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A virulent strain of tilapia lake virus and its rpa detection primer and method

A technology for tilapia and virus strains, applied in the direction of biochemical equipment and methods, methods based on microorganisms, viruses, etc., can solve problems that affect the on-site diagnosis of Luohu virus disease, pathogen monitoring, epidemiological investigation and epidemic treatment. Problems such as drug or vaccine disease, specificity and sensitivity are different, to achieve the effect of ensuring high specificity, rapid and accurate analysis and detection, and fast and simple methods

Active Publication Date: 2022-05-20
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Due to the significant geographical differences in the TiLV genome, the application of molecular biology methods to different geographical strains may have different specificity and sensitivity, and even false negative results, which will seriously affect the on-site diagnosis and pathogen monitoring of Luohu virus disease. , epidemiological investigation and epidemic management
[0005] However, so far, no effective drugs or vaccines have been developed to prevent diseases caused by TiLV

Method used

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  • A virulent strain of tilapia lake virus and its rpa detection primer and method
  • A virulent strain of tilapia lake virus and its rpa detection primer and method
  • A virulent strain of tilapia lake virus and its rpa detection primer and method

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Effect test

Embodiment 1

[0035] A tilapia lake virus GD1710 isolation and whole genome sequence determination, the main process is as follows:

[0036] (1) Under aseptic conditions, take the liver, kidney, and spleen of tilapia infected with tilapia lake virus, add them to sterilized PBS containing double antibodies to make a 1:1 homogenate, and store them at -20 After freezing and thawing three times at ℃, the tissue homogenate was centrifuged at 2000rpm / min for 10min to take the supernatant, then centrifuged at 5000rpm / min for 10min to take the supernatant, and finally centrifuged at 10000rpm / min to take the supernatant and passed it through a filter membrane with a pore size of 0.22μm. Sterilize by filtering through a pore filter, and inoculate a monolayer of tilapia brain cells (TiB cells) at a density of 80%-90%. Adsorb at room temperature for 1 hour, discard the virus solution, add maintenance solution (M199 containing 3% FBS) equal to the culture solution, incubate at a constant temperature of ...

Embodiment 2

[0061] Embodiment 2: The regression infection test of TiLV-GD1710 new isolated strain

[0062] The virus solution of TiLV-GD1710 cells passed to the third generation was treated with a concentration of 1.8x10 4 TCID50 / mL for different sizes

[0063] Nile tilapia, Egyptian red tilapia, Olia tilapia and Mozambique tilapia were intraperitoneally injected, and the cumulative mortality after 2 weeks was counted (see Table 1).

[0064] It can be seen from the table that the TiLV-GD1710 virus strain prepared by the present invention has strong toxicity to different species of tilapia and fish of different sizes. After artificially infecting TiLV-GD1710 prepared by the present invention, most tilapias have obvious abdominal swelling and a large amount of ascites in the abdominal cavity; a few fish are particularly emaciated, slight bleeding spots appear on the surface of the fish body, and the intestinal tract is full of yellow transparent fluid .

[0065] Using TaqMan qPCR to stu...

Embodiment 3

[0068] Embodiment 3: RPA amplification primer design

[0069] Based on the S2 gene segment of Luohu virus GD1710 strain (shown in SEQ ID NO.1-10), the present invention designs RPA detection primers of Luohu virus, and the nucleotide sequence is as follows:

[0070] Upstream primer TiLV-S2-F: 5'-GGCGGCTCTAGGGTACA- 3' (SEQ ID NO. 11)

[0071] Downstream primer TiLV-S2-R: 5'-CGAAGTGTTGGAACCCGTCAT- 3' (SEQ ID NO.12)

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Abstract

The invention discloses a virulent strain of tilapia lake virus and its RPA detection primers and method. The invention designs a primer set for RPA reaction; the invention isolates Luohu virus for the first time, and the detection method is fast, simple and highly specific , high sensitivity, suitable for on-site detection of farms, etc., and has extremely high application value.

Description

technical field [0001] The invention belongs to the field of virus molecular detection, and relates to a virulent strain of tilapia lake virus and its RPA (polymerase amplification technology) detection primer and method. Background technique [0002] Since 2009, Israel, Ecuador, Egypt, Thailand, India, Malaysia and other regions have successively broken out a new type of RNA virus---Tilapia Lake Virus (TiLV) for short. Tilapia farming poses enormous threats and serious challenges. Tilapia Lake Virus was preliminarily determined to belong to the Orthomyxoviridae family with influenza virus, and it is also a highly contagious and virulent virus. Infection with Tilapia Lake Virus can cause the death rate of Tilapia as high as 70%-90%. The disease caused by fish lake virus has attracted great attention from the global tilapia farming industry and related industries. [0003] Because TiLV has the characteristics of high infectivity and high mortality, it has aroused great pani...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C12N15/11C12Q1/70C12Q1/6844C12R1/93
CPCC12N7/00C12Q1/6844C12Q1/701C12N2760/16021C12Q2531/119C12Q2521/507C12Q2522/101C12Q2537/1376Y02A50/30
Inventor 曾伟伟王英英王庆尹纪元李莹莹任燕刘春张德峰石存斌
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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