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Probe, primer and kit for detecting interleukin-3 gene polymorphism

A gene polymorphism and interleukin technology, applied in the field of molecular biology, can solve the problems of high cost, time-consuming and laborious, unsuitable detection, etc., and achieve the effect of simple operation, high sensitivity, and clear genotyping

Pending Publication Date: 2021-06-22
上海利康精准医疗技术有限公司
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  • 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

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  • Probe, primer and kit for detecting interleukin-3 gene polymorphism
  • Probe, primer and kit for detecting interleukin-3 gene polymorphism
  • Probe, primer and kit for detecting interleukin-3 gene polymorphism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Design of probes and primers

[0026] The present invention designs probe and primer sequences aiming at IL3 C>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.

[0027] Design probes and primers so that when the template does not exist at the annealing temperature, the probe is in a stem-loop state, including the loop sequence and the reverse-complementary stem se...

Embodiment 2

[0034] Example 2 Detection of different genotype standard products

[0035] 1. Use the plasmid IL3 C>T to construct and prepare wild-type standard plasmids and mutant standard plasmids containing the rs31480 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) Co., Ltd. Synthesized by the company. The accuracy of the sequence was determined by sanger sequencing. The genotype of the wild-type standard plasmid rs31480 is CC;

[0036] 2. Using the probes and primers in Example 1.

[0037] 3. PCR reaction system:

[0038] 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 r...

Embodiment 3

[0041] Embodiment 3: detection method performance analysis experiment

[0042] 1. Precision experiment

[0043] 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 figure 2 . 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%).

[0044] 2. Conformity rate experiment

[0045] Select 20 cases of DNA samples from healthy volunteers in Shanghai, apply the method of Example 2 to detect the rs31480 locus, and apply the Sanger sequencing method to verify at the same time, compare the consistency of the detection results of the two methods, the results show that the method of Example 2 can be classified T...

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Abstract

The invention provides a probe for detecting interleukin-3 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 interleukin-3 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 interleukin-3 gene polymorphism are used for detecting the interleukin-3 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 interleukin-3 gene polymorphisms. Background technique [0002] Interleukin-3 (interleukin-3, IL-3), also known as multi-colony stimulating factor (multi-CSF), is one of the important members of the interleukin family, mainly produced by activated T cells, macrophages and bone marrow The generation of stromal stem cells plays an important role in the body's hematopoietic regulation and immune regulation by combining with its receptor - IL-3R. This gene is not only related to hematopoietic cell diseases such as bone marrow failure, but also related to various inflammations, such as rheumatoid arthritis, juvenile dermatitis, etc., and even schizophrenia. This genetic polymorphism may affect an individual's response to inflammation, thereby affecting the genetic susceptibility to related diseases. Therefore, the detection of IL3 gene polymorp...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11C12Q1/6851
CPCC12Q1/6851C12Q1/6883C12Q2600/156C12Q2531/113C12Q2563/107
Inventor 毛丹丹张奕黄芬芬王校
Owner 上海利康精准医疗技术有限公司
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