Probe for identifying genotype of MTHFR (Methylene Tetrahydrofolate Reductase) polymorphic site rs1801133 and application of probe
By designing specialized probe sequences and using real-time fluorescent PCR with fluorescent labels, the problem of interference between adjacent sites in the detection of the MTHFR polymorphism site rs1801133 was solved, achieving high-accuracy and low-cost genotype detection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- PEOPLES HOSPITAL PEKING UNIV
- Filing Date
- 2025-12-30
- Publication Date
- 2026-04-17
AI Technical Summary
In existing technologies, the detection of the MTHFR polymorphism site rs1801133 is easily affected by the neighboring site rs150847674, resulting in decreased detection accuracy and an inability to effectively distinguish between different genotypes.
Specific probe sequences (SEQ ID NO:1 and SEQ ID NO:2) were designed to identify wild-type and mutant types. These were combined with fluorescent labels and detected using real-time fluorescent PCR. Specialized kits and primer sets were used to eliminate interference from adjacent sites.
It enables large-scale detection of the MTHFR polymorphism site rs1801133 genotype with high accuracy and low cost, avoiding interference from adjacent sites and is easy to operate.
Smart Images
Figure CN121874335A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, and specifically relates to a probe for identifying the rs1801133 genotype of the MTHFR polymorphic site and its application. Background Technology
[0002] Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in the methionine-folate metabolic system. It can reduce 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. The former serves as a raw material for the synthesis of thymine nucleotides and participates in DNA synthesis, while the latter participates in DNA methylation, is the main methyl donor in the body, and is also the main form of folic acid metabolism.
[0003] The MTHFR gene is located at lp36.3 on chromosome 1 (NCBI ID: NC_000001.11), has 11 exons, and exhibits polymorphism. Two common mutations are at position 677 (rs1801133 (C>T)) and position 1298 (rs1801131 (A>C)). MTHFR gene mutations can lead to reduced activity of the key enzymes it encodes for folate metabolism, causing folate metabolism disorders.
[0004] The rs1801133 (C>T) site of the MTHFR gene, also known as MTHFR C677T, is a polymorphism at position 677 of exon 5 of the MTHFR gene. Macroscopically, this site is associated with a variety of diseases, including psoriasis, diabetes and diabetic neuropathy, acute coronary syndrome, hypertension, infertility, stroke, mental illness, and cancer, particularly in cancer, cardiovascular disease, and adverse pregnancies in pregnant women. Furthermore, studies show that homozygous MTHFR 677TT mutations in mothers increase the risk of neural tube defects in infants. In principle, changing from C to T at the rs1801133 site of the MTHFR gene replaces the encoded alanine with valine, leading to a decrease in the thermostability and enzymatic activity of the MTHFR enzyme.
[0005] Currently, real-time fluorescence PCR or PCR-melting curve method is mostly used for the detection of MTHFR gene polymorphism genotype. However, there are three SNP sites within three bases before and after the rs1801133 site: rs150847674, rs776956662, and rs1057519362. In particular, there is only one base between rs150847674 and rs1801133. Neither the fluorescence probe PCR method nor the PCR-melting curve method can avoid the interference of the rs150847674 site polymorphism on the detection of rs1801133. Summary of the Invention
[0006] To address the aforementioned problems in the prior art, the present invention provides a probe for identifying the genotype of the MTHFR polymorphic site rs1801133, the probe comprising: a probe for identifying the wild type (SEQ ID NO:1): 5'-AGCCRATTTCATCATC-3'; and a probe for identifying the mutant type (SEQ ID NO:2): 5'-AGTCRATTTCATCATC-3'.
[0007] The present invention also provides a kit for identifying the genotype of the MTHFR polymorphic site rs1801133, the kit comprising the probe for identifying the genotype of the MTHFR polymorphic site rs1801133.
[0008] The present invention also provides the probe for identifying the rs1801133 genotype of the MTHFR polymorphism site, and the use of the kit in the preparation of a formulation for detecting / assisting in the detection of the rs1801133 genotype of the MTHFR polymorphism site.
[0009] The present invention also provides a method for identifying the genotype of the MTHFR polymorphic site rs1801133, the method comprising using the probe for identifying the genotype of the MTHFR polymorphic site rs1801133, or the kit for identifying the genotype of the MTHFR polymorphic site rs1801133.
[0010] The probe provided by this invention for identifying the genotype of the MTHFR polymorphism site rs1801133 can effectively eliminate the interference of adjacent sites on the detection of the rs1801133 site, especially the interference of the rs150847674 site on the detection of the rs1801133 site. It has high detection accuracy, is convenient and quick to operate, and has low cost, making it suitable for large-scale promotion and use. Attached Figure Description
[0011] Figure 1 The results are the PCR amplification detection results of the four positive controls in Example 1.
[0012] Figure 2 The results are the PCR amplification detection results of sample Y99 in Example 1.
[0013] Figure 3 This is the first-generation sequencing result of sample Y99 in Example 1.
[0014] Figure 4 The results are the PCR amplification detection results of sample Y99 in Comparative Example 1.
[0015] Figure 5The image shows the melting curve of the PCR-melting curve method in Comparative Example 2.
[0016] Figure 6 This is the amplification curve of the fluorescent probe PCR method in Comparative Example 3.
[0017] Figure 7 The results are the PCR amplification detection results of sample Y772 in Example 2.
[0018] Figure 8 This is the first-generation sequencing result of sample Y772 in Example 2.
[0019] Figure 9 The results are the PCR amplification detection results of sample Y773 in Example 2.
[0020] Figure 10 This is the first-generation sequencing result of sample Y773 in Example 2.
[0021] Figure 11 The results are the PCR amplification detection results of sample Y774 in Example 2.
[0022] Figure 12 This is the first-generation sequencing result of sample Y774 in Example 2.
[0023] Figure 13 The results are the PCR amplification detection results of sample Y366 in Example 2.
[0024] Figure 14 This is the first-generation sequencing result of sample Y366 in Example 2. Detailed Implementation
[0025] To make the technical solution, objectives, and advantages of the present invention clearer, the present invention will be further described in detail below through specific embodiments. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.
[0026] The first aspect of this invention provides a probe for identifying the genotype of the MTHFR polymorphic site rs1801133, the probe comprising:
[0027] Probe used for identifying wild type (SEQ ID NO:1): 5'-AGCCRATTTCATCATC-3';
[0028] The probe used to identify the mutant (SEQ ID NO:2): 5'-AGTCRATTTCATCATC-3'.
[0029] In the above probe sequence composition, R represents purine (A or G), Y represents pyrimidine (C or T), M represents A or C, K represents G or T, and H represents A, C, or T. Unless otherwise specified, the letters R, Y, M, K, and H used in nucleic acid sequences in this invention have the above meanings.
[0030] The probe (SEQ ID NO:1) for identifying the wild type may have a fluorescent label at its 5' end, which may be any one of FAM, VIC, ROX, or CY5 fluorescent labels; the 3' end may also have a fluorescent label, for example, MGB fluorescent labels. The probe (SEQ ID NO:2) for identifying the mutant type may have a fluorescent label at its 5' end, which may be any one of FAM, VIC, ROX, or CY5 fluorescent labels, and the fluorescent label may be different from the fluorescent label at the 5' end of the probe for identifying the wild type; the 3' end may also have a fluorescent label, for example, MGB labels.
[0031] In one specific embodiment, the probe for identifying the wild type (SEQ ID NO:1) has a FAM fluorescent label at its 5' end and an MGB label at its 3' end; the probe for identifying the mutant type (SEQ ID NO:2) has a VIC fluorescent label at its 5' end and an MGB label at its 3' end.
[0032] A second aspect of the present invention provides a kit for identifying the genotype of the MTHFR polymorphic site rs1801133, the kit comprising the probe for identifying the genotype of the MTHFR polymorphic site rs1801133 provided in the first aspect of the present invention.
[0033] According to a second aspect of the invention, the kit may further include a primer set for amplifying a fragment containing the MTHFR gene, the primer set comprising:
[0034] MTHFR upstream primer (SEQ ID NO:3): 5'-AAGGCCACCCCGAAGCAG-3';
[0035] MTHFR downstream primer (SEQ ID NO:4): 5'-CCTTCACAAAGCGGAAGAATG-3'.
[0036] According to a second aspect of the invention, the kit may further include DNA polymerase, dNTPs, and Mg. 2+ At least one of PCR Bufer.
[0037] According to a second aspect of the present invention, the kit may further include at least one positive control:
[0038] Positive control 1: pcDNA3.1-MTHFR rs1801133 C / rs150847674 G (pcDNA3.1 backbone sequence omitted) (SEQ ID NO:8):
[0039]
[0040] Positive control 2: pcDNA3.1-MTHFR rs1801133 T / rs150847674 G (pcDNA3.1 backbone sequence omitted) (SEQ ID NO:9):
[0041]
[0042] Positive control 3: pcDNA3.1-MTHFR rs1801133 C / rs150847674 A (pcDNA3.1 backbone sequence omitted) (SEQ ID NO:10):
[0043]
[0044] Positive control 4: pcDNA3.1-MTHFR rs1801133 T / rs150847674 A (pcDNA3.1 backbone sequence omitted) (SEQ ID NO:11):
[0045]
[0046] In a third aspect, the present invention provides a probe for identifying the genotype of the MTHFR polymorphic site rs1801133 provided in the first aspect of the present invention, or a kit for identifying the genotype of the MTHFR polymorphic site rs1801133 provided in the second aspect of the present invention, and its use in the preparation of a formulation for detecting / assisting in the detection of the genotype of the MTHFR polymorphic site rs1801133.
[0047] In a fourth aspect, the present invention provides a method for identifying the genotype of the MTHFR polymorphic site rs1801133, the method comprising using a probe for identifying the genotype of the MTHFR polymorphic site rs1801133 provided in the first aspect of the present invention, or a kit for identifying the genotype of the MTHFR polymorphic site rs1801133 provided in the second aspect of the present invention.
[0048] Unless otherwise specified, all reagents and materials used in the following examples are products that can be obtained from commercial channels; unless otherwise specified, all testing and detection methods used in the following examples are conventional testing and detection methods in the field and can be obtained from textbooks, reference books or academic journals.
[0049] Example 1
[0050] This embodiment illustrates the method for detecting the rs1801133 genotype of the MTHFR polymorphic site using the probe provided by this invention for identifying the rs1801133 genotype of the MTHFR polymorphic site.
[0051] Genomic DNA extraction:
[0052] One EDTA-anticoagulated whole blood sample (sample Y99) carrying the rs1801133 CT / rs150847674 GA genotype was selected. Genomic DNA was extracted using an NP968-C fully automated nucleic acid extractor (purchased from Xi'an Tianlong Technology Co., Ltd.) and its accompanying reagent kit (catalog number: T146) according to the instructions. Extraction quality was assessed using a NanoDrop micro-spectrophotometer. Highly extracted DNA OD was required. 260 / OD 280 The value should be between 1.7 and 2.0, and the concentration should be between 10 and 100 ng / μL. If the concentration exceeds 100 ng / μL, it should be diluted with sterile deionized water to 10 to 100 ng / μL. If the concentration is below 10 ng / μL, the sample needs to be extracted again.
[0053] The PCR reaction system (25 μL) is as follows:
[0054] The reaction system consisted of 12.5 μL of premix, 1.0 μL of MTHFR upstream primer (10 μM), 1.0 μL of MTHFR downstream primer (10 μM), 0.5 μL of probe for identifying wild type (10 μM), 0.5 μL of probe for identifying mutant type (10 μM), 1.0 μL of ACTB upstream primer (10 μM), 1.0 μL of ACTB downstream primer (10 μM), 0.5 μL of ACTB probe (10 μM), 5.0 μL of sterile deionized water, and 2 μL of template DNA.
[0055] The probe used to identify the wild type (SEQ ID NO:1) has a FAM fluorescent label at its 5' end and an MGB label at its 3' end; the probe used to identify the mutant type (SEQ ID NO:2) has a VIC fluorescent label at its 5' end and an MGB label at its 3' end.
[0056] The reaction system premix was a 2× concentration premix of SGExcel GoldStarTaqMan Mixture (product number B532932) for real-time quantitative PCR, manufactured by Sangon Biotech, containing GoldStarTaq DNA Polymerase, PCR Buffer, dNTPs, and Mg. 2+ .
[0057] ACTB is an internal reference gene, in which:
[0058] The ACTB upstream primer (SEQ ID NO:5) has the following sequence: 5'-CCTGACGGCCAGGTCATC-3';
[0059] The ACTB downstream primer (SEQ ID NO:6) has the following sequence: 5'-CAGTCTCCACTCACCCAGGAA-3';
[0060] The ACTB fluorescent probe (SEQ ID NO:7) has the following sequence composition: 5'-TGGCAATGAGCGGTTC-3';
[0061] The ACTB fluorescent probe is ROX fluorescently labeled at the 5' end and MGB labeled at the 3' end.
[0062] The template DNA mentioned above is the genomic DNA extracted above. The blank control is deionized water. The positive controls are the positive control 1 (SEQ ID NO:8), positive control 2 (SEQ ID NO:9), positive control 3 (SEQ ID NO:10), and positive control 4 (SEQ ID NO:11) mentioned above.
[0063] Place the prepared PCR reaction system in an ABI 7500 Real-Time PCR instrument or a QuantStudio™ 5 Real-Time PCR instrument and perform the reaction according to the following program: 95℃ for 1 min; 95℃ for 15 s, 60℃ for 1 min (collect fluorescence signal), for a total of 40 cycles; 25℃ for 1 min.
[0064] rs1801133 C: Detection channel FAM, quench: MGB; rs1801133 T: Detection channel VIC, quench: MGB; ACTB (internal reference): Detection channel ROX, quench: MGB.
[0065] Result interpretation criteria:
[0066] Internal control requirements: Blank control wells must show no amplification curve, and all four positive control wells must show amplification curves for their respective corresponding fluorescence channels; otherwise, the detection is invalid. The sample internal control gene ACTB must show a significant S-shaped amplification curve and a Ct value < 38; otherwise, the detection is invalid. Genotype is determined based on the amplification curve or Ct value morphology of the corresponding channel. A typical S-shaped amplification curve and a Ct value < 38 are interpreted as positive; no amplification curve or a Ct value ≥ 38 is interpreted as negative. The result interpretation criteria are shown in Table 1.
[0067] Table 1. Criteria for interpreting PCR results
[0068]
[0069] The PCR amplification results of the four positive controls are as follows: Figure 1 As shown. By Figure 1 As can be seen, in positive controls 1 and 3, the FAM fluorescence channel (blue curve 677C, representing rs1801133 site C) showed an S-shaped amplification curve, while the VIC fluorescence channel (red curve 677T, representing rs1801133 site T) showed no amplification curve. In positive controls 2 and 4, the FAM fluorescence channel (blue curve 677C, representing rs1801133 site C) showed no amplification curve, while the VIC fluorescence channel (red curve 677T, representing rs1801133 site T) showed an S-shaped amplification curve. This indicates that all four positive controls correctly detected the genotype at the rs1801133 site, demonstrating the reliability of the operation and results of this embodiment. Since the positive controls were plasmids, the internal control ROX fluorescence channel (gray curve INT) showed no amplification curve.
[0070] The PCR amplification results of sample Y99 are as follows: Figure 2 As shown. By Figure 2As can be seen, the FAM fluorescence channel (blue curve 677C, representing rs1801133 site C), the VIC fluorescence channel (red curve 677T, representing rs1801133 site T), and the internal control ROX fluorescence channel (gray curve INT) all showed S-shaped amplification curves, indicating that the amplification process and detection results were effective, and it can be determined that the rs1801133 site of the above-mentioned sample Y99 is of the CT type.
[0071] The Y99 sample was subjected to first-generation sequencing, and the results are as follows: Figure 3 As shown, the rs1801133 locus is of the CT type ( Figure 3 The blue arrow in the image indicates that the rs150847674 locus is of the GA type. Figure 3 (As shown by the red arrow in the image), this is consistent with the detection results of the rs1801133 locus genotype obtained by the method of the present invention, indicating that the method provided by the present invention can be used to detect the rs1801133 locus genotype and is not affected by the rs150847674 locus.
[0072] Comparative Example 1
[0073] This comparative example is used to illustrate the necessity of setting the degenerate base R in the probe provided by the present invention.
[0074] Except for changing the degenerate base R to G in the probe used to identify the wild type (SEQ ID NO:1) and changing the degenerate base R to G in the probe used to identify the mutant type (SEQ ID NO:2), all other operations are the same as in Example 1.
[0075] The PCR amplification results of sample Y99 are as follows: Figure 4 As shown. By Figure 4 As can be seen, both the VIC fluorescence channel (pink curve 677T, representing the rs1801133 site T) and the internal control ROX fluorescence channel (gray curve INT) showed S-type amplification curves, while the FAM fluorescence channel (blue curve 677C, representing the rs1801133 site C) did not show an S-type amplification curve. This indicates that the amplification process and detection results were effective, and it can be interpreted that the rs1801133 site of the above-mentioned Y99 sample is of the TT type. This is obviously inconsistent with the results of the first-generation sequencing (CT type), and is a misjudgment as the TT type.
[0076] Comparative Example 2
[0077] This comparative example is used to illustrate the genotype of the MTHFR polymorphic site rs1801133 genotype identified by the PCR-melting curve method.
[0078] For sample Y99 in Example 1, genomic DNA was extracted from sample Y99 according to the method in Example 1. Then, the Human MTHFR (C677T) Gene Polymorphism Detection Kit (PCR-Melting Curve Method) (Catalog No.: P130; Registration Certificate No.: 20183400118) produced by Wuxi Ruichi Gene Biotechnology Co., Ltd. was used and the operation was carried out according to its instructions.
[0079] The PCR reaction system (25 μL) is as follows:
[0080] PCR reaction solution I (containing 20-100pM MTHFR gene and internal control gene-specific primers and probes): 8.0 μL; PCR reaction solution II (containing 0.1 U / μL Taq enzyme, 0.8 mM dNTP, 8 mM MgCl2, etc.): 5.0 μL; solution A (PCR enhancer): 5.0 μL; template DNA 2.0 μL.
[0081] PCR reaction solution I, PCR reaction solution II, and solution A are all components of the aforementioned kit from Wuxi Ruichi Gene Biotechnology Co., Ltd. The template used for the positive control reaction is the positive control in the kit (containing plasmids of the MTHFR gene 677T genotype and genomic DNA of the 677C genotype), and the template used for the blank control reaction is the blank control (purified water) provided in the kit.
[0082] Place the PCR tubes into the ABI 7500 Real-Time PCR instrument in sequence and perform amplification according to the PCR reaction program shown in Table 2:
[0083] Table 2 PCR reaction procedure
[0084]
[0085] Result interpretation: A positive result is defined as Tm value meeting the requirements of Table 3 below and having a peak height greater than 1 / 5 of the peak height of the positive control.
[0086] Table 3. Range of positive melting temperatures (Tm value)
[0087]
[0088] The FAM channel of the reaction tube is an MTHFR (677C>T) detection channel. If two peaks appear simultaneously and the Tm values are 55.12±1.5℃ and 64.97±1.5℃ respectively, the sample is C / T heterozygous; if only one melting peak appears and the Tm value is 55.12±1.5℃, the sample is T / T homozygous; if only one melting peak appears and the Tm value is 64.97±1.5℃, the sample is C / C homozygous.
[0089] Melting curve as shown Figure 5As shown, the positive control has two melting peaks after melting curve analysis, and the Tm value should conform to the Tm value range shown in the table above.
[0090] Figure 5 The results shown are from the FAM channel. The blue line represents the melting curve of the positive control provided with the kit, which has two melting peaks. The Tm value should conform to the Tm value range shown in the table above, and this is a normal melting curve for the rs1801133CT heterozygous peak. The red line represents the melting curve of sample Y99, which shows a single peak slightly shifted to the right compared to the melting peak of the rs1801133 TT genotype. This can lead to misinterpretation or failure to interpret the result as the rs1801133 TT genotype. Therefore, the PCR-melting curve method cannot eliminate the interference of the rs150847674 polymorphism on the detection of rs1801133 (C>T).
[0091] Comparative Example 3
[0092] This comparative example illustrates the use of fluorescent probe PCR to identify the genotype of the MTHFR polymorphic site rs1801133.
[0093] For sample Y99 in Example 1, genomic DNA was extracted from sample Y99 according to the method in Example 1. Then, the Human MTHFR Gene Detection Kit (PCR-Fluorescent Probe Method) produced by Wuhan Youzhiyou Medical Technology Co., Ltd. was used, and the operation was performed according to its instructions. The operation steps are as follows:
[0094] 1. Reagent preparation and sample addition
[0095] Thaw the reagents at room temperature. Add 23 μL of MTHFR 677 reaction solution (containing PCR buffer, dNTPs, specific primers and probes, internal control primers and probes, Taq enzyme, and UNG enzyme) to the PCR reaction tube. Add 2 μL of the genomic DNA extracted from sample Y99 to the PCR reaction tube. The blank control (Tris-HCl buffer (10 mM)) and the positive control (a mixture of plasmids of MTHFR 677C and MTHFR 677T) are provided with the kit. Add 2 μL of each to their respective reaction tubes.
[0096] 2. Amplification
[0097] The amplification was performed using an ABI 7500 amplification instrument according to the following program: 37℃ for 10 minutes; 95℃ for 5 minutes; cycling phase: 95℃ for 15 seconds, 60℃ for 60 seconds (fluorescence signal acquisition during this phase), for a total of 40 cycles. Fluorescence channel settings: MTHFR 677C was set to the FAM channel for fluorescence signal acquisition, MTHFR 677T was set to the VIC channel for fluorescence signal acquisition, and the internal reference gene was set to the ROX channel for fluorescence signal acquisition.
[0098] 3. Result Interpretation
[0099] The blank controls ROX, FAM, and VIC showed no amplification curves, while the positive controls FAM, VIC, and ROX had Ct values ≤32 and amplification curves showing a clear exponential growth phase. All samples' internal reference gene (ROX) had Ct values <38 and amplification curves showing a clear exponential growth phase. The interpretation criteria are shown in Table 4.
[0100] Table 4. PCR Result Interpretation Criteria
[0101]
[0102] Amplification curves as follows Figure 6 As shown, the VIC fluorescence channel (red curve Y-677T, representing rs1801133 site T) and the internal control ROX fluorescence channel (gray curve Y-INT) both show S-type amplification curves, while the FAM fluorescence channel (blue curve Y-677C, representing rs1801133 site C) does not show an S-type amplification curve. Therefore, sample Y99, which is of type rs1801133 CT, was misread as type TT.
[0103] Example 2
[0104] This embodiment illustrates the method for detecting the rs1801133 genotype of the MTHFR polymorphic site using the probe provided by this invention for identifying the rs1801133 genotype of the MTHFR polymorphic site.
[0105] Genomic DNA was extracted from samples Y772, Y773, Y774, and Y366 according to the method in Example 1, and the samples were tested according to the procedure in Example 1.
[0106] The PCR amplification results of sample Y772 are as follows: Figure 7 As shown, the VIC fluorescence channel (red curve 677T, representing the rs1801133 locus T) and the internal control ROX fluorescence channel (gray curve INT) both exhibit S-shaped amplification curves, while the FAM fluorescence channel (blue curve 677C, representing the rs1801133 locus C) does not show an S-shaped amplification curve. This indicates that the amplification process and detection results are effective, and it can be determined that the rs1801133 locus in sample Y772 is of the TT type. The results of first-generation sequencing of sample Y772 are as follows... Figure 8 As shown, the rs1801133 locus is of the TT type ( Figure 8 The blue arrow in the image indicates that the rs150847674 locus is of the GG type. Figure 8(As shown by the red arrow in the image), this is consistent with the detection results of the rs1801133 locus genotype obtained by the method of the present invention, indicating that the method provided by the present invention can be used to detect the rs1801133 locus genotype, and can normally detect the rs1801133 genotype when rs150847674 is GG wild type, without being interfered with by the rs150847674 locus.
[0107] The PCR amplification results of sample Y773 are as follows: Figure 9 As shown, the FAM fluorescence channel (blue curve 677C, representing rs1801133 site C), VIC fluorescence channel (red curve 677T, representing rs1801133 site T), and internal control ROX fluorescence channel (gray curve INT) all exhibit S-shaped amplification curves, indicating that the amplification process and detection results are effective, and it can be determined that the rs1801133 site in the above-mentioned sample Y773 is of the CT type. The above-mentioned sample Y773 was subjected to first-generation sequencing, and the results are as follows... Figure 10 As shown, the rs1801133 locus is of the CT type ( Figure 10 The blue arrow in the image indicates that the rs150847674 locus is of the GG type. Figure 10 (As shown by the red arrow in the image), this is consistent with the genotype detection results of the rs1801133 locus obtained by the method of the present invention, indicating that the method provided by the present invention can be used to detect the genotype of the rs1801133 locus, and can normally detect the rs1801133 genotype when rs150847674 is the wild type of GG, without being interfered with by the rs150847674 locus.
[0108] The PCR amplification results of sample Y774 are as follows: Figure 11 As shown, the FAM fluorescence channel (blue curve 677C, representing rs1801133 site C) and the internal control ROX fluorescence channel (gray curve INT) both exhibit S-type amplification curves, while the VIC fluorescence channel (red curve 677T, representing rs1801133 site T) does not show an S-type amplification curve. This indicates that the amplification process and detection results are effective, and it can be determined that the rs1801133 site in sample Y774 is of the CC type. The results of first-generation sequencing of sample Y774 are as follows... Figure 12 As shown, the rs1801133 locus is of the CC type ( Figure 12 The blue arrow in the image indicates that the rs150847674 locus is of the GG type. Figure 12(As shown by the red arrow in the image), this is consistent with the detection results of the rs1801133 locus genotype obtained by the method of the present invention, indicating that the method provided by the present invention can be used to detect the rs1801133 locus genotype, and can normally detect the rs1801133 genotype when rs150847674 is GG wild type, without being interfered with by the rs150847674 locus.
[0109] The PCR amplification results of specimen Y366 are as follows: Figure 13 As shown, the FAM fluorescence channel (blue curve 677C, representing rs1801133 site C), VIC fluorescence channel (red curve 677T, representing rs1801133 site T), and internal control ROX fluorescence channel (gray curve INT) all exhibit S-shaped amplification curves, indicating that the amplification process and detection results are effective, and it can be determined that the rs1801133 site in sample Y366 is of the CT type. The results of first-generation sequencing of sample Y366 are as follows... Figure 14 As shown, the rs1801133 locus is of the CT type ( Figure 14 The red arrow in the image indicates that the rs150847674 locus is of the GA type. Figure 14 (As shown by the purple-red arrow in the image), this is consistent with the detection results of the rs1801133 locus genotype obtained by the method of the present invention, indicating that the method provided by the present invention can be used to detect the rs1801133 locus genotype and is not affected by the rs150847674 locus.
[0110] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A probe for identifying the genotype of the MTHFR polymorphic site rs1801133, characterized in that, The probe includes: Probe used to identify the wild type (SEQ ID NO:1): 5'-AGCCRATTTCATCATC-3'; Probe used to identify mutants (SEQ ID NO:2): 5'-AGTCRATTTCATCATC-3'.
2. The probe according to claim 1, characterized in that: The probe (SEQ ID NO:1) used for identifying wild type has a fluorescent label at its 5' end; the fluorescent label is any one of FAM fluorescent label, VIC fluorescent label, ROX fluorescent label, and CY5 fluorescent label; The probe (SEQ ID NO:1) used for identifying the wild type has a fluorescent label at its 3' end; the fluorescent label is an MGB fluorescent label.
3. The probe according to claim 2, characterized in that: The probe (SEQ ID NO:2) used for identifying mutants has a fluorescent label at its 5' end; the fluorescent label is any one of FAM fluorescent label, VIC fluorescent label, ROX fluorescent label, and CY5 fluorescent label; The fluorescent label at the 5' end of the probe used to identify the mutant (SEQ ID NO:2) is different from the fluorescent label at the 5' end of the probe used to identify the wild type (SEQ ID NO:1); The probe (SEQ ID NO:2) used to identify mutants has a fluorescent label at its 3' end; the fluorescent label is an MGB fluorescent label.
4. The probe according to claim 1, characterized in that: The probe (SEQ ID NO:1) used for identifying wild type has a FAM fluorescent label at its 5' end and an MGB label at its 3' end; The probe (SEQ ID NO:2) used to identify mutants has a VIC fluorescent label at its 5' end and an MGB label at its 3' end.
5. A kit for identifying the genotype of the MTHFR polymorphic site rs1801133, the kit comprising the probe for identifying the genotype of the MTHFR polymorphic site rs1801133 as described in any one of claims 1-4.
6. The reagent kit according to claim 5, characterized in that: The kit also includes a primer set for amplifying the MTHFR gene fragment, the primer set comprising: MTHFR upstream primer (SEQ ID NO:3): 5'-AAGGCCACCCCGAAGCAG-3'; MTHFR downstream primer (SEQ ID NO:4): 5'-CCTTCACAAAGCGGAAGAATG-3'.
7. The kit according to claim 5 or 6, characterized in that: The kit also includes DNA polymerase, dNTPs, and Mg. 2+ At least one of PCR Bufer.
8. The reagent kit according to claim 7, characterized in that: The kit also includes at least one of the following positive controls: Positive control 1: Sequence composition as shown in SEQ ID NO:8; Positive control 2: Sequence composition as shown in SEQ ID NO:9; Positive control 3: Sequence composition as shown in SEQ ID NO:10; Positive control 4: The sequence composition is shown in SEQ ID NO:
11.
9. The probe for identifying the genotype of the MTHFR polymorphic site rs1801133 according to any one of claims 1-4, or the kit for identifying the genotype of the MTHFR polymorphic site rs1801133 according to any one of claims 5-8, used in the preparation of a formulation for detecting / assisting in the detection of the genotype of the MTHFR polymorphic site rs1801133.
10. A method for identifying the genotype of the MTHFR polymorphic site rs1801133, the method comprising using the probe for identifying the genotype of the MTHFR polymorphic site rs1801133 as described in any one of claims 1-4, or the kit for identifying the genotype of the MTHFR polymorphic site rs1801133 as described in any one of claims 5-8.