Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Probe for detecting tumor necrosis factor-alpha gene polymorphism, primer and kit

A technology of tumor necrosis factor and α gene, which is applied in the field of primers and kits and probes for detecting tumor necrosis factor-α gene polymorphism, which can solve the problems of high cost, unsuitable detection, time-consuming and labor-intensive, etc., and achieve simple operation , clear genotyping and high sensitivity

Pending Publication Date: 2021-06-22
上海利康精准医疗技术有限公司
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Probe for detecting tumor necrosis factor-alpha gene polymorphism, primer and kit
  • Probe for detecting tumor necrosis factor-alpha gene polymorphism, primer and kit
  • Probe for detecting tumor necrosis factor-alpha gene polymorphism, primer and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Design of probes and primers

[0029] The present invention designs probes and primer sequences for the TNF-α 857C>T SNP site. 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.

[0030] Design probes and primers so that in the absence of a template at the annealing temperature, the probe assumes a stem-loop state such as figure 1 , including the loop sequence and its reverse ...

Embodiment 2

[0037] Example 2 Detection of different genotype standard products

[0038] 1. Use the plasmid TNF-α 857C>T to construct and prepare wild-type standard plasmids and mutant standard plasmids containing the rs1799724 site of the target gene (the source of the plasmid and the synthesis of the plasmid containing the target gene are provided by Sangon Bioengineering (Shanghai) Synthesized by Co., Ltd. The accuracy of the sequence was determined by Sanger sequencing. The genotype of the wild-type standard plasmid rs1799724 was CC;

[0039] 2. Using the probes and primers in Example 1.

[0040] 3. PCR reaction system:

[0041] 1) Add 7.5ul of PCR Mix, 0.5uM of primer 1 solution and 0.5uM of primer 2 solution, and 0.1uM of probe into each PCR reaction well, and then add wild-type standard plasmids into 3 different PCR reaction wells respectively 2ul each of DNA, mutant standard plasmid DNA, and mixed DNA (the wild-type standard plasmid and mutant standard plasmid are mixed at a rati...

Embodiment 3

[0044] Embodiment 3: detection method performance analysis experiment

[0045] 1. Precision experiment

[0046] Take one copy of wild-type standard plasmid DNA and one mixed-type DNA (the wild-type standard plasmid and the mutant standard plasmid are mixed at a ratio of 1:1), and test 3 times a day for 5 consecutive days. The method in example 2, see the result image 3 . It shows that the fluorescent PCR amplification reaction of the present invention has good repeatability (the coincidence rate is greater than 95%, and the coefficient of variation CV of the Ct value of the test result is less than 5%).

[0047] 2. Conformity rate experiment

[0048] Select 20 cases of DNA samples from healthy volunteers in Shanghai, apply the method of Example 2 to detect the rs1799724 site, and apply the Sanger sequencing method to verify at the same time, compare the consistency of the detection results of the two methods, and the results show that the method of Example 2 can be classif...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a probe for detecting tumor necrosis factor-alpha gene polymorphism. The sequence of the probe is as shown in SEQ ID NO.: 1, or two ends of the sequence of SEQ ID NO.: 1 are modified by groups. The invention also provides a primer for detecting the polymorphism of the tumor necrosis factor-alpha gene. The sequence of the primer is shown as SEQ ID NO.: 2 (primer 1) and SEQ ID NO.: 3 (primer 2). When the probe and the primer for detecting the tumor necrosis factor-alpha gene polymorphism are used for detecting the tumor necrosis factor-alpha gene polymorphism, the probe and the primer have the advantages of high sensitivity, good specificity, rapidness, high-throughput detection and the like.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to probes, primers and kits for detecting tumor necrosis factor-alpha gene polymorphism. Background technique [0002] 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. Its dysfunction is believed to be related to many diseases, such as Alzheimer's disease, psoriasis, cancer, major depression, enteritis and so on. Therefore, the detection of TNF-α gene polymorphism can be used as an evaluation index of the disease, treatment effect and prognosis, which is of great significance. [0003] At present, the commonly used mutation detection method is DNA direct sequencing method...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/118C12Q2600/156
Inventor 黄芬芬毛丹丹王校
Owner 上海利康精准医疗技术有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com