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Fluorescent quantitation PCR kit for detecting chicken immune-related cytokines and application

An immune-related, fluorescent quantitative technology, applied in the direction of microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of unusable detection, unpublished fluorescence quantitative PCR standard curve, unable to use absolute quantitative analysis, etc., to achieve detection Wide linear range, high sensitivity and specificity

Inactive Publication Date: 2015-10-14
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the innate immune system is strictly specific to animal species, and the method for detecting duck innate immune genes cannot be used to detect the level of chicken innate immune-related genes
Moreover, they did not disclose the standard curve of the fluorescent quantitative PCR method, and their technology can only be used for relative quantitative detection of duck-related genes, not for absolute quantitative analysis

Method used

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  • Fluorescent quantitation PCR kit for detecting chicken immune-related cytokines and application
  • Fluorescent quantitation PCR kit for detecting chicken immune-related cytokines and application
  • Fluorescent quantitation PCR kit for detecting chicken immune-related cytokines and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1 Detection kit for chicken IFN-α, IFN-β, IFN-γ, IL-1β, IL-2 and IL-6

[0074] Fluorescence quantitative PCR detection kit for detection of chicken IFN-α, IFN-β, IFN-γ, IL-1β, IL-2 and IL-6, including upstream primer solution with a concentration of 10 μM and downstream primer solution with a concentration of 10 μM and corresponding plasmid standards. The nucleotide sequence of the primer pair is shown in SEQ ID NO.1~NO.12; the plasmid standard pUC57-IFN-αCh, pUC57-IFN-βCh, pUC57-IFN-γCh, pUC57-IL- The nucleotide sequences of IFN-α, IFN-β, IFN-γ, IL-1β, IL-2 and IL-6 in 1βCh, pUC57-IL-2Ch and pUC57-IL-6Ch are respectively shown in SEQ ID NO.13~NO .18 shown.

Embodiment 2

[0075] Example 2 Detection of chicken IFN-α, IFN-β, IFN-γ, IL-1β, preparation method and application of biological reagents for IL-2 and IL-6

[0076] Described preparation method comprises the following steps:

[0077] 2.1 Primer design

[0078] Download chicken IFN-α, IFN-β, IFN-γ, IL-1β, IL-2 and IL-6 gene sequences from Genbank (GenBank Accession No.: EU367971, GU119897, NM_205149, NM_204524, AF000631 and HM179640) , using Primer Premier 5.0 to design several pairs of specific PCR primers, among which the primer sequences with the best amplification effect on IFN-α, IFN-β, IFN-γ, IL-1β, IL-2 and IL-6 are shown in the attached table, The estimated amplified fragments are 188bp, 171bp, 120bp, 208bp, 162bp and 165bp respectively. Primers were synthesized using a fully automatic DNA synthesizer for OligoDNA.

[0079] 2.2 Preparation of plasmid standard

[0080] Infect 11-day-old SPF chicken embryos with attenuated Newcastle disease virus vaccine strain LaSota. The allanto...

Embodiment 3

[0084] Example 3 Detection of chicken IFN-α, IFN-β, IFN-γ, IL-1β, IL-2 and the establishment of IL-6 fluorescent quantitative PCR method

[0085] 3.1 Optimization of reaction conditions

[0086] First, use different annealing temperatures for PCR to amplify IFN-α, IFN-β, IFN-γ, IL-1β, IL-2 and IL-6, respectively, and determine the optimal annealing temperature for the reaction. Then, use different primer concentrations and optimal annealing temperature to carry out fluorescent quantitative PCR, and optimize the concentration of primers used. The primer concentration used in the reaction with the earliest exponential amplification phase (the smallest Ct value), the best amplification curve (typical S-curve) and the most amplified products (highest fluorescence increment value) was taken as the optimal primer concentration .

[0087] The test used Shanghai Biological Engineering Co., Ltd. (Sangon) fluorescent quantitative PCR premix (Master Mix) and buffer (Buffer), the instru...

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Abstract

The invention provides a fluorescent quantitation PCR kit for detecting chicken immune-related cytokines and application. The fluorescent quantitation PCR kit is used for SYBR Green I fluorescent quantitation PCR of gene transcription levels of chicken's IFN-alpha, IFN-beta, IFN-gamma, IL-1(beta), IL-2 and IL-6 genes. The kit and a detection method provided by the invention are high in sensitivity, high in specificity, wide in linear concentration range, good in repeatability, and simple and convenient to operate, saves time, and can be used for absolute or relative quantitative detection of chicken immune-related cytokines in various sample types.

Description

technical field [0001] The invention relates to a detection method in the field of veterinary biotechnology, in particular to a fluorescent quantitative PCR kit for detecting six kinds of cytokines related to chicken immunity and an application thereof. Background technique [0002] Cytokines are low-molecular-weight glycoproteins produced by reticuloendothelial cells, lymphoid / macrophages, and somatic cells under the stimulation of cytokine-inducing agents such as viruses, and have anti-viral, anti-tumor, and immune-regulating functions. Cytokines play an important role in the growth and differentiation of organisms, immune regulation and disease response. For example, when a pathogen invades an organism, the pattern recognition receptors of the body's cells are stimulated by the pathogen-associated molecular pattern to activate the cell signal transduction pathway, which eventually leads to the up-regulation of the expression of cytokines and effector molecules, thereby pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q2531/113C12Q2545/113C12Q2563/107
Inventor 黄艳艳许传田吴家强杨少华黄庆华张琳朱曼玲高丹丹
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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