating folic acid metabolism ability |
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| CN103184269A (en) * | 2011-12-28 | 2013-07-03 | 协和干细胞基因工程有限公司 | Kit for detecting SNP sites related to homocysteine metabolism and amplification method and detection method thereof |
| CN105463116A (en) * | 2016-01-15 | 2016-04-06 | 中南大学 | Forensic medicine composite detection reagent kit based on 20 triallelic SNP genetic markers and detection method |
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| CN112522375A (en) * | 2020-12-11 | 2021-03-19 | 武汉博杰生物医学科技有限公司 | Detection kit and detection method for gene mutation of folate metabolism related molecular marker |
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