CN108753944B - Primer, probe and kit for detecting gene locus genotype related to folic acid metabolism - Google Patents
Primer, probe and kit for detecting gene locus genotype related to folic acid metabolism Download PDFInfo
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Abstract
The invention provides a primer, a probe and a kit for detecting gene loci genotype of folic acid metabolism related genes, which aim at least 4 loci among 8 loci of the following 4 folic acid metabolism related genes: 677C > T site, 1298A > C site of MTHFR gene, 66A > G site, 56A > C site of MTRR gene, 186T > R site, 905G > A site, 2756A > G site of MTR gene and 551C > G site of CBS gene, wherein each primer is shown in SEQ ID NO.1-16, each probe is shown in SEQ ID NO.17-24, and both ends of each probe are respectively marked with a fluorescent group and a quenching group. The primer and the probe have higher specificity, and can detect the genotype of the gene locus related to folic acid metabolism by a fluorescence melting curve method, thereby evaluating the folic acid metabolism capacity of a tested person.
Description
Technical Field
The invention relates to detection of gene locus genotypes related to folic acid metabolism, in particular to primers, probes and a kit for detecting gene locus genotypes related to folic acid metabolism.
Background
Folic acid is one of water-soluble vitamin and vitamin B complex, corresponds to pteroylglutamic acid (Pteroylglutamic acid, PGA), and has the effect of promoting the maturation of young cells in bone marrow. In humans, deficiency of folic acid, which is particularly important for pregnant women, can cause megaloblastic anemia and leukopenia. Folic acid acts as a coenzyme of a single-carbon unit transferase system in-vivo biochemical reaction, plays a role of a single-carbon unit transporter, and can participate in the synthesis of purine and thymine to further synthesize DNA and RNA. Folic acid has been reported to play a critical role in the synthesis of proteins, nucleic acids and the metabolism of various amino acids, for example: during the synthesis of purine and pyrimidine, folic acid can be used as a coenzyme to participate in the formation of purine and pyrimidine during nucleic acid synthesis, thereby functioning in DNA biosynthesis. In the amino acid conversion process, folic acid can participate in the mutual conversion of two-carbon amino acid and three-carbon amino acid, and can promote the conversion of phenylalanine and tyrosine, histidine and glutamic acid, and cysteine and methionine. In addition, folic acid is also a component of iron-containing hemoglobin, which is a substance essential for human body in utilizing sugar and amino acids, and is a substance essential for growth and reproduction of body cells. Folic acid acts in vivo in the form of tetrahydrofolate, which is involved in the synthesis and conversion of purine nucleic acids and pyrimidine nucleotides in vivo. Folic acid plays an important role in the process of producing nucleic acids (ribonucleic acid, deoxyribonucleic acid). Folic acid, which promotes protein metabolism and promotes erythrocyte production and maturation together with vitamin B12, is an essential substance for producing erythrocytes. Folic acid also plays a role as a proliferation promoting factor for lactobacillus casei (Lactobacillus casei) and other microorganisms, and plays an important role in cell division growth and synthesis of nucleic acids, amino acids, and proteins, and various synthetases, transferases, and oxidoreductases are involved in a large network of their metabolism, and genetic polymorphisms of these enzymes may cause imbalance in the entire metabolic network.
Folate metabolism is mainly regulated by four key enzymes, namely methylene tetrahydrofolate reductase (5, 10-methylenetetrahydrofolate reductase, MTHFR), methionine synthetase (MTR), methylthionine synthase reductase (5-methyl formate-homocysteine methyltransferase reductase, MTRR) and Cystathionine Beta Synthase (CBS), and mutation of the gene loci of the 4 enzymes leads to structural instability and enzyme activity reduction, thereby affecting the metabolism of folic acid.
Disclosure of Invention
The invention aims to solve the technical problem of providing a primer, a probe and a kit for detecting gene locus genotypes of folic acid metabolism related genes.
The technical scheme of the invention is as follows:
a primer and probe for detecting the genotype of the locus of a gene related to folate metabolism, the primer comprising a primer pair for detecting at least 4 loci of 8 loci of the following 4 genes related to folate metabolism:
a primer pair which consists of two sequences shown in SEQ ID NO.1 and SEQ ID NO.2 in a sequence table and aims at 677C > T locus of an MTHFR gene;
a primer pair which is composed of two sequences shown in SEQ ID NO.3 and SEQ ID NO.4 in a sequence table and aims at 1298A > C locus of the MTHFR gene;
primer pairs for 66A > G locus of MTRR gene, which consists of two sequences shown in SEQ ID NO.5 and SEQ ID NO.6 in the sequence table;
a primer pair which consists of two sequences shown in SEQ ID NO.7 and SEQ ID NO.8 in a sequence table and aims at 56A > C locus of the MTRR gene;
primer pairs for 186T > R locus of MTR gene, which consists of two sequences shown in SEQ ID NO.9 and SEQ ID NO.10 in the sequence list;
primer pairs which are formed by two sequences shown in SEQ ID NO.11 and SEQ ID NO.12 in a sequence table and aim at 905G > A locus of the MTR gene;
a primer pair which consists of two sequences shown in SEQ ID NO.13 and SEQ ID NO.14 in a sequence table and aims at 2756A > G locus of the MTR gene;
primer pairs for 551C > G locus of CBS gene, which consists of two sequences shown in SEQ ID NO.15 and SEQ ID NO.16 in the sequence table;
accordingly, the probes include probes that detect at least 4 of the 8 sites of the following 4 folate metabolism related genes:
a probe for 677C > T locus of MTHFR gene consisting of a sequence shown in SEQ ID NO.17 or a reverse complement sequence thereof;
a probe for 1298A > C locus of MTHFR gene consisting of a sequence shown in SEQ ID NO.18 in the sequence table or a reverse complement sequence thereof;
a probe for 66A > G locus of MTRR gene composed of a sequence shown in SEQ ID NO.19 in the sequence table or a reverse complementary sequence thereof;
a probe for 56A > C locus of MTRR gene composed of a sequence shown in SEQ ID NO.20 in the sequence table or a reverse complementary sequence thereof;
a probe for the 186T > R locus of the MTR gene, which consists of a sequence shown in SEQ ID NO.21 in the sequence table or a reverse complement sequence thereof;
a probe for a site 905G > A of the MTR gene, which consists of a sequence shown by SEQ ID NO.22 in a sequence table or a reverse complementary sequence thereof;
a probe for 2756A > G locus of MTR gene composed of a sequence shown in SEQ ID NO.23 or reverse complement sequence thereof in the sequence table;
a probe for a 551C > G site of a CBS gene, which consists of a sequence shown by SEQ ID NO.24 in a sequence table or a reverse complement sequence thereof;
the two ends of each probe are respectively marked with a fluorescent group and a quenching group.
Preferably, the primer is a primer pair for detecting all 8 sites, and correspondingly, the probe is a probe for detecting all 8 sites.
Preferably, one end of the probe for 4 of the 8 sites is labeled with a different fluorescent group, and independently, one end of the probe for the other 4 sites is also labeled with a different fluorescent group.
Preferably, the fluorescent group is selected from FAM, ROX, HEX, CY5.
A kit for detecting gene locus genotypes related to folic acid metabolism comprises the primer and the probe.
Preferably, the primers in the kit are primer pairs for detecting all 8 sites, and correspondingly, the probes are probes for detecting all 8 sites, and the kit comprises two reaction tubes, wherein one reaction tube is provided with primers and probes for detecting 4 sites, and the other reaction tube is provided with primers and probes for detecting the other 4 sites.
Preferably, one end of the probe for 4 sites in the same reaction tube is labeled with different fluorophores.
Preferably, the concentration of each primer in the reaction system in the kit is 0.2. Mu. Mol/L.
Preferably, the concentration of each probe in the reaction system in the kit is 0.25. Mu. Mol/L.
The beneficial effects of the invention include:
by adopting the technical scheme of the invention, the gene type of the gene locus related to folic acid metabolism can be determined, the product is directly subjected to melting curve analysis after PCR amplification by using a multiplex PCR fluorescent melting curve method, uncapping analysis is not needed, the pollution of the product can be effectively avoided, multicolor fluorescence and melting curve are combined, the sufficient flux of detection is ensured, the detection specificity is high, the operation is simple and convenient, the detection is accurate, sensitive and rapid, the cost is low, and the method is suitable for being popularized and used in a large amount in clinic. Furthermore, the primer and the probe have higher specificity, and can detect 8 sites of 4 genes simultaneously.
Drawings
FIG. 1 is a graph showing HEX fluorescence melting curves of the genotype of the 66A > G locus of the MTRR gene in the specific embodiment of the invention, which is the AA normal genotype;
FIG. 2 is a graph showing HEX fluorescence melting curves of AG heterozygous genotypes at the locus 66A > G of the MTRR gene in the embodiment of the present invention;
FIG. 3 is a graph showing HEX fluorescence melting curves of the genotype of the G/66A locus of MTRR gene in the embodiment of the present invention, which is GG mutant genotype.
Detailed Description
The present invention will be described in further detail with reference to the following detailed description and the accompanying drawings. It should be emphasized that the following description is merely exemplary in nature and is in no way intended to limit the scope of the invention or its applications.
According to an embodiment of the present invention, the PCR primers and probes for detecting the folate metabolism related gene locus genotype are directed to at least 4 loci out of 8 loci of the following 4 folate metabolism related genes: 677C > T site, 1298A > C site, 66A > G site, 56A > C site of MTRR gene, 186T > R site, 255G > A site, 2756A > G site of MTR gene, and 551C > G site of CBS gene.
The PCR primers for each site are shown in table 1 below:
TABLE 1
Wherein "F" represents the upstream primer and "R" represents the downstream primer.
Specific probes for each site are shown in table 2 below:
TABLE 2
| Sequence numbering | Sequence (5 '-3') | Site of interest |
| SEQ ID NO.17 | gtctgcgggagccgattt | 677C of MTHFR Gene>T site |
| SEQ ID NO.18 | ccagtgaagcaagtgtct | 1298A of MTHFR gene>C site |
| SEQ ID NO.19 | gcagaagaaatatgtgag | 66A of MTRR Gene>G site |
| SEQ ID NO.20 | agccaagcttttggtttgg | 56A of MTRR Gene>C site |
| SEQ ID NO.21 | ggagctcgggcagaccaat | 186T of MTR Gene>R site |
| SEQ ID NO.22 | aggtgcccgccaccaca | 905G of MTR Gene>A site A |
| SEQ ID NO.23 | ttagacaggaccattatg | 2756A of MTR Gene>G site |
| SEQ ID NO.24 | gcagagaccgcggcggagc | 551C of CBS Gene>G site |
Wherein, the two ends of each probe are respectively marked with a fluorescent group and a quenching group. The fluorophore may be selected from, but is not limited to FAM, ROX, HEX, CY5, and the fluorophore may be labeled at the 5 'end (accordingly, the quencher is at the 3' end) or at the 3 'end (accordingly, the quencher is at the 5' end). In this example, the 5 'end of the probe is labeled with a fluorescent group and the 3' end is labeled with a quenching group.
In other embodiments, the sequence of the probe may also be the reverse complement of each of the sequences in Table 2.
In a preferred embodiment, the primer is a primer pair for detecting all 8 sites, and accordingly the probe is a probe for detecting all 8 sites, one end of the probe for 4 sites out of 8 sites is labeled with a different fluorophore, and one end of the probe for the other 4 sites is also labeled with a different fluorophore independently.
The invention also provides a kit for detecting gene locus genotypes related to folic acid metabolism, which comprises the primer and the probe.
In a preferred embodiment, the primers in the kit are primer pairs for detecting all 8 sites, and correspondingly, the probes are probes for detecting all 8 sites, and the kit comprises two reaction tubes, wherein one reaction tube is provided with the primers and probes for detecting 4 sites, and the other reaction tube is provided with the primers and probes for detecting the other 4 sites, and one end of each of the probes for detecting the other 4 sites in the same reaction tube is marked by different fluorescent groups, so that mutual interference and influence among the primers, among the probes, among the primers and the probes, and among products formed by the primers and the probes can be reduced as far as possible.
In the multiplex PCR reaction, the concentration of the primers is tested in different gradients, and when the concentration of each primer in the reaction system is 0.2 mu mol/L, the preferable reaction result is achieved. The probe concentration is also tested in different gradients, and when the concentration of each probe in the reaction system is 0.25 mu mol/L, the reaction result is better.
The following will describe in detail an example of simultaneous detection of 8 loci of 4 folate metabolism-related genes, and the genotype of the folate metabolism-related locus genes was detected by a fluorescence melting curve method, whereby the folate metabolism ability of the subject was evaluated.
PCR primer: as shown in table 1 above.
The reaction tube: white and blue tubes are one each.
The fluorescence detection channel used: FAM, CY5, ROX and HEX
And (3) probe: as shown in table 2 above.
Wherein, the 5' end of the probe aiming at 677C > T site of MTHFR gene is marked with FAM fluorescent group, the 5' end of the probe of 66A > G site of MTRR gene is marked with HEX fluorescent group, the 5' end of the probe of 255G > A site of MTR gene is marked with ROX fluorescent group, the 5' end of the probe of 551C > G site of CBS gene is marked with CY5 fluorescent group, the 3' end of each probe is marked with DABCY1 quenching group, and the 4 probes and the primer pairs (4 pairs and 8) of the corresponding sites are filled in white tube. Aiming at the 5' end of the probe of 1298A > C site of MTHFR gene, the 5' end of the probe of 2756A > G site of MTR gene, the HEX fluorescent group of MTRR gene, the ROX fluorescent group of the 5' end of the probe of 56A > C site of MTRR gene, the CY5 fluorescent group of the 5' end of the probe of 186T > R site of MTR gene, the DABCY1 quenching group of the 3' end of each probe, the 4 probes and the primer pairs of the corresponding sites are arranged in a blue tube.
Each reaction tube also comprises: deionized water, dNTPs, taq enzyme and MgCl 2 PCR buffer, etc.
The method comprises the following specific steps:
(1) The DNA of the sample to be detected is extracted, and the specific process refers to the related specification, and the sample DNA source can be blood, tissue, body fluid and the like.
(2) Taking DNA of a sample to be detected as a template, and respectively constructing the following fluorescent quantitative reaction system by a white tube and a blue tube: 10 Xbuffer, 2.5. Mu.L; 25mM MgCl 2 2.5. Mu.L; 2.5mM dNTPs, 2. Mu.L; taq, 0.5. Mu.L; 10. Mu.M (0.4. Mu.L each) of the corresponding primer; 10. Mu.M probe (0.5. Mu.L each); template DNA 2. Mu.L, the reaction system was made up to 20. Mu.L with deionized water.
(3) The reaction system was subjected to PCR amplification using the following procedure: pre-denaturation at 95 ℃ for 5min, amplification stage: denaturation at 95℃for 10s, annealing at 60℃for 30s, extension at 72℃for 30s for 38 cycles. Finally, the mixture is thoroughly extended for 5min at 72 ℃. Wherein the amplified target sequence is as follows:
SEQ ID NO.25, 677C > T site for the MTHFR gene:
ctgactgtcatccctattggcaggttaccccaaaggccaccccgaagcagggagctttgaggctgacctgaagcacttgaaggagaaggtgtctgcgggagccgatttcatcatcacgcagcttttctttgaggctgacacattcttccgctttgtgaaggcatgcaccgacatgggcatcactt
SEQ ID NO.26, 1298A > C locus for the MTHFR gene:
aggagctgctgaagatgtggggggaggagctgaccagtgaagcaagtgtctttgaagtctttgttctttacctctcgggagaaccaaaccggaatggtcacaaagtgagtgatgctggaagtggggaccctggttcatcccctgcccctggcctcagggtgccaaacctgatggtcgccccagccagctcaccgtctc
SEQ ID NO.27, 66A > G locus for the MTRR gene:
tctgttactatatgctacacagcagggacaggcaaaggccatcgcagaagaaatatgtgagcaagctgtggtacatggattttctgcagatcttcactgtattagtgaatccgataaggttagagccgttacagtggattttaccgttttgtgctttgaagaattttggttgggaagtSEQ ID NO.28, 56A > C locus for MTRR gene:
gtttccaccgttttctgtgagctgtcaatgtgtagtgttttcatattgtttattcactcattcagataacttatcgagtaccagtttcatgccaggtgccgttctgggcggttcattcaccgaaagccaagcttttggtttgggtttcagtttaaatctgtctctgcaaattgacagcccaccaattttagccgttagatgagta
SEQ ID NO.29, directed against the 186T > R site of the MTR gene:
gatcctgggcacacagagacgcgaaaagctcccgcgggaaactggggagcgggactacatctcccagagagccccggggctaaacagcaggtgattggttgatattgacagccaatgcggctgcgaggagctcgggcagaccaatcacaggccggaaggggcgggacctcccacgccgaggatagattgagcgcagaactaaccgcgctctgaaaggttctaa
SEQ ID NO.30, directed to site 255G > A of the MTR gene:
gcaatctcggctcatagcaagctccgcctcctgggttcatgccattctcctgcctcagcctccagagtagctgggactacaggtgcccgccaccacacccggctaattttttgtgtttttacaaaatacaaaaaagtagagacaggatttcactgtgttagccaggatggtcttgatctc
SEQ ID NO.31, 2756A > G site for MTR gene:
gctcatctatggctatcttgcattttcagtgttcccagctgttagatgaaaatctaaaggatgaatactttgaggaaatcatggaagaatatgaagatattagacaggaccattatgagtctctcaaggtaagtggtagaaacagatttttgcttgtttttaatgtgactgttttttatgatcctagtttttaatgtgactttttaaaatggttttgaggagtgt
SEQ ID NO.32, directed against the 551C > G site of the CBS gene:
gtcagttcctgccagtggacatttaattctaattcacgtctcactccctcccctactgtgagccccacgtggcagagaccgcggcggagctgcgcgggccggaggaggctgcggagcatcgctctgacctgagccggggacgctgcggaggcgcagcacgttcctctgcccagagctgatgggcac
after amplification, a melting curve reaction procedure was performed: denaturation at 95℃for 2min at 40℃for 3min, gradual heating from 40℃to 80℃with a rate set as inherent in the instrument, and signal collection. In the process, probes with different labels can be respectively and specifically combined with four amplification product sequences, fluorescent light with different colors is emitted under the irradiation of different excitation wavelengths, the intensity of the fluorescent light is changed according to the change of temperature, the peak values of melting peaks are different due to the difference of base sites, and finally, the base type of a gene can be judged according to the curve shape formed by the fluorescent light in the melting process.
The above relevant reaction procedure can be performed on an ABI 7500 or Roche z480 fluorescent quantitative PCR instrument.
(4) And judging the result according to the temperature when the peak value appears and the number of the peak value, and determining the genotype of the related gene locus. The relevant decision criteria are shown in table 3 below:
table 3: peak interpretation of fluorescence curves
For example, when the genotype at the 66A > G locus of the MTRR gene of the sample to be tested is the AA normal genotype, the HEX fluorescence melting curve is shown in FIG. 1 (peak temperature 55.409 ℃), when the genotype at the 66A > G locus of the MTRR gene of the sample to be tested is the AG heterozygous genotype, the HEX fluorescence melting curve is shown in FIG. 2 (two peaks occur, and each peak temperature corresponds to the peak temperature range of the normal and mutant shown in the table), and when the genotype at the 66A > G locus of the MTRR gene of the sample to be tested is the GG mutant genotype, the HEX fluorescence melting curve is shown in FIG. 3 (peak temperature 60.079 ℃).
In the preferred embodiment, the genotyping of 8 loci of 4 genes related to folic acid metabolism of one sample can be completed by one-time detection, and the method has the characteristics of strong specificity, accurate result, simple process operation and the like, and has positive significance in evaluating the clinical diagnosis of folic acid metabolism, cerebral apoplexy, coronary heart disease and the like.
The foregoing is a further detailed description of the invention in connection with specific/preferred embodiments, and is not intended to limit the practice of the invention to such description. It will be apparent to those skilled in the art that several alternatives or modifications can be made to the described embodiments without departing from the spirit of the invention, and these alternatives or modifications should be considered to be within the scope of the invention.
Claims (1)
1. The application of the primer and probe combination in preparing a kit for detecting gene locus genotypes related to folic acid metabolism by a multiplex PCR fluorescent melting curve method is characterized in that the primer and the probe are divided into two groups, wherein one group is a primer pair and a probe for detecting the following 4 loci:
a probe consisting of a primer pair consisting of two sequences shown in SEQ ID NO.1 and SEQ ID NO.2 and a sequence shown in SEQ ID NO.17 or a reverse complement thereof aiming at 677C > T locus of the MTHFR gene, wherein the 5' end of the probe is marked with a FAM fluorescent group;
a probe consisting of a primer pair consisting of two sequences shown as SEQ ID NO.5 and SEQ ID NO.6 and a sequence shown as SEQ ID NO.19 or a reverse complement sequence thereof aiming at a 66A > G locus of the MTRR gene, wherein the 5' end of the probe is marked with HEX fluorescent groups;
a probe consisting of a primer pair consisting of two sequences shown as SEQ ID NO.11 and SEQ ID NO.12 and a sequence shown as SEQ ID NO.22 or a reverse complement sequence thereof aiming at a site 905G > A of the MTR gene, wherein the 5' end of the probe is marked with a ROX fluorescent group;
a probe consisting of a primer pair consisting of two sequences shown as SEQ ID NO.15 and SEQ ID NO.16 aiming at a 551C > G site of a CBS gene and a sequence shown as SEQ ID NO.24 or a reverse complement sequence thereof, wherein the 5' end of the probe is marked with a CY5 fluorescent group;
the other set is a primer pair and probe that detects the following 4 sites:
a probe consisting of a primer pair consisting of two sequences shown in SEQ ID NO.3 and SEQ ID NO.4 and a sequence shown in SEQ ID NO.18 or a reverse complement sequence thereof aiming at 1298A > C locus of the MTHFR gene, wherein the 5' end of the probe is marked with a FAM fluorescent group;
a probe consisting of a primer pair consisting of two sequences shown as SEQ ID NO.7 and SEQ ID NO.8 and a sequence shown as SEQ ID NO.20 or a reverse complement sequence thereof aiming at a 56A > C site of the MTRR gene, wherein the 5' end of the probe is marked with a ROX fluorescent group;
a probe consisting of a primer pair consisting of two sequences shown in SEQ ID NO.9 and SEQ ID NO.10 and a sequence shown in SEQ ID NO.21 or a reverse complement sequence thereof aiming at the 186T > R site of the MTR gene, wherein the 5' end of the probe is marked with a CY5 fluorescent group;
a probe consisting of a primer pair consisting of two sequences shown in SEQ ID NO.13 and SEQ ID NO.14 and a sequence shown in SEQ ID NO.23 or a reverse complement thereof aiming at 2756A > G locus of the MTR gene, wherein the 5' end of the probe is marked with HEX fluorescent groups;
wherein the 3' end of each probe is marked with a DABCY1 quenching group;
and judging the result according to the temperature and the number of the peaks when the peaks appear by adopting a multiplex PCR fluorescence melting curve method, and determining the genotype of the related gene locus, wherein the related judgment standard is as follows:
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