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Improved sample processing method for detecting polymorphism of C677T site of MTHFR gene

A technology of locus polymorphism and processing method, which is applied in the field of genetic engineering, can solve problems such as the background of the detection line, the influence of the result judgment, and the long detection time, so as to improve the yield and quality of DNA, improve the effect, and improve the specificity Effect

Pending Publication Date: 2020-12-04
JILIN JINYU MEDICAL SCI INSPECTION CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the current kit sample processing methods are manual operation, the detection time is long, and the final judgment result is partly chromatographic method, but the detection line often has background, which affects the judgment of the result and leads to the phenomenon of misjudgment of the result

Method used

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  • Improved sample processing method for detecting polymorphism of C677T site of MTHFR gene
  • Improved sample processing method for detecting polymorphism of C677T site of MTHFR gene
  • Improved sample processing method for detecting polymorphism of C677T site of MTHFR gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] In this embodiment, a sample processing method for polymorphism detection of C677T site of MTHFR gene comprises the following steps:

[0043] (1) Collect 100ul of the whole blood sample of the subject to be tested, then add 20ul of proteinase K and 200ul of lysate, vortex and mix, and place in a metal bath in a dry bath at 70°C for 5 minutes;

[0044](2) Add 300 ul of binding solution and 25 ul of magnetic particles capable of adsorbing DNA to the product obtained in step (1), vortex and mix, then directly put it on the magnetic device for separation for 5 minutes, and discard the supernatant;

[0045] (3) Wash the magnetic particles obtained in step (2): first add 700ul cleaning solution I, vortex and mix, put it directly on the magnetic device for separation for 5min, discard the supernatant; then add 500ul cleaning solution II, vortex Mix well, put it directly on the magnetic device for separation for 5 minutes, and discard the supernatant; place the sample in a cent...

Embodiment 2

[0054] A method for detecting the polymorphism of the C677T site of the MTHFR gene in this embodiment comprises the following steps:

[0055] S1. Sample processing: use the sample processing method described in Example 1 to process the sample to be tested to obtain the corresponding PCR amplification product, that is, the processed sample;

[0056] S2. Detect the processed sample obtained in step S1: take out the test card from the sealed bag, drop the PCR products in the mutant PCR amplification tube and the wild-type PCR amplification tube of the sample to be tested respectively on the corresponding test card. Wait for 2-5 minutes to interpret the results, and the results are unreliable after 20 minutes.

[0057] In this embodiment, the polymorphism of the MTHFR gene C677T site of the same sample to be detected as the detection method in this embodiment is detected by using the existing detection method, and the existing detection method includes the following steps:

[005...

Embodiment 3

[0070] The sample processing method of the present invention optimizes the conditions of sample lysis and PCR amplification at the same time, thereby effectively improving the quality of the detection results, avoiding the background of the detection line, shortening the detection time, and improving the detection efficiency.

[0071] This example mainly studies the influence of the incubation conditions during sample lysis, the amount of DNA used during PCR amplification, and the number of PCR amplification cycles on the detection effect. Whole blood samples from 30 cases of MTHFR gene C677T polymorphism. The incubation conditions during sample lysis, the amount of PCR amplified DNA, and the number of PCR amplification cycles in the sample processing methods of each group are shown in Table 2, and the other methods are the same as the sample processing methods described in Example 1.

[0072] Table 2 Processing methods of samples in each group

[0073]

[0074] The 30 cas...

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Abstract

The present invention provides a sample processing method for detecting polymorphism of a C677T site of an MTHFR gene. The sample processing method comprises the following steps: (1) adding a lysis solution into a sample to be detected and conducting incubating at 68-75 DEG C for 4-6 min; (2) adding magnetic particles into a product obtained in the step (1), conducting uniform mixing and magneticseparation, and discarding a supernatant; (3) washing the magnetic particles obtained in the step (2) and discarding the supernatant after the magnetic separation; (4) adding an eluent into the magnetic particles obtained in the step (3), conducting uniform mixing, then conducting incubating for 4-6 min at 70-75 DEG C, and collecting a supernatant after the magnetic separation; and (5) conductingPCR amplification on the supernatant obtained in the step (4) to obtain a treated sample. After optimization of sample lysing, PCR amplification and other varous parameters, the processing method caneffectively improve quality of detection results, avoid background of a detection line, meanwhile, shorten detection time and improve detection efficiency.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an improved sample processing method for polymorphism detection of C677T site of MTHFR gene. Background technique [0002] Methylenetetrahydrofolate reductase (5,10-methylenetetrahydrofolate reductase, MTHFR) is a key enzyme in folic acid metabolism. The MTHFR gene is 20.374 kb in length, with 12 exons in total, encoding a protein consisting of 656 amino acid residues. The function is to convert 5,10-methylenetetrahydrofolate into 5-methyltetrahydrofolate, which is a methyl donor for the remethylation of homocysteine ​​(HCY) to methionine . The C677T site polymorphism of MTHFR gene is 677CC type (wild type), 677CT type (heterozygous mutant type) and 677TT (homozygous mutant type). The mutation of the C677T site makes the enzyme intolerable to heat and reduces its activity. Compared with CC individuals, the enzyme activity of CT individuals is only 65%, w...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6886C12Q1/6806C12Q1/6858
CPCC12Q1/6883C12Q1/6886C12Q1/6806C12Q1/6858C12Q2600/156C12Q2527/101C12Q2523/308C12Q2531/113
Inventor 宋丽影蒙裕欢周婷婷李雨艳王双阁范喜杰
Owner JILIN JINYU MEDICAL SCI INSPECTION CO LTD
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