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Composition, kit and method for detecting human folate metabolism gene polymorphism

A technology of folic acid metabolism and composition, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of failure to detect SLC19A1, limitation of testing samples on the machine, inability to realize multiple tests in one tube, etc. problems, to achieve the effect of convenient PCR amplification operation, high accuracy and simple method

Pending Publication Date: 2020-05-22
SANSURE BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it uses multiple PCR-LDR technology combined with capillary electrophoresis technology of genetic analyzer for detection. This method requires professionals to carry out, and the detection time is long and the cost is high.
Therefore, in response to this problem, fluorescent quantitative PCR is used for detection in the prior art. Chinese publication CN108034710A discloses a method for detecting MTHFR C677T sites, MTHFR A1298C sites, and MTRR A66G sites based on fluorescent quantitative PCR, but one channel Only one type of SNP can be detected, and it can only achieve one test for one tube, not multiple tests for one tube, which limits the number of samples tested on the machine, and it also fails to detect the SLC19A1 A80G locus

Method used

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  • Composition, kit and method for detecting human folate metabolism gene polymorphism
  • Composition, kit and method for detecting human folate metabolism gene polymorphism
  • Composition, kit and method for detecting human folate metabolism gene polymorphism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1. Primers and probes used in the present invention

[0073] The upstream and downstream primers used for target polynucleotide amplification and probes used for target polynucleotide detection are derived from MTHFR gene rs1801133 (C677T) and rs1801131 sites (A1298C), MTRR gene rs1801394 site (A66G) and SLC19A1 The 150bp before and after the gene rs1051266 (A80G) was used as the target region, respectively.

[0074] The sequence of the MTHFR gene C677T primer probe is as follows:

[0075]MTHFR-677-F: 5'-CTGACCTGAAGCACTTGAAGGAG-3' (SEQ ID NO: 1);

[0076] MTHFR-677-R: 5'-CGGTGCATGCCTTCACAAA-3' (SEQ ID NO: 2);

[0077] MTHFR-677C-P: 5'-ATGAAATCGGCTCCC-3' (SEQ ID NO: 3);

[0078] MTHFR-677T-P: 5'-TGGAGTCGATTTCA-3' (SEQ ID NO: 4);

[0079] Determine the MTRR gene primer A66G probe sequence as follows:

[0080] MTRR-66-F: 5'-GTTGAAGTGATGAGGAGGTTTCTGTT-3' (SEQ ID NO: 5);

[0081] MTRR-66-R: 5'-GATCTGCAGAACATCCATGTACCAC-3' (SEQ ID NO: 6);

[0082] MTRR-66A-P: 5...

Embodiment 2

[0093] Embodiment 2, the method that detects folic acid metabolism ability

[0094] 1. Reagent preparation:

[0095] According to the number of samples to be tested, negative controls, and positive controls, take corresponding amounts of PCR reaction solution and enzyme mixture in proportion (42-46 μL / person of reaction solution + 2-3 μL / person of enzyme mixture), and mix thoroughly. into PCR-mix, and centrifuged briefly for later use.

[0096] 2. Sample processing

[0097] (1) Sample collection

[0098] a. Fingertip blood collection: first disinfect the inside of the ring finger with an alcohol cotton ball, then puncture with a disposable blood collection needle to allow the blood to flow out naturally, and collect the blood into an anticoagulation tube, with a total volume of about 0.2 to 0.3 ml. After blood collection, press the puncture site with a dry cotton ball for 2 to 3 minutes;

[0099] b. Oral swab: rinse the mouth with water twice before sampling to remove fore...

Embodiment 3

[0122] Example 3. Comparative test between detection results and second-generation sequencing results

[0123] According to the experimental scheme provided by the present invention, 8 peripheral blood samples with unknown detection results were compared with the Sanger sequencing results, and the results were as follows:

[0124]

[0125]

[0126] Conclusion: The present invention can detect various genotypes of peripheral blood samples well. The detection results of human MTHFR gene, MTRR gene and SLC19A1 gene polymorphism nucleic acid detection kit are consistent with the MTHFR gene rs1801133 (C677T) and rs1801131 loci ( A1298C), MTRR gene rs1801394 locus (A66G) and SLC19A1 gene rs1051266 locus (A80G) gene polymorphism detection Sanger sequencing results were completely consistent.

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Abstract

The invention relates to the field of detection of molecular biology, in particular to the field of detection of folate metabolism capability, provides composition for detecting the folate metabolismcapability and also provides an application of the composition for detecting the folate metabolism capability, a kit containing the composition and a method for detecting the folate metabolism capability. With the adoption of the composition and the method, two SNP types can be detected through one channel, multiple detection through one tube is realized, time and cost are saved, and samples for computer detection can be increased.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, and more specifically, to the field of detection of human folic acid metabolism gene polymorphism. Background technique [0002] Folic acid is a water-soluble B vitamin, and the active structure produced by its metabolism, tetrahydrofolate, is the most important factor affecting the metabolism of homocysteine ​​in the body. It has been recognized all over the world that folic acid supplementation during perinatal period can prevent the initial and recurrence of fetal neural tube defects (NTDs). Since folic acid is closely related to DNA synthesis, if women take insufficient folic acid during peri-pregnancy, it will lead to obstacles in the conversion of homocysteine ​​(HCY) to methionine, resulting in hyper-HCY hyperemia or hypomethionine hyperemia disease. Hyper-HCY hyperemia can cause vascular endothelial injury, promote the proliferation of vascular smooth muscle cells, and cause d...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6851C12N15/11
CPCC12Q1/6883C12Q1/6851C12Q2600/156C12Q2600/106C12Q2531/113C12Q2563/107
Inventor 龙凤英吴康缪为民戴立忠
Owner SANSURE BIOTECH INC
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