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Method for detecting bastard halibut LITAF gene expression by applying reverse transcription-polymerase chain reaction (RT-PCR)

A technology of RT-PCR and gene expression, which is applied in the field of detection of LITAF gene expression in flounder with fluorescent RT-PCR technology, and can solve the problems of no immune control technology and treatment methods

Inactive Publication Date: 2013-09-04
TIANJIN NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the lack of understanding of the immune regulation mechanism of flounder, there is no effective immune control technology and treatment

Method used

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  • Method for detecting bastard halibut LITAF gene expression by applying reverse transcription-polymerase chain reaction (RT-PCR)
  • Method for detecting bastard halibut LITAF gene expression by applying reverse transcription-polymerase chain reaction (RT-PCR)
  • Method for detecting bastard halibut LITAF gene expression by applying reverse transcription-polymerase chain reaction (RT-PCR)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] A kit for detecting the expression of the flounder LITAF gene:

[0073] Component I SYBR Premix Ex Taq II * (Ex Taq fluorescent PCR mix) Component II LITAF gene upstream specific primer LITAF-q-F Component III LITAF gene downstream specific primer LITAF-q-R Component IV β-actin gene-specific upstream primer β-actin-F Component V β-actin gene-specific downstream primer β-actin-R

[0074] *Purchased from Dalian Bao Biological Company (product number: DRR420A), containing SYBR Green I, Ex Taq enzyme, dNTP and reaction buffer.

Embodiment 2

[0076] Flounder infection experiment:

[0077] Edwardsiella tarda (provided by the Tianjin Aquatic Animal Disease Prevention and Control Center, available externally) was inoculated in Luriai-Bertani liquid medium with a pH value of 7.5 and 2% NaCl, and cultured at 28°C with constant temperature shaking (150rpm / min) 24h. The OD value was detected at 550nm, and the concentration of the bacterial solution and the total number of bacterial cells were calculated. Centrifuge (3000rpm / min) for 5 minutes to collect the bacteria, wash the bacteria three times with PBS, and prepare 10 7 cells / mL concentration.

[0078] Fifty healthy flounder (body length 9-11cm) that had not been vaccinated recently were kept in the aquarium for a week and then injected with bacteria. Each fish was intraperitoneally injected with 100 μL of live bacteria (10 6 tarda), the flounder was sacrificed at different time periods and the head kidney tissue was dissected.

[0079] Stimulation experiment of f...

Embodiment 3

[0082] Extraction and purification of total RNA

[0083] (1) Take flounder head kidney tissue 100 mg, put it into a homogenizer, and add 1000 μL Trizol reagent (invitrogen company) to it, and grind it thoroughly. If it is a cell, add 1000 μL Trizol to the culture well and pipette repeatedly to lyse the cells. Then transfer it to a 1.5 mL RNase-free centrifuge tube;

[0084] (2) Centrifuge at 12000rcf for 10min at 4°C;

[0085] (3) Take the supernatant, add it to a new centrifuge tube, and let it rest for a few minutes to completely lyse the nucleoprotein;

[0086] (4) Add 200 μL of chloroform to the supernatant, shake vigorously for 15 sec, and let stand at room temperature for 2-3 min;

[0087] (5) Centrifuge at 12000 rcf for 15 min at 4°C. After centrifugation, the product is divided into three layers: the lower phase is mainly protein, the middle phase is mainly DNA, and the upper phase is RNA;

[0088] (6) Extract the upper phase (rather less than more, do not touch t...

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Abstract

The invention discloses a method for detecting bastard halibut LITAF gene expression by applying reverse transcription-polymerase chain reaction (RT-PCR). The method lays a foundation for studying bastard halibut LITAF gene expression regulation mechanism and immunology function. TNF-alpha plays an important role in the process of immune response and foreign pathogenic bacteria killing, and the LITAF is a transcription factor of TNF-alpha gene and directly controls the expression of the TNF-alpha gene. Therefore, the expression abundance of the LITAF gene reflects the activity of a bastard halibut immune system to a certain extent and can be used as an immune monitoring index and immune agent quality evaluation index in prevention and treatment of bastard halibut diseases. By detecting the variation of the bastard halibut LITAF gene expression quantity, whether the bastard halibut is infected with a disease can be judged in advance and a measure of prevention and treatment can be taken timely so as to avoid unavoidable loss resulting from serious situation; and besides, the advantages and disadvantages of a fish immune agent can be evaluated so as to judge which immune agent has better effect during prevention and treatment of flounder diseases. The invention provides a technology platform for relative quantitative analysis of LITAF gene at the mRNA level.

Description

[0001] This application was supported by the National High-tech Development Program (863) Project (2012AA10A401), the National Science and Technology Support Program Project (2011BAD13B07, 2012BAD26B04) and the Tianjin Normal University Talent Research Startup Fund (Project No.: 5RL120). technical field [0002] The invention relates to the field of biological technology, and relates to a specific primer for detecting the expression of the flounder LITAF gene, and more specifically to a method for detecting the expression of the flounder LITAF gene by using a fluorescent RT-PCR technique. It is mainly used for the study of the expression regulation mechanism of the LITAF gene of the flounder and the study of the immunological function, as well as the detection of the early pathogenic infection of the flounder and the evaluation of the effect of the fish immune preparation. Background technique [0003] In recent years, with the improvement of people's living standards, fish ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 孙金生李硕李雪静耿绪云张亦陈
Owner TIANJIN NORMAL UNIVERSITY
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