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Extraction-free direct-amplification rapid detection method for SNP typing of human methylenetetrahydrofolate reductase (MTHFR) gene via test strips

A detection method and technology of test strips, applied in the field of genetic detection, can solve the problems of being unsuitable for promotion and use in clinical hospitals, increasing sample cross-contamination, and wasting a lot of time, and achieving the effects of being easy to promote and use, reducing cross-contamination, and saving costs

Inactive Publication Date: 2017-03-15
金磁(苏州)纳米科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods generally have limitations such as complicated operation, long detection time, or the need for expensive large-scale instruments and equipment, and these methods need to purify DNA from samples. The complicated and cumbersome purification steps not only consume a lot of time, but also increase the cost. The risk of cross-contamination between samples, so it is not suitable for most clinical hospitals to promote the use in routine clinical tests

Method used

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  • Extraction-free direct-amplification rapid detection method for SNP typing of human methylenetetrahydrofolate reductase (MTHFR) gene via test strips
  • Extraction-free direct-amplification rapid detection method for SNP typing of human methylenetetrahydrofolate reductase (MTHFR) gene via test strips
  • Extraction-free direct-amplification rapid detection method for SNP typing of human methylenetetrahydrofolate reductase (MTHFR) gene via test strips

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] 1. Take a small amount of whole blood sample and add it to a PCR centrifuge tube, use a pipette to absorb 10 μL of the sample treatment solution, add it to the centrifuge tube, and let it stand for 5 minutes for later use;

[0048] 2. Take the two PCR centrifuge tubes A and B and add the following substances respectively:

[0049]

[0050] 3. Put the two tubes A and B into the PCR instrument, and proceed according to the following amplification procedure:

[0051] 31 cycles of UNG enzyme action at 50°C for 2min, pre-denaturation at 95°C for 3min, denaturation at 94°C for 5s, annealing at 60°C for 10s, extension at 72°C for 30s, 31 cycles, and final extension at 65°C for 10min. Total time: 1h20min.

[0052] 4. Drop the amplification products of tubes A and B onto two test strips respectively, the results are as follows: figure 2 shown. according to figure 2 According to the color interpretation of the middle T line, the sample is a heterozygous mutant of MTHFR C...

Embodiment 2

[0054] 1. Take a small amount of oral swab sample and add it to the PCR centrifuge tube, use a pipette to absorb 10 μL of the sample treatment solution, add it to the centrifuge tube, and let it stand for 5 minutes for later use;

[0055] 2. Take C and D two PCR centrifuge tubes and add the following substances respectively:

[0056]

[0057] 3. Put the two tubes C and D into the PCR instrument, and proceed according to the following amplification procedure:

[0058] 31 cycles of UNG enzyme action at 50°C for 2min, pre-denaturation at 95°C for 3min, denaturation at 94°C for 5s, annealing at 60°C for 10s, extension at 72°C for 30s, 31 cycles, and final extension at 65°C for 10min. Total time: 1h20min.

[0059] 4. Drop the amplified products of tubes C and D onto two test strips respectively, and the results are as follows: image 3 shown. according to image 3 The middle T line color interpretation, the sample is MTHFR C677T wild type.

Embodiment 3

[0061] 1. Take a small amount of fingertip blood spot sample and add it to a PCR centrifuge tube, use a pipette to absorb 10 μL of the sample treatment solution, add it to the centrifuge tube, and let it stand for 5 minutes for later use;

[0062] 2. Take the two PCR centrifuge tubes A and B and add the following substances respectively:

[0063]

[0064] 3. Put the two tubes A and B into the PCR instrument, and proceed according to the following amplification procedure:

[0065] 31 cycles of UNG enzyme action at 50°C for 2min, pre-denaturation at 95°C for 3min, denaturation at 94°C for 5s, annealing at 60°C for 10s, extension at 72°C for 30s, 31 cycles, and final extension at 65°C for 10min. Total time: 1h20min.

[0066] 4. Drop the amplification products of tubes A and B onto two test strips respectively, the results are as follows: Figure 4 shown. according to Figure 4 According to the color interpretation of the middle T line, the sample is a homozygous mutant of ...

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Abstract

The invention provides an extraction-free direct-amplification rapid detection method for SNP typing of a human MTHFR gene via test strips. The method comprises the following steps: step 1, mixing samples with a sample treatment fluid and carrying out standing for 5 min, wherein obtained mixtures are used as templates; step 2, separately designing a wild specific primer, a mutant specific primer and a shared primer for identification and amplification of the C677T SNP site of a to-be-detected MTHFR gene, wherein the 5'-terminals of the wild specific primer and the mutant specific primer are both labeled with digoxin and the 5'-terminal of the shared primer is both labeled with biotin; step 3, carrying out parallel two-tube PCR reactions on each sample so as to obtain amplification products; and step 4, subjecting the amplification products obtained in the step 3 to typing detection via the test strips by using chromatographic techniques and action between digoxin and a digoxin monoclonal antibody and between biotin and streptavidin, and then judging and determining genotypes.

Description

technical field [0001] The invention belongs to the technical field of gene detection, in particular to a rapid test strip detection method for human MTHFR gene SNP typing without extraction and direct amplification. Background technique [0002] Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in the metabolism of folic acid in the human body. Its main function is to convert 5,10-methylenetetrahydrofolate into 5-methyltetrahydrofolate, 5-methyltetrahydrofolate Hydrofolate is a methyl donor for the remethylation of homocysteine ​​to methionine. Studies have shown that methylenetetrahydrofolate reductase activity is related to the single nucleotide polymorphism (SNP) at C677T site of MTHFR gene. The single nucleotide polymorphisms of C677T site of MTHFR gene are wild type (CC type), heterozygous mutant type (CT type) and homozygous mutant type (TT type). The mutation at the C677T site reduces the activity of the enzyme due to heat intolerance. Compared with wild-...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6804C12Q2531/113C12Q2563/131C12Q2563/143C12Q2563/149C12Q2565/625
Inventor 张朝崔亚丽朱娟莉惠文利张秦鲁魏华刘克武
Owner 金磁(苏州)纳米科技有限公司
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