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CN-121992087-A - LAMP primer probe composition for detecting folic acid MTHFR gene polymorphism, kit and application thereof

CN121992087ACN 121992087 ACN121992087 ACN 121992087ACN-121992087-A

Abstract

The application belongs to the technical field of molecular biology and in-vitro diagnosis, and particularly discloses an LAMP primer probe composition for detecting folic acid MTHFR gene polymorphism, a kit and application thereof. The application constructs a probe-mediated LAMP melting curve analysis system, realizes high-efficiency nucleic acid amplification under the constant temperature condition by utilizing LAMP reaction, and combines the high-specificity distinguishing characteristics of a double-standard probe melting curve to realize quick and accurate typing of a C677T mutation site. Compared with the traditional method, the method has the advantages of short reaction time, high sensitivity, strong specificity, no need of complex instruments and subsequent operation and the like, and is suitable for clinical rapid parting detection and basic molecular diagnosis application.

Inventors

  • ZHANG ZHANG
  • YAO JUAN
  • Chang Chuohao
  • LI LIAN

Assignees

  • 西南医科大学附属中医医院

Dates

Publication Date
20260508
Application Date
20260409

Claims (10)

  1. 1. The LAMP primer probe composition for detecting the polymorphism of the folic acid MTHFR gene is characterized by comprising an outer primer pair, an inner primer pair and a fluorescent probe; The outer primer pair comprises a forward outer primer F3: CCCTCACCTGGATGGGAA with a nucleotide sequence shown as SEQ ID NO.1 and a reverse outer primer B3: TCTTCATCCCTCGCCTTGA with a nucleotide sequence shown as SEQ ID NO. 2; The inner primer pair comprises a forward inner primer FIP with a nucleotide sequence shown as SEQ ID NO.3, AGGCTGACACATTCTTCCGCTTT-CATGTCGGTGCATGCCT and a reverse inner primer BIP with a nucleotide sequence shown as SEQ ID NO.4, TGCTTCAGGTCAGCCTCAAAGC-TACCCCAAAGGCCACCC; The fluorescent probe comprises a nucleotide ATGAAATCGACTCCCGCAG with a sequence shown as SEQ ID NO. 5.
  2. 2. The LAMP primer probe composition of claim 1, characterized in that: The 5 'end of the fluorescent probe is marked with a fluorescent group, the fluorescent group is selected from ROX, FAM, HEX, CY or CY5, the 3' end of the fluorescent probe is marked with a quenching group, and the quenching group is selected from BHQ series.
  3. 3. The LAMP primer probe composition according to claim 2, wherein said fluorescent probe is ROX-ATGAAATCGACTCCCGCAG-BHQ2.
  4. 4. The LAMP kit for detecting the polymorphism of the folic acid MTHFR gene is characterized by comprising the LAMP primer probe composition according to any one of claims 1-3.
  5. 5. The LAMP kit of claim 4, further comprising at least one of a reaction solution, a DNA polymerase, a fluorescent dye, water (preferably ultrapure water), and a negative control.
  6. 6. The LAMP kit of claim 5, wherein said DNA polymerase is at least one selected from Bst 2.0 polymerase and Bst 3.0 polymerase; and/or, the LAMP kit further comprises 2× LAMP MASTER Mix.
  7. 7. The LAMP kit of any one of claims 4 to 6, wherein in the LAMP kit, the molar ratio of the outer primer pair, the inner primer pair and the fluorescent probe is 1:4 to 8:1.
  8. 8. The use of the LAMP primer probe composition as claimed in any one of claims 1 to 3 or the LAMP kit as claimed in any one of claims 4 to 7 in the preparation of a reagent for detecting a folic acid MTHFR gene polymorphism.
  9. 9. The use according to claim 8, characterized in that: Detecting the folic acid MTHFR gene polymorphism by using a loop-mediated isothermal amplification melting curve method, wherein the loop-mediated isothermal amplification melting curve method comprises a loop-mediated isothermal amplification reaction and a melting curve analysis; the temperature of the loop-mediated isothermal amplification reaction is 55-65 ℃, and the amplification time is equal to or longer than 30 minutes; and the melting curve analysis is performed at 50-70 ℃.
  10. 10. The use according to claim 1, characterized in that: And after the loop-mediated isothermal amplification reaction is finished, performing melting curve analysis, and parting the MTHFR gene according to the melting curve: When only one characteristic peak appears in the melting curve and the Tm peak is 55+/-0.3 ℃, the target MTHFR gene is of CC type; when the melting curve peak only shows one characteristic peak and the Tm peak is at 61+/-0.3 ℃, the target MTHFR gene is TT type; When two characteristic peaks appear at the same time in the melting curve peak and the two Tm peaks are respectively 55+ -0.3 and 61+ -0.3 ℃, the target MTHFR gene is CT heterozygous.

Description

LAMP primer probe composition for detecting folic acid MTHFR gene polymorphism, kit and application thereof Technical Field The application relates to the technical fields of molecular biology and in-vitro diagnosis, in particular to an LAMP primer probe composition for detecting folic acid MTHFR gene polymorphism, a kit and application thereof. Background Folic acid, one of the most important water-soluble vitamins, plays a vital role in the human body by participating in synthesis and metabolism of biomolecules such as DNA synthesis, methylation reaction, amino acid metabolism, and the like. Folate deficiency may lead to pregnancy neural tube defects, megaloblastic anemia, cardiovascular disease and other chronic dysfunctions. Generally, a person may ingest sufficient folic acid through a normal daily diet. However, for those carrying a variant of the folate metabolism gene, additional supplementation with folic acid is required to prevent folate-related diseases. Therefore, folate metabolism-related genotyping is of great importance for the identification, prevention and treatment of folate deficiency. Methylene tetrahydrofolate reductase (MTHFR) is two key enzymes involved in folate metabolism. The MTHFR gene exhibits a gene polymorphism, and the most studied gene mutation is C677T. Physiologically, MTHFR is responsible for the reduction of 5, 10-methylene tetrahydrofolate to 5-methyl tetrahydrofolate, providing a methyl donor for the re-methylation of homocysteine to methionine. When the C677T mutation results in substitution of alanine at position 222 of the enzyme with valine, the thermostability of the enzyme is reduced and the activity is significantly reduced, especially in TT homozygous individuals only about 30% of normal persons, thereby causing folate metabolism disorder and homocysteine accumulation. Therefore, the MTHFR C677T locus detection has important clinical significance in genetic risk assessment, disease prevention, pre-pregnancy screening and accurate nutrition intervention. Currently, methods for genotyping folate metabolism related genes mainly comprise techniques such as first generation Sanger sequencing, gene chip, PCR and the like. Despite the great progress of these techniques, there are still drawbacks of high cost, time and effort consumption, false positives, etc. Sequencing is used as the most accurate mutation detection method, but the method is complex in operation, time-consuming and labor-consuming, and PCR is used as a nucleic acid index amplification technology and has been widely applied to clinical molecular detection. PCR is typically combined with other techniques (e.g., PCR sequencing, PCR-RFLP, PCR-fluorescent probes) to effect detection of mutations. Among these techniques, PCR-fluorescent probes are the most commonly used SNPs analysis method in clinic. However, this method still has some disadvantages in that the PCR-fluorescent probe method relies on a stringent annealing temperature, and an excessively high annealing temperature leads to dissociation of the probe from the target and an excessively low annealing temperature leads to non-specific hybridization of the probe. Isothermal amplification techniques have the characteristics of rapid, efficient and constant temperature amplification and ease of field detection, and many scholars consider isothermal amplification techniques as an alternative to PCR. Among them, loop-mediated isothermal amplification (LAMP) shows high potential for alternative PCR. However, the polymerase used in LAMP lacks 5 'to 3' exonuclease activity and cannot use TaqMan hydrolysis probes like in PCR. In addition, the real-time LAMP method mainly relies on fluorescent dye or Cas system combination to generate signals, and risks of poor specificity, aerosol pollution and the like exist. Disclosure of Invention In view of the shortcomings of the prior art, the application aims to provide an LAMP primer probe composition for detecting folic acid MTHFR gene polymorphism, a kit and application thereof, and the LAMP isothermal amplification technology is combined with a double-standard probe-mediated melting curve to realize the parting of C677T under closed-tube reaction conditions, so that the problems of long detection period, high cost, complex operation, high false positive rate and the like in SNPs detection technology in the prior art are solved. To achieve the above and other related objects, the present application provides a LAMP primer probe composition for detecting a polymorphism of a folic acid MTHFR gene, the LAMP primer probe composition including an outer primer pair, an inner primer pair, and a fluorescent probe; The outer primer pair comprises a forward outer primer (F3) with a nucleotide sequence shown as SEQ ID NO.1, CCCTCACCTGGATGGGAA, and a reverse outer primer (B3) with a nucleotide sequence shown as SEQ ID NO.2, TCTTCATCCCTCGCCTTGA; the inner primer pair comprises a Forward Inner Primer (FIP) with a nucleotide