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A detection primer and application of tilapia parvovirus tipv CPA

A parvovirus and tilapia technology, applied in the direction of virus, application, virus peptide, etc., can solve the problems of tilapia farming and export economic loss, tilapia death, etc., to ensure high specificity, fast and simple method, The effect of improving detection efficiency

Active Publication Date: 2021-04-16
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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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] A tilapia parvovirus TiPV, its separation process is as follows:

[0020] 1) After the virus-carrying tilapia spleen, kidney and other tissues are taken out and ground, the culture medium is diluted 100 times, the supernatant is filtered after centrifugation at 2000 rpm, and inoculated into the tilapia kidney cell line (Tilapia Kidney, TiK) monolayer, Cultured at 25°C for 7 days, resulting in cytopathic effect (CPE), which showed that TiK cells shrank and became round, the refractive index increased, some cells began to fall off, and the cell monolayer began to rupture.

[0021] Medium: M199, 10% fetal bovine serum, pH 7.0~7.2

[0022] 2) The diseased cells were collected and morphologically identified by transmission electron microscopy.

[0023] 3) Collect the virus, sequence it, and obtain the specific gene of tilapia parvovirus TiPV, which is shown in SEQ ID NO.1.

[0024] Morphological characteristics of tilapia parvovirus: the virus is spherical, and the virus h...

Embodiment 2

[0026] Primers designed based on the difference between the specific gene of tilapia parvovirus TiPV (shown in SEQ ID NO. 1) and other viral genes:

[0027] A tilapia parvovirus TiPV LAMP detection primer, comprising 2a1s: GAACCGGGTCTTGAGTTGTGCCGACAAATTACAGTC; 2a: GAACCGGGTCTTGAGTTGT; 3a: CGCTGCGGAGAGCTTATCGG; 4s: TGCCACCGATAGTGGACG; 5a: CGGAATTGCTGGATCCGTG.

Embodiment 3

[0029] Using tilapia parvovirus TiPV CPA detection primers to detect tilapia parvovirus, the methods include:

[0030] 1. Extraction of total DNA from tilapia tissue or infected cells: Extract tissue DNA using Omega (USA) DNA extraction kit.

[0031] 2. CPA reaction amplification:

[0032] Using 25μl reaction system: 10× Reaction Buffer 2.5μL, Betaine 0.7mol / L, Bst DNA polymerase 8U, primer 2a1s 1.0μmol / L, primers 2a and 3a each 0.5μmol / L, primers 4s and 5a each 0.6μmol / L, Mg 2+ 8.0 mmol / L, 1.2 mmol / L of dNTPs, 1 μL of template DNA, and nuclease-free water to make up to 25 μL. The optimal reaction conditions were isothermal amplification at 63 °C for 60 min.

[0033] 2a1s: GAACCGGGTCTTGAGTTGTGCCGACAAATTACAGTC; 2a: GAACCGGGTCTTGAGTTGT; 3a: CGCTGCGGAGAGCTTATCGG; 4s: TGCCACCGATAGTGGACG; 5a: CGGAATTGCTGGATCCGTG.

[0034] Analysis of CPA reaction amplification products:

[0035] 1. Take 5 μL of the amplified product, electrophoresis it on a 2% agarose gel, and place it in a g...

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Abstract

The invention belongs to the field of virus detection, and specifically discloses a detection primer of Tilapia parvovirus (Tilapia parvovirus) TiPV CPA and its application. It is Tilapia parvovirus (Tilapia parvovirus) TiPV, and the preservation number is: CCTCC NO: V201856, which has pioneering significance for the research of Tilapia parvovirus. The primers designed for the virus sequence can detect the tilapia parvovirus, and the primers have good specificity and high sensitivity.

Description

technical field [0001] The invention belongs to the technical field of virus molecular detection, in particular to a tilapia parvovirus TiPV CPA detection primer and application. [0002] technical background [0003] Parvovirus is the smallest single-stranded DNA virus discovered so far, with icosahedral virions 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 a variety of diseases. Among them, human parvovirus B19 (B19) is well-known and can cause a series of serious autoimmune diseases in young children. Infection during pregnancy can lead to fetal edema, 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 parvovirus. In addition, parvovirus can also cause a variety of acute infectious diseases in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12N15/35C12R1/93
CPCC07K14/005C12N2750/14322C12Q1/6844C12Q1/701
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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