A kind of tilapia parvovirus tipv and pcr detection primer and its application

A parvovirus and tilapia technology, applied in the direction of virus, virus/bacteriophage, single-stranded DNA virus, etc., can solve the problems of tilapia death, tilapia farming and export economic loss, etc., to ensure high specificity, The method is fast, simple, and highly specific

Active Publication Date: 2020-06-19
YANGTZE RIVER FISHERIES 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

Currently, there is no relevant report of parvovirus infection in fish among the currently divided parvovirus genus. The newly identified tilapia parvovirus in this patent application belongs to a new virus, which caused a large number of tilapias in my country to die in a short period of time. Huge economic losses from fish farming and export

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  • A kind of tilapia parvovirus tipv and pcr detection primer and its application
  • A kind of tilapia parvovirus tipv and pcr detection primer and its application

Examples

Experimental program
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Embodiment 1

[0025] A kind of tilapia parvovirus TiPV, its isolation process is as follows:

[0026] 1) Take out and grind the tilapia spleen, kidney and other tissues carrying the virus, dilute the medium by 100 times, centrifuge at 2000rpm, take the supernatant and filter, inoculate into a single layer of tilapia kidney cell line (Tilapia Kidney, TiK), After culturing at 25°C for 7 days, cytopathic effect (CPE) occurred. It can be seen that the TiK cells shrank and became round, the refractive index increased, some cells began to fall off, and the cell monolayer began to rupture.

[0027] Medium: M199, 10% fetal bovine serum, pH 7.0-7.2

[0028] 2) Collect the diseased cells, and use transmission electron microscopy for morphological identification.

[0029] 3) The virus is collected and sequenced to obtain the specific gene of tilapia parvovirus TiPV, which is shown in SEQ ID NO.1.

[0030] Morphological characteristics of tilapia parvovirus: the virus is spherical, and the virus has ...

Embodiment 2

[0032] Primers designed based on the difference between the specific gene of tilapia parvovirus TiPV (shown in SEQ ID NO.1) and other viral genes:

[0033] Including F1: 5′-TATCCAGTCCCGACCTACTCAAA-3′, R1: 5′-AGTTAAAACTAATCGCGTTCTTCCC-3′; F2: 5′–CACGTTAAGGGCGTATCGTGTAA-3′, R2: 5′-TATCTGCTGCTGTTGGTGTTGGT-3′.

Embodiment 3

[0035] Utilize Tilapia parvovirus TiPV PCR detection primers to detect Tilapia parvovirus, the method comprises:

[0036] 1. Extraction of total DNA from tilapia tissues or infected cells: Omega (USA) DNA extraction kit was used to extract tissue DNA.

[0037] 2. Polymerase chain reaction amplification:

[0038] Use 25μl reaction system: add 10μM primers F1 and R1 to each PCR reaction tube, add 1μl each, then add 2.5mMdNTPs 2μl, 25mM MgCl 2 1.5 μl, 0.5 μl of 5U Taq polymerase, 1 μl of template DNA, 2.5 μl of 10×Thermo PolReaction Buffer, make up the system to 25 μl with sterilized double distilled water, and perform PCR reaction.

[0039] Using the reaction product as a template, take 1 μl, add 1 μl of 10 μM primers F2 and R2, then add 2 μl of 2.5mM dNTPs, 25mM MgCl21.5μl, 5U Taq polymerase 0.5μl, 10×ThermoPol Reaction Buffer 2.5μl, and use sterilized Make up the system to 25 μl with double distilled water, and carry out PCR reaction.

[0040] Reaction conditions: pre-dena...

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Abstract

The invention belongs to the field of virus detection, and specifically discloses a TiPV and PCR detection primer for tilapia parvovirus and application. The inventors isolated a parvovirus from the infected tilapia adult fish, which was named tilapia parvovirus (Tilapia parvovirus) TiPV, and its deposit number was CCTCC NO: V201856. It is of pioneering significance for the research of tilapia parvovirus. The primer designed for the specific sequence of the virus can be used to detect tilapia parvovirus. The primer has good specificity and high sensitivity.

Description

technical field [0001] The invention belongs to the technical field of virus molecular detection, and in particular relates to a tilapia parvovirus TiPV and a PCR detection primer and application thereof. [0002] technical background [0003] Parvovirus (Rhabdovirus) is the smallest single-stranded DNA virus found so far. The virion is an icosahedron with a diameter of 18-30 nm, no envelope, and equiaxed symmetry. Parvoviruses are widely distributed in nature, can infect humans, vertebrates and invertebrates, and are associated with many diseases. Among them, human parvovirus B19 (Human parvovirus B19, B19) is well-known and can cause a series of severe autoimmune diseases in young children. Infection during pregnancy can lead to hydrops fetalis, miscarriage or congenital infection, which is more harmful to newborns and young children. attracted worldwide attention. Therefore, it is of great significance to conduct in-depth research on parvoviruses. In addition, parvoviru...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C12Q1/686C12Q1/70C12N15/11
CPCC12N7/00C12N2750/14321C12Q1/686C12Q1/701
Inventor 刘文枝曾令兵范玉顶
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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