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Kit for detecting chicken body antiviral protein and application of kit

A kit and anti-virus technology, applied in the detection/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of different reaction systems and cycle parameters, incomparability of experimental results, etc., and achieve detection linearity Wide-ranging, high-sensitivity, versatile effects

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

Fluorescent quantitative PCR technology to detect these proteins is only mastered by some research laboratories
Moreover, the primers, reaction systems and cycle parameters used in each laboratory are different, resulting in the incomparability of the experimental results.

Method used

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  • Kit for detecting chicken body antiviral protein and application of kit
  • Kit for detecting chicken body antiviral protein and application of kit
  • Kit for detecting chicken body antiviral protein and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Detection kit for chicken IFIT5, PKR and OASL

[0059] Fluorescence quantitative PCR detection kits for detecting chicken IFIT5, PKR and OASL respectively include an upstream primer solution with a concentration of 10 μM, a 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.6; the IFIT5, PKR and OASL nucleotides in the plasmid standard pUC57-IFIT5Ch, pUC57-PKR Ch and pUC57-OASL Ch The sequences are respectively shown in SEQ ID NO.7-NO.9.

Embodiment 2

[0060] Embodiment 2 detects chicken IFIT5, the preparation method and application of the biological reagent of PKR and OASL

[0061] Described preparation method comprises the following steps:

[0062] 2.1 Primer design

[0063] Download chicken IFIT5, PKR and OASL gene sequences from Genbank (GenBank Accession No.: XM_421662, NM_204487 and NM_205041, respectively), and use Primer Premier 5.0 to design several pairs of specific PCR primers, which have the best amplification effect on IFIT5, PKR and OASL Good primer sequences are shown in the attached table, and the predicted amplified fragments are 151bp, 94bp and 81bp respectively. Primers were synthesized using a fully automatic DNA synthesizer for OligoDNA.

[0064] 2.2 Preparation of plasmid standard

[0065] Infect 11-day-old SPF chicken embryos with attenuated Newcastle disease virus vaccine strain LaSota. The allantoic fluid of chicken embryos was collected 24 hours after virus infection, and the total RNA was extra...

Embodiment 3

[0069] Embodiment 3 detects the establishment of the fluorescent quantitative PCR method of chicken IFIT5, PKR and OASL

[0070] 3.1 Optimization of reaction conditions

[0071] First, PCR was performed with different annealing temperatures to amplify IFIT5, PKR and OASL respectively, and the optimal annealing temperature for the reaction was determined. 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 .

[0072] The test used Shanghai Biological Engineering Co., Ltd. (Sangon) fluorescent quantitative PCR premix (Master Mix) and buffer (Buffer), the instrument is: Roche LightCycl...

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Abstract

The invention provides a kit for detecting chicken body antiviral protein and application of the kit. The kit is used for SYBR Green I fluorescent quantitation of chicken's IFIT5, PKR and OASL genes of transcription levels. The kit and a detection method provided by the invention are high in sensitivity, high in sensitivity, wide in linear concentration range, good in repeatability, and convenient to operate, saves time, and can be used for absolute or relative quantitative detection of chicken body antiviral protein of 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 chicken body antiviral protein and an application thereof. Background technique [0002] When infected by the virus, the pathogen-associated molecular pattern (PAMP) is recognized by the pattern-recognition receptor (PRR) of chicken somatic cells. PRR activates the corresponding ligand, initiates the signal transduction of the chicken's innate immune pathway, and induces the production of interferon (Interferon, IFN) and pro-inflammatory cytokines (pro-inflammatory cytokines). Interferon further stimulates the expression of interferon-stimulated genes (ISGs), produces a variety of antiviral proteins, and exerts anti-infection effects. [0003] Among the proteins expressed by ISG, three proteins play an important antagonistic role in various viral infections, which are introduced as follows. [0004] (...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
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