A method for judging whether tilapia is in a state of hypoxic stress and its primers and kit

A stress state, tilapia technology, applied in the field of biochemistry and molecular biology, can solve problems affecting the growth and reproduction of tilapia, achieve good specificity and sensitivity, and good amplification efficiency

Inactive Publication Date: 2020-01-24
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Tilapia has strong adaptability to low oxygen conditions, but too low oxygen concentration will also affect the growth and reproduction of tilapia

Method used

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  • A method for judging whether tilapia is in a state of hypoxic stress and its primers and kit
  • A method for judging whether tilapia is in a state of hypoxic stress and its primers and kit
  • A method for judging whether tilapia is in a state of hypoxic stress and its primers and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The PCR detection method of the specificity of GABA transporter 2 gene in the spleen of embodiment 1 tilapia

[0032] The samples are the 6h, 12h and 24h samples of the control group and the hypoxic treatment group. The total RNA in the spleen of tilapia was extracted using Trizol, and the first strand of cDNA was synthesized using a reverse transcription kit (Dongsheng), which was used as a template for PCR detection of GABA transporter 2 gene and fluorescent quantitative PCR.

[0033] 1.1.1 Experimental treatment of tilapia and extraction of total RNA from spleen

[0034] (1) tilapia experimental treatment: the treatment method of control group (N=5) is to continuously fill the fish tank water body containing tilapia with oxygen, and the oxygen concentration is maintained at about 7.5-7.8mg / l; hypoxic group ( N=5) The treatment method is to continuously fill the fish tank water containing tilapia with gas, the gas is composed of oxygen and nitrogen, adjust the fillin...

Embodiment 2

[0050] Example 2 Utilize the specific primers of the EF1α gene synthesized in Example 1 and the specific primers of the GABA transporter 2 gene to carry out fluorescent quantitative PCR amplification, and analyze the expression of GABA transporter 2 in the spleen of the hypoxic group and the control group tilapia expression volume.

[0051] 1.1 Sample source

[0052] The samples used in Example 2 were 6h, 12h and 24h control groups and spleen total RNA of the hypoxic treatment group, and each treatment was repeated three times.

[0053] 1.1 cDNA synthesis and dilution for fluorescent quantitative PCR

[0054] The cDNA was synthesized as in Example 1, and the obtained cDNA product was diluted 10 times and then used for fluorescent quantitative PCR (2 μg RNA sample was reverse-transcribed and then diluted to 200 μl).

[0055] 1.2 Fluorescence quantitative PCR

[0056] Fluorescent quantitative PCR reaction system is calculated in 10 μl:

[0057]

[0058] Fluorescent quanti...

Embodiment 3

[0069] Example 3 analyzes primer specificity and sensitivity.

[0070] 1.1 Primer specificity experiment

[0071] 1.1.1 Ligation of PCR product to T carrier

[0072] The amplified products of the specific primers of the GABA transporter 2 gene of the control group in Example 1 were connected to the pGEM-TEasy vector (Promega), and the connection reaction system was 5 μl of 2x connection buffer, 0.5 μl of pGEM-T Easy vector, and T4DNA Ligase 1 μl, PCR product 3.5 μl, total volume 10 μl. After mixing, place in a 4°C refrigerator overnight.

[0073] 1.1.2 Preparation of ampicillin plate containing X-gal and IPTG

[0074] X-gal was prepared as a 20mg / ml stock solution in DMSO. Store in foil-wrapped tubes at -20°C to prevent damage from exposure to light. Preparation of IPTG: After dissolving 2g of IPTG in 8ml of distilled water, dilute to 10ml with distilled water. After preparation, filter-sterilize with a 0.22 μm filter and store at -20°C. Mix 40 μl X-gal and 5 μl IPTG an...

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Abstract

The invention discloses a method for determining whether a tilapia is in a hypoxia stress state or not. The method comprises the steps of measuring expression quantity of a GABA transport protein 2 gene of a spleen of a normal tilapia and that of a spleen of a to-be-measured hypoxia-processed tilapia separately; if the expression quantity of the GABA transport protein 2 gene of the spleen of the to-be-measured hypoxia-processed tilapia is obviously lower than that of the spleen of the normal tilapia, representing that the to-be-measured hypoxia-processed tilapia is in the hypoxia stress state; and if obvious difference does not exist between the two sides, representing that the to-be-measured hypoxia-processed tilapia is not in the hypoxia stress state. Experiments prove that a specific primer used for detecting the GABA transport protein 2 gene of the spleen of the tilapia has high specificity and sensitivity and high amplification efficiency, and can be used for detecting the level of the expression efficiency of the GABA transport protein 2 gene.

Description

Technical field: [0001] The invention belongs to the field of biochemistry and molecular biology, and specifically relates to a method for judging whether tilapia is in a hypoxic stress state, primers and a kit thereof. Background technique: [0002] Tilapia, commonly known as African crucian carp, is a freshwater farmed fish cultivated by key scientific research in the world's aquaculture industry, and is known as one of the main sources of animal protein in the future. Tilapia has the advantages of fast growth and strong adaptability, and is the main farmed fish in the world. Tilapia has a strong ability to adapt to low oxygen conditions, but low oxygen concentration will also affect the growth and reproduction of tilapia. Therefore, the study on tilapia hypoxia is of great significance. When studying hypoxia-related problems in tilapia, it is first necessary to be able to accurately determine whether tilapia is under hypoxic stress conditions. [0003] Real-time fluore...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/6888C12Q2600/124C12Q2600/166C12Q2531/113C12Q2545/101C12Q2563/107
Inventor 夏军红李红莲
Owner SUN YAT SEN UNIV
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