Fluorescent quantitative PCR detection method for TLR3 (toll-like receptors) gene of duck

A fluorescence quantitative and detection method technology, applied in the field of life sciences, to achieve the effect of simple operation, wide linear range and improved reliability

Inactive Publication Date: 2015-05-27
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Claims
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At present, there is no report on the application of fluoresce...

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  • Fluorescent quantitative PCR detection method for TLR3 (toll-like receptors) gene of duck
  • Fluorescent quantitative PCR detection method for TLR3 (toll-like receptors) gene of duck
  • Fluorescent quantitative PCR detection method for TLR3 (toll-like receptors) gene of duck

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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 duck TLR3 gene, which belongs to the field of life sciences. Background technique [0002] Toll-like receptors (Toll-like receptors, TLRs) are a class of evolutionarily conserved type I transmembrane receptors, including leucine rich (Leucine Rich Repeats, LRRs)-rich extracellular region, transmembrane region and intracellular TIR region (Toll / interleukin-1 receptor). As the first line of defense of innate immunity against microbial infection, TLRs play an important role in the detection of pathogen-associated molecular patterns (Pathogen-associated molecular patterns, PAMPs), and ultimately activate the adaptive immune response. TLR3 can recognize double-stranded RNA (dsRNA), through the non-MyD88-dependent signaling pathway, combined with TRIF to mediate downstream signal transduction, activate IRF-3 and nuclear factor kappa-B (nuclear factor kappa-B, NF-κB), stimulate induc...

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Application Information

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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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