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Probes, primers and kits for detecting gene polymorphism of immune antioxidant genes

A gene polymorphism and anti-oxidation technology, applied in the field of molecular biology, can solve the problems of high cost, inapplicability to large-scale sample detection, time-consuming and labor-intensive problems, and achieve the effects of simple operation, clear genotyping and high sensitivity

Pending Publication Date: 2021-02-02
上海利康精准医疗技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the commonly used mutation detection method is direct DNA sequencing method. PCR products can be directly analyzed by DNA sequence, which can clarify the mutation site, but it has the disadvantages of time-consuming, laborious, expensive, and not suitable for detection of a large number of samples.

Method used

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  • Probes, primers and kits for detecting gene polymorphism of immune antioxidant genes
  • Probes, primers and kits for detecting gene polymorphism of immune antioxidant genes
  • Probes, primers and kits for detecting gene polymorphism of immune antioxidant genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1: probe and primer design

[0053] The present invention designs probes and primer sequences for the SNP sites of TNF-α, CTLA4, IL3 and SOD-2. The specific principle is to use the conformational change of the fluorescent probe and the target sequence after hybridization to release the fluorescent dye, and judge the genotyping results according to the peak diagrams and Tm values ​​at different temperatures after hybridization. In the absence of target DNA, the fluorophore and quencher can be stably combined together, and no fluorescent signal can be detected; when there is target DNA, the structure of the fluorescently labeled probe is destroyed, and the fluorophore and quencher The extinguishing groups are separated from each other, and the fluorescent signal can be detected.

[0054] Design the probe and primers so that the probe is in a stem-loop state in the absence of the template at the annealing temperature. Take TNF as an example, such as figure 1 ...

Embodiment 2

[0076] Embodiment 2: Detect different genotype standard products

[0077] 1. Construct and prepare wild-type standard plasmids and mutant standard plasmids containing the target gene TNF, CTLA4, IL3, and SOD-2 gene loci with plasmid immune anti-oxidation (the source of the plasmid, and the synthesis of the plasmid containing the target gene Synthesized by Gong Biological Engineering (Shanghai) Co., Ltd. The accuracy of the sequence was determined by sanger sequencing. The wild-type standard plasmid TNF genotype is CC, CTLA4 genotype is AA, IL3 genotype is CC, SOD-2 genotype is TT ;The TNF genotype of the mutant standard plasmid is TT, the CTLA4 genotype is GG, the IL3 genotype is TT, and the SOD-2 genotype is CC. The standard plasmid DNA concentration is standardized to 10ng / ul.

[0078] 2. Using the probes and primers in Example 1.

[0079] 3. PCR reaction system:

[0080] 1) Add 7.5ul of PCR Mix, 0.5uM of 4 sets of forward primer solutions and 0.5uM of each of 4 sets of re...

Embodiment 3

[0083] Example 3: Double-blind experimental investigation using the immune antioxidant detection kit

[0084] 1. The genomic DNA of oral epithelial cells of 75 cases of healthy volunteers in Shanghai was extracted by silica gel adsorption method, and the concentration and purity of DNA were detected by electrophoresis gel imaging, and the DNA concentration of the samples to be tested was standardized to 10ng / ul.

[0085] 2. The detection method is as follows: Add 7.5ul of PCR Mix, 0.5uM of the forward primer solution, 0.5uM of the reverse primer solution, and 0.1uM of the probe in each PCR reaction well, and carry out a weak positive control (TNF gene genotype CT, CTLA4 genotype AG, IL3 genotype CT and SOD-2 genotype TT), negative control (TNF genotype CC, CTLA4 genotype AA, IL3 genotype CC and SOD-2 genotype Type TT) and the detection of the sample to be tested, add 2ul of DNA to each reaction well, make up 15ul with sterilized double distilled water; perform the reaction on ...

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Abstract

For different polymorphism of immune antioxidant genes, four groups of specific primer and probe combinations are designed in the present invention, and the sequences of the probes are shown as SEQ IDNO.: 1, 4, 7 and 10. The probes and primers with different polymorphism for immune antioxidant gene loci have the advantages of high sensitivity, high specificity, and rapid high-throughput detectionwhen being used for detecting the polymorphism of immune antioxidant genes.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to probes, primers and kits for detecting polymorphisms of immune and antioxidant genes. Background technique [0002] The two root causes of diseases—antioxidation and immunity are closely related to cancer; the immune and antioxidative function of the body is related to gene polymorphisms. [0003] Tumor Necrosis Factor-α (Tumor Necrosis Factor-α, TNF-α) is a cytokine involved in systemic inflammation, mainly secreted by macrophages. Its main function is to regulate immune cells. As an endogenous pyrogen, it can cause fever, induce apoptosis, prevent tumorigenesis and virus replication, etc., and play an important role in inflammation and immune response. Genetic variation can lead to impairment of the ability to regulate immune cell activity in the body, and excessive specific immune function can easily lead to inflammatory reactions, resulting in increased susceptibility to var...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2600/156C12Q2531/113C12Q2563/107C12Q2545/114
Inventor 傅咏南黄芬芬毛丹丹张奕
Owner 上海利康精准医疗技术有限公司
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