Kit and method for detecting STAT3 gene mutation of high IgE (Immunoglobulin E) syndrome

A kit and gene technology, applied in the field of high IgE syndrome STAT3 gene mutation detection kits

Inactive Publication Date: 2017-07-21
济南艾迪康医学检验中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is currently no cure for HIES, and treatment focus

Method used

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  • Kit and method for detecting STAT3 gene mutation of high IgE (Immunoglobulin E) syndrome
  • Kit and method for detecting STAT3 gene mutation of high IgE (Immunoglobulin E) syndrome
  • Kit and method for detecting STAT3 gene mutation of high IgE (Immunoglobulin E) syndrome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0146] A primer for detecting the polymorphic mutation site of STAT3 gene, the design of the primer is an amplification primer designed for the whole exon of STAT3, including:

[0147] The primers for amplifying the whole exon sequence of STAT3 gene, its base sequence is:

[0148] ST-E1-F: TGTAAAACGACGGCCAGTCTCCGCCCTTTTCTCCTAGC

[0149] ST-E1-R: AACAGCTATGACCATGGCGTCCATCACAACATCCC

[0150] ST-E2-F: TGTAAAACGACGGCCAGT AGCTGTGATTATCCCAAGGTGG

[0151] ST-E2-R: AACAGCTATGACCATG TGCCCAAGTAAATGAGGTCCA

[0152] ST-E3-F: TGTAAAACGACGGCCAGTGTGTATGCGTCGGCTTCA

[0153] ST-E3-R: AACAGCTATGACCATGGACTCTGCGGGTCCTGTT

[0154] ST-E4-F: TGTAAAACGACGGCCAGT AGTAACGACCTCCCCTTCGC

[0155] ST-E4-R: AACAGCTATGACCATGTTGGGTCTGTTGGATTCTTTTGGTG

[0156] ST-E5 / 6-F: TGTAAAACGACGGCCAGT GACCTGTGTAGTGTCACCTGT

[0157] ST-E5 / 6-R: AACAGCTATGACCATG GGACCCGTACCTCCCTTGC

[0158] ST-E7 / 8-F: TGTAAAACGACGGCCAGT TCCCTCAGGTCAAGGAGT

[0159] ST-E7 / 8-R: AACAGCTATGACCATG CTGGCCGAAATAAAGTAAAA

[0160] ST-E9 / 10-F:...

Embodiment 2

[0192] Operation process of blood / cell / tissue genomic DNA extraction kit (Tiangen Biology):

[0193] (1) Extract tissue DNA from blood: 1) Extract 300 μl of blood and add 900 μl of erythrocyte lysate, mix by inverting, and place at room temperature for 5 minutes, during which time, invert and mix several times. Centrifuge at 12,000rpm for 1min, suck off the supernatant, leave the white blood cell pellet, add 200μl buffer GA, shake until thoroughly mixed. 2) Add 20 μl proteinase K solution and mix well. 3) Add 200 μl of buffer GB, mix thoroughly by inversion, place at 70°C for 10 minutes, the solution should become clear, and briefly centrifuge to remove water droplets on the inner wall of the tube cap. 4) Add 200 μl of absolute ethanol, vortex and mix well for 15 seconds. At this time, flocculent precipitates may appear. Briefly centrifuge to remove water droplets on the inner wall of the tube cap. 5) Add the solution and flocculent precipitate obtained in the previous step ...

Embodiment 3

[0220] Three cases of clinical samples (sample number 1-3) were taken according to the reagents and methods of Examples 1 and 2 to extract genomes, prepare reagents, amplify and sequence. Add 1 μl of each sample to the detection system PCR reaction solution. Electrophoresis results such as Figure 2-4 Shown, show that the primers ST-E1-F / R, ST-E2-F / R, ST-E3-F / R, ST-E4-F / R, ST-E5 / 6-F / R of the present invention , ST-E7 / 8-F / R, ST-E9 / 10-F / R, ST-E11-F / R, ST-E12 / 13 / 14-F / R, ST-E15 / 16-F / R , ST-E17 / 18-F / R, ST-E19 / 20-F / R, ST-E21-F / R, ST-E22 / 23-F / R, ST-E24-1-F / R, ST -E24-2-F / R, ST-E24-3-F / R, ST-E24-4-F / R can effectively amplify blood samples with a single band.

[0221] Some test results of sample 1 are as follows: Figure 5 , 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19:

[0222] Figure 5 It shows the wild-type sequencing screenshot of STAT3 exon 1 of sample 1,

[0223] Figure 6 It shows the wild-type sequencing screenshot of STAT3 exon 2 of sample 1,

[0224] Figur...

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Abstract

The invention discloses a kit and method for detecting STAT3 gene mutation of high IgE (Immunoglobulin E) syndrome. The kit comprises (i) a blood DNA (Deoxyribonucleic Acid) extraction reagent; (ii) PCR (Polymerase Chain Reaction) amplification reaction liquid for detecting a system; (iii) a reagent for sequencing the system; wherein primers comprise amplification primers and sequencing primers for amplifying whole exon sequences of STAT3 genes. In addition, a Sanger sequencing technology is used. According to the method disclosed by the invention, the mutation of whole exons of the STAT3 genes in the body of a patient with the high IgE syndrome can be quickly detected out. The detecting results obtained by through the invention can be used for assisting in diagnosis of the high IgE syndrome, which has an important reference significance for early intervention and early treatment.

Description

Background technique [0001] The invention belongs to the biotechnical field of medical detection, in particular to a detection kit and a detection method for hyper IgE syndrome STAT3 gene mutation. Background technique [0002] Hyper IgE syndrome (HIES) is a rare and complex primary immunodeficiency disease, clinically characterized by stubborn eczema-like rash, skin and lung abscesses caused by repeated bacterial infections, and accompanied by significantly elevated serum IgE levels . It can be divided into 2 types, type Ⅰ is autosomal dominant inheritance (AD-HIES), which is caused by STAT3 gene mutation; type Ⅱ is autosomal recessive inheritance (AR-HIES), which is caused by TYK-2 or Dock8 gene mutation Sincerely. STAT3 is a signaling transcription factor that is activated by a variety of cytokines and growth factors, including IL-6, IL-10, IL-22, IL-23, and macrophage colony-stimulating factor. STAT3 mutations in AD-HIES interfere with the above cytokine-activated s...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q1/6883C12Q2600/156C12Q2535/122C12Q2531/113
Inventor 刘赵玲桑志高王淑一
Owner 济南艾迪康医学检验中心有限公司
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