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Kit for simultaneously detecting human MTHFR and MTGG gene sequences

A gene sequence and kit technology, which is applied in the field of kits for detecting MTHFR and MTGG gene sequences, can solve the problems of high detection throughput, limited instruments, and expensive equipment in the chip hybridization method

Pending Publication Date: 2019-05-21
广东安科华南生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

5,10-methylenetetrahydrofolate reductase (MTHFR) and methionine synthetase (MTRR) are the key metabolic enzymes in the folate metabolism pathway, MTHFR single nucleotide polymorphisms can lead to changes in MTHFR enzyme activity, It leads to the body's folic acid metabolism disorder, which leads to the relative deficiency of folic acid in the body, that is, the intake of normal doses of folic acid still cannot meet the body's demand for folic acid, which in turn leads to a decrease in the level of folic acid in the body and hyperhomocysteinemia (Hcy)
However, the disadvantage of this method is that cross-reactions and false-positive reactions sometimes occur, tissue samples are not processed quickly enough, and degradation enzymes cannot be inactivated, and salt and pH sometimes affect the results, etc.
[0008] Although the fluorescent PCR melting curve method is easy to operate, it is limited by the instrument itself and is not suitable for the test of direct expansion samples, nor is it suitable for the detection of multiple sites in the same sample or the detection of large sample volumes.
In contrast, the chip hybridization method has high detection throughput, but the equipment required is expensive and difficult to popularize

Method used

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  • Kit for simultaneously detecting human MTHFR and MTGG gene sequences
  • Kit for simultaneously detecting human MTHFR and MTGG gene sequences
  • Kit for simultaneously detecting human MTHFR and MTGG gene sequences

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: A kit for simultaneously detecting human MTHFR and MTGG gene sequences.

[0029] Including a composite amplification system and a composite connection system, the composite amplification system includes: 10×PCR BufferMixture, 10 μM specific amplification primers, Template, Taq DNA Polymerase, sdH 2 O; the composite ligation system includes: 10×Ligase Buffer, probes, Ligase, label ligation reaction template, and the probes include shared probes, specific probes and label probes.

[0030] The amplification primer sequences are shown in Table 1:

[0031] Table 1 Specific amplification primer sequence list

[0032]

[0033] The common probe sequence is as described in Table 2:

[0034] Table 2 Common probe sequence list

[0035]

[0036]

[0037]The shared probe is composed of an external region (an exogenous sequence base-complementary to the external probe binding region of the label ligation reaction template, which has been marked in italics) a...

Embodiment 2

[0046] Example 2: The method of using the kit for simultaneous detection of human MTHFR and MTGG gene sequences.

[0047] 1. Extraction of sample DNA

[0048] According to the actual situation of the sample received, choose the appropriate method to extract the sample DNA. Common methods include: magnetic bead method, alkali lysis method, anion exchange resin method, etc. If the sample exists in the form of blood filter paper, FTA card, saliva card, etc., the steps can be simplified, and an appropriate amount of sample material can be directly amplified. However, it needs to be clear that whether it is extracting DNA samples or directly amplifying test materials, when using fragment compound amplification, it is necessary to ensure that the DNA content in each reaction reaches 2-4ng / μL.

[0049] 2. Fragment compound amplification

[0050] Design appropriate fragment amplification primers at 90 to 130 bp upstream and downstream of the SNP site to be tested, and test the speci...

Embodiment 3

[0067] Embodiment 3: the establishment of standard map.

[0068] Use the kit and method of implementation 1 to detect the 5 gene loci (C677, MTRR, A1298, G1793, T1317) of the known synthetic mutant samples, and design the typing according to the peak arrangement of each locus, For details, see figure 1 , the map of the detection results of the 5 gene loci is shown below Figure 2-6 .

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Abstract

The invention discloses a kit for simultaneously detecting human MTHFR and MTGG gene sequences. The kit comprises specific amplification primers and probes for identifying C677T, A1298C, G1793A and T1317C sites of MTHFR genes and A66G site mutations of MTRR genes. Compared with other existing detection products, the kit can accurately judge the genotype of samples, and it is confirmed that the samples are heterozygous mutations or homozygous mutations.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a kit for detecting MTHFR and MTGG gene sequences. Background technique [0002] Folic acid is widely distributed in food. After it is absorbed by the human intestine, it is reduced to tetrahydrofolate with physiological activity by the action of folate reductase. Tetrahydrofolate, as the carrier of one-carbon unit in the cell, participates in the synthesis of purine and pyrimidine into Methylation of DNA, proteins provides methyl groups. Folic acid metabolism disorder refers to the reduction of enzyme activity due to key gene mutations in the folic acid metabolic pathway, so that the folic acid absorbed by the body cannot normally perform physiological functions, and the transmission of one-carbon units is blocked, resulting in neural tube defects, miscarriage, hypertension, etc. Increased risk. In the process of folic acid metabolism, a variety of enzymes are involved in the tran...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6886C12N15/11
Inventor 黄源坚郑文彦王邦超杜蔚安郑阳阳周咏松
Owner 广东安科华南生物科技有限公司
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