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Kit for simultaneously detecting human MTHFR and MTRR genes

A kit and technology for human detection, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of inability to realize automation and incomplete detection.

Pending Publication Date: 2020-11-03
AUTOBIO DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the mainstream manufacturers on the market, such as Wuhan Youzhiyou and Suzhou Kuangyuan, etc., use the fluorescence method, they are all MTHFR (C677T) single-point, the detection is not comprehensive, and it cannot be automated.

Method used

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  • Kit for simultaneously detecting human MTHFR and MTRR genes
  • Kit for simultaneously detecting human MTHFR and MTRR genes
  • Kit for simultaneously detecting human MTHFR and MTRR genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Embodiment 1: the composition of kit

[0081] The composition of the kit of the present invention is shown in Table 1:

[0082] Table 1

[0083]

[0084]

[0085]

Embodiment 2

[0086] Example 2: DNA detection

[0087] 1. Whole blood pretreatment: use the whole blood sample processing reagent independently developed by Zhengzhou Antu Bioengineering Co., Ltd. to process the whole blood sample, and the specific operation is carried out according to the instructions.

[0088] 2. Nucleic acid extraction: Use nucleic acid extraction or purification reagents independently developed by Zhengzhou Antu Bioengineering Co., Ltd., and follow the instructions for specific operations.

[0089] 3. Build a reaction system

[0090] Calculate the number of samples required, and increase the amount of reagents in proportion to the amount in the table below, and only make one copy of negative and positive quality controls.

[0091] Table 2

[0092]

[0093] 4. On-board testing

[0094] Directly use the fluorescent quantitative PCR instrument for detection, the amplification program is set to 50°C for 2min, 60°C for 15min, 95°C for 1min, (95°C for 5s,

[0095] 60°C...

Embodiment 3

[0100] Embodiment 3: the main performance index of this kit

[0101] 66, 677, and 1298 wild, mutant, and heterozygous whole blood clinical samples were screened by sequencing, purified using whole blood processing reagents and nucleic acid extraction and purification reagents independently developed by Zhengzhou Antu Bioengineering Co., Ltd., and diluted with normal saline to 1ng / μL, using this kit for detection, the results are shown in Tables 4-5, where "no Ct" means that there is still no amplification signal value after 45 cycles of amplification.

[0102] Table 4

[0103]

[0104]

[0105] table 5

[0106]

[0107]

[0108] Table 6

[0109]

[0110]

[0111] It can be seen from Tables 4 to 6 that the detection results of known samples show that the detection results of the kit provided by the present invention are completely consistent with the gold standard sequencing results, and the accuracy rate can reach 100%.

[0112] Table 7

[0113]

[011...

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Abstract

The invention relates to the technical field of gene detection, in particular to a kit for simultaneously detecting human MTHFR and MTRR genes. The primer provided by the invention has good specificity, and the kit provided by the invention can simultaneously obtain detection results of three sites of C677T, A1298C and A66G in primary detection, so that sample preparation is saved, manual operation and secondary repeated detection are avoided, and the detection efficiency is greatly improved. By adding PCR protective agents namely Tween20 and glycerol, the denaturation of bifunctional DNA polymerase is effectively prevented, the enzyme stability and activity are improved, and the long-term stable preservation of an amplification reagent at 2-8 DEG C is realized. By adding a reinforcing agent PEG6000, the melting point and melting temperature of DNA are functionally reduced, non-specific amplification is reduced, the amplification efficiency is improved, and the detection of a sample iscompleted within 50 minutes.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a kit for simultaneously detecting human MTHFR and MTRR genes. Background technique [0002] There are individual differences in the mutation frequency of the key enzymes methylenetetrahydrofolate reductase (MTHFR) C677T, A1298C and methionine synthesis reductase (MTRR) A66G in the process of folic acid metabolism in vivo, and each person's ability to utilize folic acid different. The mutation at the C677T site of MTHFR will change the alanine in the active region of MTHFR protease to valine, thereby causing changes in the enzyme activity, in which the enzyme activity of heterozygotes is reduced to 65%, and that of homozygotes is reduced to 30%; A1298C is MTHFR Another polymorphic site in the gene, the mutation that converts glutamic acid to alanine, will reduce the enzyme activity to 83% for heterozygotes and 61% for homozygotes. MTRR is a flavoprotein-related enzyme th...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6883C12Q1/6858C12Q2600/156C12Q2531/113C12Q2521/531C12Q2563/107C12Q2545/113
Inventor 李静静李新阳赵倩李振红付光宇
Owner AUTOBIO DIAGNOSTICS CO LTD
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