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Fluorescent quantitative PCR method for detecting interleukin 17 in pig intestinal tissue

A technology for interleukin and fluorescence quantification, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, recombinant DNA technology, etc. Detection effect

Active Publication Date: 2014-06-18
昆明云中美农牧科技有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method has strong specificity, the operation process is cumbersome and expensive, and it is difficult to promote
(2) Western-blot method, that is, the enzyme-labeled anti-cytokine antibody is hybridized with the protein sample immobilized on the denaturing gel. This method is mostly used for qualitative detection. When used for quantification, the operation level is high and the accuracy is low
(3) ELISA method, which is mainly used to detect secreted cytokines, such as protein levels in serum, is difficult to reflect the situation in cells
However, there are few reports on the fluorescence quantitative detection method of interleukin-17 in porcine intestinal tract by dye method

Method used

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  • Fluorescent quantitative PCR method for detecting interleukin 17 in pig intestinal tissue
  • Fluorescent quantitative PCR method for detecting interleukin 17 in pig intestinal tissue
  • Fluorescent quantitative PCR method for detecting interleukin 17 in pig intestinal tissue

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Preparation of a standard curve containing IL-17 target gene

[0021] 1. Primer design and synthesis

[0022] First search the porcine IL-17 sequence (AB102693.1) from http: / / www.ncbi.nlm.nig.gov / GenBank, and then use Perlprimer software to design primers according to the porcine IL-17 mRNA conserved sequence (CDS region) and send them to Shanghai Synthesized by Yingjun Bioengineering Co., Ltd. Primers and template sequences are as follows:

[0023] Forward primer: 5'-CTGGAGAAAGTGATGGTGAC-3' (SEQ ID No.1);

[0024] Reverse primer: 5'-CCTGAAAGCCTAACTGATTTGG-3' (SEQ ID No.2);

[0025] Template (SEQ ID No.3):

[0026] 5'-CTGGAGAAAGTGATGGTGACAGTGGGCTGCACCTGTGTCACCCCCATCGTCCGCCATATTTCTAAGAGCTTCTAGTCTGACCCCTGCTCCCCAAATCAGTTAGGCTTTCAGG-3'.

[0027] 2. Collection of intestinal samples from differently treated piglets

[0028] In order to detect the effect of Lactobacillus casei Zhang on the expression level of intestinal IL-17 in piglets after adding Lactobacill...

Embodiment 2

[0040] Example 2 Detection of IL-17 Gene Expression in Sample Pig Intestinal Tissue

[0041] 1. Pig intestinal samples were prepared into cDNA according to the aforementioned method, and then PCR reaction was carried out simultaneously with the internal reference gene GAPDH, and finally according to the relative quantitative △△C T Methods To determine the relative expression of IL-17 gene in the samples.

[0042] The detection results of the relative expression of IL-17 transcripts in the ileum and colon tissues of 4 blank group (Control) and 4 probiotic group (Lc Zhang) piglets (using GAPDH as an internal reference gene) are as follows:

[0043]

[0044]

[0045] 2. The expression level of IL-17 in pig intestinal tract was detected by ELISA method and compared with the detection results of fluorescent quantitative PCR method.

[0046] Collect 10 mg of porcine intestinal tissue sample, and add 200 μl of phosphate buffer saline containing 5mM DTT, 1mMPMSF (i.e. PBS solut...

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Abstract

The invention relates to a fluorescent quantitative PCR method for detecting interleukin 17 in pig intestinal tissue. The method comprises the following steps: designing a specific fluorescent quantitative PCR primer, establishing a fluorescent quantitative PCR reaction system and a reaction process, preparing plasmids as standard plasmids for drawing standard curve, finally detecting the gene expression quantity according to relative quantification triangle triangle CT method after target genes and reference genes GAPDH of samples to be tested are amplified simultaneously, wherein the plasmids contain target fragment IL-17. Due to the adoption of the characteristics of high sensitivity, strong specificity, no pollution, real time, accuracy and high speed of the fluorescent quantitative PCR, the invention provides a practical method for detecting IL-17 expression level of pig intestinal part, so that evaluation of immune state or disease process of the animal intestinal part is assisted.

Description

technical field [0001] The invention relates to a fluorescent quantitative PCR detection method for interleukin 17 in pig intestinal tissue, belonging to the field of biotechnology. Background technique [0002] Interleukin 17 (IL-17) is an important pro-inflammatory cytokine, which is secreted by helper T cells (Th17) and innate immune cells, and plays a role in various inflammatory reactions and pathological processes of autoimmune diseases. play a key role in. [0003] Under the action of the microenvironment in animals, naive T cells can differentiate into Th1, Th2, Th17, Th22, follicular helper T cells and Treg cell subtypes. Among them, Th17 cells are a unique subtype capable of secreting IL17 cytokine. IL-17 is a pro-inflammatory cytokine expressed by Th17 cells, natural killer T cells, γσT cells, etc., and plays a key role in autoimmune diseases and various inflammatory responses. Studies related to human IL-17 have shown that this factor is an important bridge co...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 董晓丽苏华荔王安如闫轶洁
Owner 昆明云中美农牧科技有限公司
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