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Reference gene of procambarus clarkia in-vivo tissue and primer thereof

A technology of Procambarus clarkii and internal reference genes, which is applied in the field of molecular biology to achieve high amplification efficiency, strong specificity, and improved repeatability

Active Publication Date: 2022-04-15
西北农林科技大学深圳研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In view of the fact that the internal reference genes of Procambarus clarkii have not been reported at different times (stages) of WSSV infection, different drug treatments, and different tissues, in order to further study the influence of WSSV infection on the expression of related functional genes in the tissues of Procambarus clarkii, it is necessary to Screening of internal reference genes of Procambarus clarkii infected with WSSV virus

Method used

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  • Reference gene of procambarus clarkia in-vivo tissue and primer thereof
  • Reference gene of procambarus clarkia in-vivo tissue and primer thereof
  • Reference gene of procambarus clarkia in-vivo tissue and primer thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] This example presents the screening method and results of the optimal internal reference genes in different tissues of Procambarus clarkii infected with WSSV at different times.

[0031] 1. Test materials

[0032] Select healthy Procambarus clarkii (17.07±2.15g) with basically the same body weight as the aquaculture animals to be tested, and raise them in a 30L water tank at 28°C, and feed them twice a day until the formal experiment. Each shrimp in the experimental group was injected with 100 μL of WSSV virus solution (prepared with L-15 medium containing 5% bovine embryonic serum as a solvent), and samples were taken at 24h, 48h, and 72h after injection. Healthy Procambarus clarkii. Shrimp in each group were dissected under aseptic conditions to obtain 6 different tissues including gills, muscles, brain, liver, intestines and gonads.

[0033] 2. Total RNA extraction and cDNA synthesis of Procambarus clarkii

[0034] Total RNA was extracted from different tissues ac...

Embodiment 2

[0061] This example presents the screening method and results of the most suitable internal reference genes in different tissues of Procambarus clarkii infected with WSSV and treated with different drugs.

[0062] 1. Test materials

[0063] Operations such as selection, feeding and tissue dissection of Procambarus clarkii are the same as in Example 1. The difference from Example 1 is that the treatment groups of Procambarus clarkii are shown in Table 5. In Table 5, "W" represents the WSSV virus liquid, and "M" represents the matrine solution. PBS (pH=7.4) formula: potassium dihydrogen phosphate 0.24g, disodium hydrogen phosphate 1.44g, sodium chloride 8.0g, potassium chloride 0.2g, add water to 1000mL. The preparation method of WSSV virus solution: the virus stock solution was prepared with L-15 medium containing 5% bovine embryonic serum as a solvent, and diluted with TM (100 mM Tris-HCl, 10 mM MgCl2, pH=7.5) buffer solution to the desired value when used. demand. Prepara...

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Abstract

The invention discloses a reference gene of an in-vivo tissue of procambarus clarkia infected with white spot syndrome virus. The reference genes are an EIF gene and a TBP gene, the nucleotide sequence of the EIF gene is shown as SEQ ID NO.8, and the nucleotide sequence of the TBP gene is shown as SEQ ID NO.7. The invention further discloses a kit for detecting the TBP gene. The invention also designs a primer of the reference gene, and the primer has strong specificity and high amplification efficiency. According to the invention, the technical blank that no stable reference gene exists in different tissues in different periods of WSSV infected by the procambarus clarkia, different drug treatment and the like is filled up, and a reliable analysis basis is provided for quantitative expression of related functional genes of the tissues under the condition that the procambarus clarkia is infected by the WSSV.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and relates to the prevention and treatment of aquatic animal diseases, in particular to the screening method of the optimal internal reference gene of Procambarus clarkii infected with WSSV at different times, different drug treatments, and different tissues, as well as its primers and application. Background technique [0002] Procambarus clarkii ( Procambarus clarkii ) Commonly known as crayfish, it belongs to the phylum Arthropoda, the class Crustacea, the order Decapoda, and the family Cambaridae. Originally produced in the United States, it was introduced to China from the 1930s to the 1940s. It caused great harm to the environment as an invasive species at the beginning, and then became an aquatic economic animal after domestication. At present, it is widely cultivated all over the country and the scale is increasing day by day. In 2019, the output value of Procambarus clarkii h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
Inventor 王二龙王高学杜辉刘天强凌飞刘韬刘佳陈诚
Owner 西北农林科技大学深圳研究院
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