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Fluorescence quantitative pcr detection method of a kind of duck tlr3 gene

A fluorescence quantitative and detection method technology, applied in the field of life sciences, to achieve the effects of reduced selectivity, small inhibitory effect, and wide linear range

Inactive Publication Date: 2017-01-11
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Application Information

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Problems solved by technology

At present, there is no report on the application of fluorescent quantitative PCR method to the detection of Peking duck TLR3 gene

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  • Fluorescence quantitative pcr detection method of a kind of duck tlr3 gene
  • Fluorescence quantitative pcr detection method of a kind of duck tlr3 gene
  • Fluorescence quantitative pcr detection method of a kind of duck tlr3 gene

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Embodiment Construction

[0055] The present invention will be described in detail below in conjunction with specific embodiments. The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0056] 1 Materials and methods

[0057] 1.1 Strains and main reagents

[0058] Escherichia coli Trans5α competent cells were purchased, and the pEASY-T5-zero vector was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; qPCR Master Mix, Trizol reagent, and reverse transcriptase M-MLV were purchased from Promega. Fluorescent quantitative PCR tubes were purchased from Ai Sijin Biotechnology (Hangzhou) Co., Ltd. The gel recovery kit was purchased from Kangwei Century Biotechnology Co., Lt...

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Abstract

The invention provides a fluorescent quantitative PCR detection method for a TLR3 (toll-like receptors) gene of a duck. The detection method comprises the following steps: by taking a recombinant plasmid Peasy-T5-TLR3 as a template, screening the optimum concentration of a primer, a fluorescent quantitative PCR reaction system and a reaction condition; by taking the recombinant plasmid as a standard substance, determining the OD260 value; according to the molecular weight and the mass concentration, calculating the copy concentration, diluting, carrying out FQ-PCR amplification; by taking logarithm of the recombinant plasmid copy number as axis X and circulating number of times as axis Y, establishing a standard curve; repeatedly verifying 1.0*10<3>-1*10<8> copy / microliter recombinant plasmid; carrying out FQ-PCR detection on the recombinant plasmid; and carrying out double verification on the specificity of the detection method by virtue of sequence alignment of a sample and the standard substance and a peak pattern of a solubility curve of the sample.

Description

technical field [0001] The invention relates to a fluorescent quantitative PCR detection method of the TLR3 gene of ducks, belonging to the field of life sciences. Background technique [0002] Toll-like receptors (TLRs) are a class of evolutionarily conserved type I transmembrane receptors, including leucine rich (Leucine Rich Repeats, LRRs) extracellular domain, transmembrane domain and intracellular domain TIR region (Toll / interleukin-1 receptor). As the first line of defense against microbial infections in innate immunity, TLRs play an important role in detecting pathogen-associated molecular patterns (PAMPs), ultimately activating adaptive immune responses. TLR3 can recognize double-stranded RNA (dsRNA), mediate downstream signal transduction through a MyD88-independent signaling pathway, combined with TRIF, activate IRF-3 and nuclear factor kappa-B (NF-κB), stimulate and induce a large amount of The expression of IFN-α / β and other cytokines, and the secreted interfer...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/63C12N15/11
CPCC12Q1/6851C12Q1/6888C12Q2600/158C12Q2531/113C12Q2545/113C12Q2563/107
Inventor 刘光清宋凯杰陈宗艳李传峰孟春春
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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