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CN-122012735-A - Human MTHFR, MTRR and SLC19A1 gene detection kit, detection method and application thereof

CN122012735ACN 122012735 ACN122012735 ACN 122012735ACN-122012735-A

Abstract

The invention discloses a human MTHFR, MTRR and SLC19A1 gene detection kit. The kit is based on a high-resolution melting curve analysis technology and comprises four primer pairs aiming at MTHFR gene C677T and A1298C loci, MTRR gene A66G locus and SLC19A1 gene A80G locus. The kit can rapidly, sensitively and accurately identify the genotypes of the four key sites by detecting the fusion temperature and curve shape difference of the PCR products under the same reaction program. The kit is not only suitable for risk assessment of cardiovascular diseases and birth defects, but also more creatively applied to donor screening and receptor assessment of fecal fungus transplantation, fills up the blank of genetic background detection tools in the field, and has the advantages of low cost, high sensitivity, strong specificity, simple and convenient operation, strong repeatability, rapid and objective detection results and the like.

Inventors

  • ZHOU JIALIN
  • WANG DONGCHAO

Assignees

  • 上海偿道生物医药科技有限公司

Dates

Publication Date
20260512
Application Date
20260226

Claims (10)

  1. 1. The human MTHFR, MTRR and SLC19A1 gene detection kit is characterized by comprising four detection solutions of MTHFR 677/MTHFR 1298/MTRR 66/SLC19A 180, a nucleic acid amplification reaction solution, an enzyme mixed solution, a positive control standard substance and a blank control substance; Wherein, four detection solutions of MTHFR 677/MTHFR 1298/MTRR 66/SLC19A 180 comprise the following 4 groups of primer pairs: the 1 st primer pair is a forward primer and a reverse primer aiming at MTHFR 677 locus, and the nucleotide sequences of the forward primer and the reverse primer are respectively shown as SEQ ID No.1 and SEQ ID No. 2; The 2 nd primer pair is a forward primer and a reverse primer aiming at MTHFR 1298 locus, and the nucleotide sequences of the forward primer and the reverse primer are respectively shown as SEQ ID No.3 and SEQ ID No. 4; the 3 rd primer pair is a forward primer and a reverse primer aiming at MTRR 66 locus, and the nucleotide sequences of the forward primer and the reverse primer are respectively shown as SEQ ID No.5 and SEQ ID No. 6; The 4 th primer pair is a forward primer and a reverse primer aiming at SLC19A1 locus, and the nucleotide sequences of the forward primer and the reverse primer are respectively shown as SEQ ID No.7 and SEQ ID No. 8; the nucleic acid amplification reaction solution comprises dNTP, magnesium ions, buffer solution and saturated fluorescent dye.
  2. 2. The gene detection kit according to claim 1, wherein: The enzyme mixed solution is DNA polymerase; the saturated fluorescent dye is selected from one of SYBR Green, LC Green, eva Green or Reso Light.
  3. 3. The gene assaying kit according to claim 1, wherein the positive control standard comprises wild-type homozygous genomic DNA, mutant heterozygous genomic DNA and mutant homozygous genomic DNA for four sites.
  4. 4. A detection method for detecting genes of human MTHFR, MTRR and SLC19A1 by using the gene detection kit as set forth in any one of claims 1 to 3, comprising the steps of: Step1, extracting human genome DNA of a sample to be detected; Step 2, preparing a PCR reaction system, namely respectively mixing each detection solution with the nucleic acid amplification reaction solution, the enzyme mixed solution, the purified water and the sample to be detected obtained by extraction in the step 1; step 3, performing real-time fluorescence quantitative PCR amplification; Step 4, performing high-resolution melting curve analysis on the amplified product obtained in the step 3; And 5, data processing and result judging, namely determining genotypes of the sample to be detected at four sites by analyzing the peak shape and melting temperature change of the melting curve.
  5. 5. The method according to claim 4, wherein the PCR amplification in the step 3 comprises the steps of: Step 31, pre-denaturation, namely keeping at 95 ℃ for 2 to 10 minutes; Step 32, amplification cycle, 10 seconds at 95 ℃, 25 seconds at 60 ℃,10 seconds at 72 ℃, is performed for 35-50 cycles.
  6. 6. The method according to claim 4, wherein the high resolution melting curve analysis in step 4 comprises the steps of: Step 41, melting analysis, namely heating to 65 ℃ at the temperature of 95 ℃ for 1 minute at the temperature of 40 ℃ for 1 minute, heating to 95 ℃ at the speed of 0.01 ℃/s to 1 ℃/s, and continuously collecting fluorescent signals.
  7. 7. The method according to claim 4, wherein the result determination criteria in step 5 are: the wild-type homozygote exhibits a single melting peak, and the melting temperature value is consistent with that of a wild-type standard; mutant homozygotes exhibit a single melting peak and have a melting temperature value consistent with that of the mutant standard; the heterozygote is shown as a double melting peak or a characteristic change in shape of the melting curve, and the curve morphology is consistent with that of the heterozygote standard.
  8. 8. Use of a gene detection kit according to any one of claims 1 to 3 for genotyping human MTHFR, MTRR and SLC19A1 for non-disease diagnostic purposes.
  9. 9. Use of a gene detection kit according to any one of claims 1 to 3 in the manufacture of a product for screening fecal transplant donors or assessing the suitability of fecal transplant recipients.
  10. 10. The use according to claim 9, wherein the screening or evaluation comprises detecting the MTHFR, MTRR and SLC19A1 genotypes of the individual to be tested to evaluate the folate metabolism ability and homocysteine level risk of the individual, thereby guiding recipient pairing for fecal transplantation or nutritional intervention strategy after transplantation.

Description

Human MTHFR, MTRR and SLC19A1 gene detection kit, detection method and application thereof Technical Field The invention belongs to the technical field of biological medicine detection, in particular to a molecular diagnosis technology, and particularly relates to a kit for detecting polymorphism of human MTHFR, MTRR and SLC19A1 genes and application of the kit in high resolution melting curve (HRM) analysis, which is particularly suitable for genetic background screening related to fecal bacteria transplantation. Background Folic acid is a B-group vitamin essential to the human body and plays a central role in DNA synthesis, repair and methylation modification. Folate deficiency causes uracil misinsertion in human DNA, and chromosome breakage, resulting in increased genomic instability and mutation probability. Abnormalities in the folate metabolic pathway can lead to hyperhomocysteinemia, which in turn increases the risk of cardiovascular disease, neurological deficit, recurrent abortion, and various cancers. The human body cannot synthesize folic acid by itself, needs to be obtained from dietary or intestinal flora metabolites, and depends on a series of key enzymes and transport proteins for metabolism and utilization. In the folate metabolic pathway, the polymorphisms of the following three genes are particularly critical: (1) The MTHFR (5, 10-methylenetetrahydrofolatereductase) gene, which encodes a 5,10-methylenetetrahydrofolate reductase, is located at position 1p36.3 of chromosome I. MTHFR has a total length of 19.3kb, contains 12 exons, has a total length of 7,105bp of mRNA and encodes a protein consisting of 657 amino acid residues, and can catalyze the reduction of 5,10-MTHFR into 5-methyltetrahydrofolate as a methyl donor which participates in methylation of homocysteine to methionine. The MTHFR gene mutation mainly occurs at two sites of 677 and 1298, the mutation at the 677 site (C > T) can change the activity of the 5,10-methylene tetrahydrofolate reductase coded by the gene to different degrees, the 677TT can reduce the enzyme activity by about 70%, and the 677CT can reduce the activity by 35%. The 1298 locus of the MTHFR gene, the polymorphism of which results in replacement of isoleucine by methionine, resulting in a reduced rate of homocysteine re-methylation. According to the research of the related literature, the mutation at 1298 site and the mutation at 677 site can have a synergistic effect, so that the activity of the 5,10-methylene tetrahydrofolate reductase is further reduced. (2) The MTRR (5-methyltetrahydrofolate-homocysteinemethyltransferasereductase) gene, which codes for methionine synthase reductase, is located at chromosome 5p15.3-p15.2, the total length of MTRR gene is 32021kb, the total length of mRNA is 3274bp, 15 exons are all contained, and the protein with 726 amino acids is coded. MTRR-encoded methionine synthase reductase is capable of regenerating functionally active methionine synthase by reductive methylation. Mutations at position 66 (A > G) of the MTRR gene have an effect on the folate DNA methylation metabolic pathway. (3) SLC19A1 is a reduced folic acid carrier gene, and the fat-soluble carrier 19A1 on the coded cell membrane is related to the active transportation of various substances into cells, and the common polymorphic site of SLC19A1 is 80 (A > G), which can make the coded 27 th histidine replaced by arginine, so that the expressed protein function is changed. The only in vitro diagnostic kit for MTHFR and MTRR gene detection in the prior market is not the kit for MTHFR, MTRR and SLC19A1 gene detection, and most of the in vitro diagnostic kits adopt a probe method or directly adopt a direct sequencing method, a pyrosequencing method and the like. In the face of the current public demands and the popularization of large-scale sample detection, on one hand, a large amount of manpower and material resources are consumed, the price is high, the diagnosis time is prolonged, and the sequencing rule is complex in operation, time-consuming and low in sensitivity. Therefore, it is highly desirable to establish a method with good specificity and high sensitivity, which can be realized at low cost and can detect genotypes at four sites, so as to be better and faster for detecting genotypes at 4 sites of MTHFR, MTRR and SLC19A1 genes. Thus, the prior art has, in summary, the following disadvantages: (1) Most of the kits only cover MTHFR and MTRR, and neglect the importance of the folic acid transporter SLC19A 1. Studies have shown that evaluation of metabolic enzymes alone, but not transport vectors, does not fully reflect the folic acid availability of individuals. (2) The method has the limitations of complicated operation, long period and high cost of the sequencing method, and the TaqMan probe method has good specificity, but has high synthesis cost, is limited by the number of fluorescent channels and has high multiple detection difficulty. (3) The applicati