Kit and detection method of folic acid metabolic capability and genotyping as well as application of kit

A technology for folic acid metabolism and genotyping, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of increasing the workload of inspection operators, increasing the cost and time of inspection, and being unfavorable for large-scale promotion and use. Achieve the effect of optimizing the design method, reducing the inspection cost and reducing the inspection cost

Pending Publication Date: 2018-07-06
PRO MED BEIJING TECH
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0007] However, in the existing technology, the fluorescent PCR method is used to detect the genotyping of folic acid metabolism ability, and it is necessary to extract blood genomic DNA as a template for corresponding detection. cost and time
In addition, the existing fluorescent PCR method to detect the genotype of folic acid metabolism capacity is mostly to set up a detection tube for each locus, such as detecting the MTHFR gene 677C>T mutation, MTHFR gene 1298A>C mutation and MTRR gene 66A>C mutation in a sample. There are three sites for G mutation, and three detection tubes need to be set up for corresponding detection. The operation is time-consuming and laborious, which is not conducive to large-scale clinical promotion and use.

Method used

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  • Kit and detection method of folic acid metabolic capability and genotyping as well as application of kit
  • Kit and detection method of folic acid metabolic capability and genotyping as well as application of kit
  • Kit and detection method of folic acid metabolic capability and genotyping as well as application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Preparation of Folate Metabolism Ability Genotyping Detection Kit

[0051] PCR reagent: final concentration 0.1M Tris-HCl buffer, 10mmol / L MgCl 2 , 0.6mg / mL of BSA, 1% of gelatin, 0.5% of Triton X-100 and 0.5mmol / L of KCl mixture, stored in 1mL / branch. The mixture of 5U Taq DNA polymerase and 2.5mmol / L dNTPs is stored in 0.3mL / cartridge. ddH2O, stored in 1mL / cartridge aliquots.

[0052] Specific primers and specific mutation detection probes: the nucleotide sequence is SEQ ID NO.1-9, used for PCR reaction, and packed in 0.25mL / branch.

[0053] Negative quality control, sterile purified water.

[0054] Wild-type quality control products, artificially synthesized wild-type DNA at position 677 of the MTHFR gene, wild-type DNA at position 1298 of the MTHFR gene, and wild-type DNA at position 66 of the MTRR gene, used at a final concentration of 0.2mM, divided into 0.1mL / branch Pack storage.

[0055] Mutant quality control products, artificially synthesized MT...

Embodiment 2

[0057] Example 2 Genotyping Detection of Folic Acid Metabolism Ability

[0058] The method comprises the steps of:

[0059] 1) Take 2.5 μL specific primers and specific mutation detection probes, 10 μL RT-PCR reaction buffer, 2.5 μL RT-PCR reaction enzyme and 8 μL ddH 2 O, mix and oscillate evenly on ice; and set up a routine RT-PCR reaction buffer control group;

[0060] 2) Take 2 μL of whole blood sample, negative quality control, wild-type quality control or heterozygous mutant quality control, and add them to the reaction tube in step 1), in which the wild-type quality control and homozygous mutant quality control Mix equal amounts of the products into heterozygous mutant quality control products; and extract the blood genome DNA template from the experimental group samples for control experiments;

[0061] 3) Set PCR reaction conditions: 95°C for 5 minutes; 95°C for 30 seconds, 55°C for 35 seconds, 42 cycles; 72°C for 30 seconds;

[0062] 4) Put the reaction tube into ...

Embodiment 3

[0064] Example 3 Interpretation of detection results

[0065] Such as figure 1 As shown, the negative quality control product has no amplification and no Ct value, indicating that there is no contamination in the experimental operation; the wild-type and heterozygous quality control products have amplification, and the Ct value is <36, indicating that the experimental results are credible.

[0066] Such as figure 2 As shown, in the conventional RT-PCR reaction buffer control group, whole blood samples did not significantly amplify, indicating that there was no de-inhibition effect on whole blood inhibitors.

[0067] Depend on image 3 It can be seen that the Ct values ​​of the whole blood sample group and the genomic DNA template control group have a good correlation, and R 2 =0.9775; the significance analysis results showed that P<0.05, the difference was not significant, indicating that the genotyping results of direct PCR detection of folic acid metabolism in whole bloo...

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Abstract

The invention relates to a folic acid metabolic capability and genotyping detection technology and in particular relates to a kit and a detection method of a folic acid metabolic capability and genotyping as well as application of the kit. The kit comprises specific primers aiming at 677C>T mutation and 1298A>C mutation of an MTHFR (Methylene Tetrahydrofolate Reductase) gene, and 66A>G mutation ofan MTRR (Methionine Synthase Reductase) gene, and a specific mutation detection probe, RT-PCR (Reverse Transcription-Polymerase Chain Reaction) buffer, an RT-PCR reaction Taq enzyme, sterile purifiedwater, a negative quality control product, a wild type quality control product, a mutant quality control product and a packaging box for separating and packaging a reagent bottle or pipe. The detection method of the folic acid metabolic capability and the genotyping, provided by the invention, has the advantages of strong specificity, high sensitivity, small pollution, simplicity and rapidness inoperation, high safety performance and the like; a detection result has relatively good accuracy and repeatability; the detection method is especially suitable for directly taking whole blood as a detection sample and related gene mutation detection is directly carried out; a genome DNA (Deoxyribonucleic Acid) template does not need to be extracted. The folic acid metabolic capability can be accurately judged and individualized folic acid oral administration and supplementing dosages are provided; the kit and the detection method have important value.

Description

technical field [0001] The invention relates to a folic acid metabolic ability genotyping detection technology, in particular to a kit, a detection method and an application thereof for the folic acid metabolic ability genotyping. Background technique [0002] Folic acid is a water-soluble vitamin found in spinach, also known as vitamin B9. Subsequently, scientists discovered that folic acid is contained in various animal and plant foods, and this type of folic acid is called natural folic acid. The human body can obtain folic acid supplements through the consumption of food, especially green leafy vegetables. In addition, you can also supplement folic acid by taking synthetic folic acid tablets, which has a higher bioavailability, which is about twice that of natural folic acid. Folic acid plays an important role in the division and growth of cells and the synthesis of nucleic acids, amino acids, and proteins, and is also an indispensable nutrient for fetal growth and dev...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2527/125C12Q2531/113C12Q2563/107
Inventor 陈勇田茹田媛李西兰余占江陈永强
Owner PRO MED BEIJING TECH
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