Human MTHFR and MTRR gene detection method and kit
By using the Type IIS restriction endonuclease FokI to design primers, the MTHFR and MTRR gene loci were PCR amplified and then digested, combined with resin desalting purification and mass spectrometry detection, the problem of complex operation of the nucleic acid mass spectrometry platform was solved, and efficient and low-cost gene detection was achieved.
Patent Information
- Application Number
- CN202510635403.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-16
- Publication Date
- 2025-09-26
AI Technical Summary
The genetic testing process of existing nucleic acid mass spectrometry platforms is complex, the detection cycle is long, it is susceptible to aerosol contamination, and the cost is high.
Primers were designed using the Type IIS restriction endonuclease FokI. PCR amplification of the MTHFR and MTRR gene loci was followed by direct enzyme digestion. Mass spectrometry analysis was performed directly by combining resin desalting purification with mass spectrometry detection, avoiding dephosphorylation and single-base extension steps.
It simplifies the detection process, reduces aerosol pollution, shortens detection time, reduces costs, and improves detection efficiency and accuracy.
Smart Images
Figure CN120700129A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of gene detection, and in particular to a method and a kit for detecting human MTHFR and MTRR genes. Background Art
[0002] Folic acid is a water-soluble B vitamin composed of pteridine, para-aminobenzoic acid and glutamic acid residues. The mutation frequencies of three key enzymes in the metabolism of folic acid in the body, namely methylenetetrahydrofolate reductase (MTHFR) C677T, A1298C and methionine bioreductase (MTRR) A66G, vary among individuals, and each person has different abilities to utilize folic acid.
[0003] The MTHFR C677T mutation occurs in 45.2% of people in China and manifests as three genotypes: CC, CT, and TT. The C677T mutation converts the encoded amino acid alanine to valine, leading to decreased heat tolerance and enzyme activity. The "Expert Consensus on the Diagnosis and Treatment of Type H Hypertension" clearly states that individuals with the TT genotype have a 60% decrease in MTHFR activity. The A1298C polymorphism, in which adenine (A) is replaced by cytosine (C) at position 1298, results in a mutation of glutamate to alanine, resulting in three genotypes: AA, AC, and CC. These mutations reduce enzyme activity less than the C677T mutation. Studies have shown that the C677T mutations (CT and TT) and the A1298C mutations (AC and CC) are not only associated with decreased MTHFR enzyme activity but can also cause folate metabolism disorders, significantly reducing plasma folate levels and inducing hyperhomocysteinemia.
[0004] MTRR is a flavoprotein-related enzyme that maintains the activity of methionine synthase and plays a crucial regulatory role in maintaining the methionine cycle. The A66G polymorphism in the gene encoding this enzyme leads to reduced methionine synthase activity and accumulation of homocysteine in the body. Elevated homocysteine concentrations can cause a range of vascular endothelial damage, including fetal malformations and miscarriage in early pregnancy and pregnancy complications such as premature birth, pregnancy-induced hypertension, and gestational diabetes mellitus in the second and third trimesters. Different patients may have different MTHFR and MTRR genotypes, and their responses to medications vary. Therefore, distinguishing these genotypes is crucial to predict an individual's ability to metabolize folate, thereby assisting in the development of personalized treatment plans for patients requiring exogenous folate supplementation.
[0005] The nucleic acid mass spectrometry analysis system integrates multiplex PCR, chip technology, and time-of-flight mass spectrometry to achieve high-throughput, high-sensitivity, and high-precision nucleic acid sequence analysis. This technology platform is currently widely used in genotyping, methylation analysis, microbial detection and typing, and copy number variation detection.
[0006] While nucleic acid mass spectrometry platforms offer significant advantages in application, they often have complex experimental procedures and longer detection cycles compared to fluorescent quantitative PCR platforms. These typically involve three thermal cycler (PCR) steps: 1) multiplex PCR reaction, 2) dephosphorylation reaction, and 3) single-base extension reaction. Each step requires opening the instrument lid, significantly increasing the potential for aerosol contamination. Furthermore, the three-step reaction process increases reaction and detection time. Therefore, a new method is needed to address the complex operational procedures of nucleic acid mass spectrometry and thereby shorten the detection cycle.
[0007] In view of this, the present invention is proposed. Summary of the Invention
[0008] In response to the above technical problems, the present invention provides a method and kit for detecting human MTHFR and MTRR genes.
[0009] In order to achieve the above object, the technical solution of the present invention is as follows:
[0010] A method for detecting human MTHFR and MTRR genes comprises: designing a primer pair containing a Type IIS restriction endonuclease recognition sequence for the MTHFR gene sites C677T and A1298C, and for the MTRR gene site A66G, performing a PCR amplification reaction, enzymatically digesting the amplified product with the Type IIS restriction endonuclease, and desalting and purifying the obtained product with a resin before mass spectrometry detection.
[0011] Wherein, both the forward primer and the reverse primer of the primer pair contain Type IIS restriction endonuclease recognition sequences.
[0012] Wherein, the Type IIS restriction endonuclease is FokI endonuclease.
[0013] Before the mass spectrometry detection is performed, the enzyme cleavage product is purified. Purification of the enzyme cleavage product can reduce interference with the detection result and improve the resolution.
[0014] Wherein, the purification treatment includes resin desalting purification.
[0015] The mass spectrometry detection includes performing molecular weight detection on the purified product using a matrix-assisted laser desorption / ionization time-of-flight mass spectrometry system, determining the genotype of the sample based on the difference in molecular weight, and determining whether a drug-resistant mutation occurs at a certain site in the sample to be tested.
[0016] Wherein, the primer pair consists of primers whose sequences are shown as SEQ ID NO.1 to SEQ ID NO.6.
[0017] The present invention also provides a human MTHFR and MTRR gene detection kit, which comprises the primer pair according to claim 7.
[0018] The kit also contains a PCR premix, which includes a hot start Taq enzyme, dNTPs and Mg 2+ One or more of .
[0019] The kit also contains Type IIS restriction endonuclease: FokI endonuclease.
[0020] The beneficial effects of the present invention are:
[0021] 1. Conventional nucleic acid mass spectrometry protocols require multiple openings during the detection process, which can easily lead to aerosol contamination and other phenomena. The present invention uses direct enzyme digestion after PCR, a nearly one-step method for detecting mutations at three sites in the human MTHFR and MTRR genes. After the PCR reaction, enzyme digestion is performed using Type IIS restriction enzymes (such as Fok I) to generate target fragments with a nucleotide sequence range of 15-30 nt. This is then directly analyzed on a mass spectrometer, avoiding the dephosphorylation and single-base extension steps required by traditional nucleic acid mass spectrometry, reducing the number of openings, and minimizing aerosol contamination and other problems. This also shortens detection time and improves detection efficiency.
[0022] 2. The present invention employs a strategy of using Type IIS restriction endonucleases to shorten the detection process, reducing the possibility of aerosol contamination and shortening the detection time. A typical characteristic of Type IIS restriction endonucleases is that their cleavage sequence is different from their recognition sequence, and the cleavage sequence is often located far from the recognition sequence.
[0023] 3. The present invention uses Fok I endonuclease as the reaction enzyme. This enzyme recognizes the sequences GGATG (5' end of the top strand) and CCTAC (3' end of the bottom strand). The cleavage sequences are located after the ninth base following the recognition sequence at the 5' end of the top strand and the 13th base following the recognition sequence at the 3' end of the bottom strand, respectively. If the Fok I endonuclease recognition sequence is introduced at both ends of the primer sequence, the cleavage sites at both ends are closer to the middle region, easily generating small fragments of 15-30 bp.
[0024] 4. Compared with similar technologies for existing human MTHFR and MTRR gene detection, the technical solution of the present invention avoids aerosol contamination caused by repeated opening of the lid during the detection process and has the advantage of short detection time, that is, there is no dephosphorylation and single-base extension steps.
[0025] 5. The PCR process of the present invention does not require high-fidelity enzymes or SAP enzymes, and eliminates the single-base extension step, thereby reducing the detection cost by about 30%.
[0026] 6. The entire process of the present invention only requires a common PCR reaction procedure, which is simple and easy to operate; and the nucleic acid fragments are directly detected without the need for isotope or fluorescence signal amplification, so it is sensitive and accurate.
[0027] 7. The specific fragment amplification design technology of the present invention can completely eliminate the false positive problems caused by PCR product contamination and homologous sequence probe mismatch, providing a reliable experimental method for human MTHFR and MTRR gene detection.
[0028] 8. The present method can simultaneously detect the MTHFR gene C677T site, the A1298C site, and the MTRR gene A66G site in the same test well. For each mutation site, the present method can generate two DNA fragments with different molecular weights, which can verify the accuracy of the test and reduce false positive results. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 This is the mass spectrum of the MTHFR gene C677T mutation;
[0030] Figure 2 This is the mass spectrum of the A1298C mutation of the MTHFR gene;
[0031] Figure 3 This is the mass spectrum of the MTRR gene A66G site mutation;
[0032] Figure 4 It is a mutation diagram of multiple systems. DETAILED DESCRIPTION
[0033] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that these descriptions are merely exemplary and are not intended to limit the scope of the present invention. In addition, in the following description, descriptions of well-known structures and technologies are omitted to avoid unnecessary confusion of the concepts of the present invention.
[0034] A method for detecting human MTHFR and MTRR genes, comprising:
[0035] PCR reaction: Design primer pairs containing Type IIS restriction endonuclease recognition sequences for the MTHFR gene sites C677T and A1298C, and the MTRR gene site A66G, and perform PCR amplification reactions to obtain target sequence amplification products in the sample to be tested;
[0036] Endonuclease digestion reaction: The amplified product is digested with the Type IIS restriction endonuclease to cut the target fragment into three segments;
[0037] Resin desalting: The obtained product is purified by resin desalting;
[0038] Mass spectrometry detection: The purified product is subjected to molecular weight detection using a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MASS) system. The molecular weight marker is used to determine whether the sample to be tested has mutated, and the typing category can be determined. The software automatically processes and reports the typing results.
[0039] The amplification procedure was 95°C for 10 min, followed by PCR cycle amplification for a total of 45 cycles. The reaction conditions were denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 1 minute; the final extension was at 72°C for 5 min, and the temperature was kept constant at 4°C after completion.
[0040] Wherein, both the forward primer and the reverse primer of the primer pair contain Type IIS restriction endonuclease recognition sequences.
[0041] Wherein, the Type IIS restriction endonuclease is FokI endonuclease.
[0042] Before the mass spectrometry detection is performed, the enzyme cleavage product is purified. Purification of the enzyme cleavage product can reduce interference with the detection result and improve the resolution.
[0043] Wherein, the purification treatment includes resin desalting purification.
[0044] The mass spectrometry detection includes performing molecular weight detection on the purified product using a matrix-assisted laser desorption / ionization time-of-flight mass spectrometry system, determining the genotype of the sample based on the difference in molecular weight, and determining whether a drug-resistant mutation occurs at a certain site in the sample to be tested.
[0045] Wherein, the primer pair consists of primers whose sequences are shown as SEQ ID NO.1 to SEQ ID NO.6.
[0046] The present invention also provides a human MTHFR and MTRR gene detection kit, which comprises the primer pair according to claim 7.
[0047] The kit also contains a PCR premix, which includes a hot start Taq enzyme, dNTPs and Mg 2+ One or more of .
[0048] The kit also contains Type IIS restriction endonuclease: FokI endonuclease.
[0049] Example 1
[0050] Design of primer sets for MTHFR and MTRR gene detection
[0051] Two genes closely related to folate metabolism are MTHFR and MTRR. Three polymorphic sites within these two genes directly influence folate metabolism: sites 677 and 1298 of MTHFR, and site 66 of MTRR. MTHFR site 677 has three genotypes: CC / CT / TT; MTHFR site 1298 has three genotypes: AA / AC / CC; and MTRR site 66 has three genotypes: AA / AG / GG.
[0052] Primer design and screening
[0053] Specific site primers were designed for the C677T and A1298C sites of MTHFR and the A66G site of MTRR, and a Fok I restriction endonuclease recognition site was designed.
[0054] The Fok I recognition sequences are GGATG (5' end) and CCTAC (3' end), and the cleavage sequences are located 9 bases after the 5' recognition sequence and 13 bases after the 3' recognition sequence, respectively. Fok I restriction enzyme cleavage site recognition sequences are incorporated into both upstream and downstream primers to ensure that the target site is within 15-30 bp after cleavage.
[0055] The primer sequences designed by the present invention are as follows:
[0056] C677T forward primer (SEQ ID NO. 1): 5'-GCACTGGATGGAGAAGGTGTCTGCGGGA-3'
[0057] C677T reverse primer (SEQ ID NO. 2): 5'-AGCCTGGATGAAAAGCTGCGTGATGATGAA-3'
[0058] A1298C forward primer (SEQ ID NO. 3): 5'-AGATGGGATGGGAGGAGCTGACCAGTGAA-3'
[0059] A1298 reverse primer (SEQ ID NO. 4): 5'-GAGAGGGGATGAACGAAGACTTCAAAGACA-3'
[0060] A66G forward primer (SEQ ID NO. 5): 5'-GCAGGGGGATGCAAAGGCCATCGCAGAAGAA-3'
[0061] A66G reverse primer (SEQ ID NO. 6): 5'-CTGCAGGGATGCCATGTACCACAGCTTGCTC-3'
[0062] Example 2
[0063] MTHFR and MTRR Gene Detection Kit
[0064] The components of the MTHFR and MTRR gene detection kit include the amplification primer mixture described in Example 1, as well as Multiplex PCR Mix, 10x Buffer, Fok I enzyme, RNase Free Water, positive control substances, and negative control substances.
[0065] Reaction reagents: Multiplex PCR Mix, 10x Buffer, Fok I enzyme, and RNase Free Water are all commercial reagents, and the reaction system was added according to the instructions.
[0066] Positive control: a plasmid mixture containing all loci of the three detectable folate genes; negative control: a human wild-type sample.
[0067] Example 3
[0068] The MTHFR and MTRR gene detection kit prepared in Example 2 was used to detect the sample to be tested.
[0069] 1. DNA sample extraction: Select a blood sample with known results and follow the instructions.
[0070] 2. PCR amplification: Use PCR primers to amplify the sample DNA to obtain PCR products.
[0071] 3. PCR reaction system configuration is shown in Table 1:
[0072] Table 1. PCR reaction system
[0073] Reagents Volume 1×(μL) PCR Primer Mix 1 Multiplex PCR Mix 2 DNA Template 1 RNase-Free Water 1 Total 55
[0074] 4. The PCR reaction procedure is as follows:
[0075]
[0076] 5. Restriction enzyme digestion reaction
[0077] Try to use FokI restriction endonuclease to digest the PCR product in step 2. The enzyme digestion reaction system configuration is shown in Table 2:
[0078] Table 2. Enzyme digestion reaction system
[0079] Reagents Volume 1×(μL) 10xBuffer 2.5 Fok I enzyme 1 PCR products 2.5 RNase-Free Water 19 Total 25
[0080] 6. Enzyme digestion reaction conditions: 37°C, 60 min, then constant temperature at 4°C.
[0081] 7. Resin purification
[0082] 8. Add 16 μL of water and 9 mg of resin to each reaction well and shake for 15 minutes to facilitate the full binding of cations in the reaction system and achieve desalting and purification. Then centrifuge at 4000 rpm for 5 minutes and use directly for mass spectrometry analysis.
[0083] 9. Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry
[0084] 10. The product after enzyme digestion is tested for molecular weight using matrix-assisted laser desorption ionization time-of-flight mass spectrometry system. The molecular weight marker is used to determine whether a mutation occurs at a certain site in the sample to be tested. The test results are as follows: Figures 1-4 shown.
[0085] Example 4
[0086] Interpretation of MTHFR and MTRR gene test kit results
[0087] The molecular weight distribution of each fragment after FokI digestion (T represents TOP and B represents Bottom) is shown in Table 3:
[0088] Table 3. Molecular weight of enzyme-digested fragments
[0089]
[0090] The specific instructions are as follows:
[0091] 1. The middle region is used to distinguish the molecular weight of the target area, including 12 middle region molecular weights.
[0092] 2. The T chain and B chain have different molecular weights, but detect the same target site, and can confirm the accuracy of the test results.
[0093] 3. During the nucleic acid mass spectrometry detection process, the laser excitation separates the double strands, so the double strands do not affect the detection results.
[0094] 4. According to the above instructions Figures 1 to 4 Interpret the results.
[0095] It should be understood that the above-described specific embodiments of the present invention are merely illustrative or illustrative of the principles of the present invention and do not constitute limitations of the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included within the scope of protection of the present invention. In addition, the appended claims are intended to cover all variations and modifications that fall within the scope and metes and bounds of the appended claims, or equivalents thereof.
Claims
1. A method for detecting human MTHFR and MTRR genes, characterized in that: include: Primer pairs containing Type IIS restriction endonuclease recognition sequences were designed for the MTHFR gene sites C677T and A1298C and the MTRR gene site A66G. PCR amplification reactions were performed, and the amplified products were digested with the Type IIS restriction endonucleases. The obtained products were purified by resin desalting and then detected by mass spectrometry.
2. A method for detecting human MTHFR and MTRR genes according to claim 1, characterized in that: The forward primer and the reverse primer of the primer pair both contain Type IIS restriction endonuclease recognition sequences.
3. A method for detecting human MTHFR and MTRR genes according to claim 1, characterized in that: The Type IIS restriction endonuclease is FokI endonuclease.
4. A method for detecting human MTHFR and MTRR genes according to claim 1, characterized in that: Before the mass spectrometry detection, the enzyme cleavage product is purified.
5. A method for detecting human MTHFR and MTRR genes according to claim 4, characterized in that: The purification process includes resin desalting purification.
6. A method for detecting human MTHFR and MTRR genes according to claim 1, characterized in that: The mass spectrometry detection includes performing molecular weight detection on the purified product using a matrix-assisted laser desorption / ionization time-of-flight mass spectrometry system.
7. A method for detecting human MTHFR and MTRR genes according to claim 1, characterized in that: The primer pair consists of primers whose sequences are shown as SEQ ID NO.1 to SEQ ID NO.
6.
8. A human MTHFR and MTRR gene detection kit, characterized in that: The kit comprises the primer pair according to claim 7.
9. A human MTHFR and MTRR gene detection kit according to claim 8, characterized in that: The kit also contains a PCR premix, which includes a hot start Taq enzyme, dNTPs, and Mg. 2+ One or more of .
10. A human MTHFR and MTRR gene detection kit according to claim 9, characterized in that: The kit also contains the Type IIS restriction endonuclease: FokI endonuclease.