TiPV and PCR detection primer for tilapia parvovirus and application

A parvovirus, tilapia technology, applied in the direction of virus, virus/phage, single-stranded DNA virus, etc., can solve the problems of tilapia breeding and export economic loss, tilapia death, etc., to ensure high specificity, The method is quick and easy, and the effect is highly specific

Active Publication Date: 2019-01-18
YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

Method used

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  • TiPV and PCR detection primer for tilapia parvovirus and application
  • TiPV and PCR detection primer for tilapia parvovirus and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] TiPV, a kind of tilapia parvovirus, is separated as follows:

[0025] 1) After removing the virus-carrying tilapia spleen, kidney and other tissues and milling, the medium is diluted 100 times, centrifuged at 2000 rpm, and the supernatant is filtered and inoculated into the tilapia kidney cell line (TilapiaKidney, TiK) monolayer. Culturing for 7 days at 25°C produced a cytopathic effect (CPE). It can be seen that TiK cells contracted and rounded, the refractive index increased, some cells began to fall off, and the cell monolayer began to break.

[0026] Medium: M199, 10% fetal bovine serum, pH 7.0~7.2

[0027] 2) Collect diseased cells and use transmission electron microscopy for morphological identification.

[0028] 3) The virus is collected and sequenced to obtain the specific gene of tilapia parvovirus TiPV, which is shown in SEQ ID NO.1.

[0029] Morphological characteristics of tilapia parvovirus: The virus has a spherical shape and has no envelope, which is consistent wi...

Embodiment 2

[0031] Primers designed based on the difference between the specific gene of tilapia parvovirus TiPV (shown in SEQ ID NO.1) and other viral genes:

[0032] Including F1: 5'-TATCCAGTCCCGACCTACTCAAA-3', R1: 5'-AGTTAAAACTAATCGCGTTCTTCCC-3'; F2: 5'–CACGTTAAGGGCGTATCGTGTAA-3', R2: 5'-TATCTGCTGCTGTTGGTGTTGGT-3'.

Embodiment 3

[0034] The detection of tilapia parvovirus using TiPV PCR primers includes:

[0035] 1. Extraction of total DNA from tilapia tissue or infected cells: Use Omega (USA) DNA extraction kit to extract tissue DNA.

[0036] 2. Polymerase chain reaction amplification:

[0037] Using 25μl reaction system: In the PCR reaction tube, add 10μM primers F1 and R1 each plus 1μl, then add 2.5mMdNTPs 2μl, 25mM MgCl 2 1.5μl, 5U Taq polymerase 0.5μl, template DNA 1μl, 10×Thermo PolReaction Buffer 2.5μl, fill up the system to 25μl with sterilized double distilled water, and perform PCR reaction.

[0038] Using the reaction product as a template, take 1μl, 10μM primers F2 and R2 and add 1μl each, then add 2μl 2.5mM dNTPs, 25mM MgCl21.5μl, 5U Taq polymerase 0.5μl, 10×ThermoPol Reaction Buffer 2.5μl, and sterilize Double distilled water to make up the system to 25μl, and perform PCR reaction.

[0039] Reaction conditions: 95°C pre-denaturation for 5 minutes; 95°C for 30s, 55°C for 30s, 72°C for 30s, 30 cycl...

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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 specifically relates to a TiPV and PCR detection primer and application of tilapia parvovirus. technical background [0002] Rhabdovirus (Rhabdovirus) is the smallest single-stranded DNA virus discovered so far. It has an icosahedron with a diameter of 18-30nm, no capsule, and isometric symmetry. Parvoviruses are extremely widespread 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 serious autoimmune diseases in young children. Infection during pregnancy can cause fetal edema, miscarriage or congenital infection, which is more harmful to newborns and infants. Aroused widespread concern worldwide. Therefore, in-depth research on parvovirus is of great significance. In addition, parvovirus can also cause a variety of acute infect...

Claims

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Application Information

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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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