LAMP primer for detecting tilapia parvovirus and application thereof

A parvovirus, tilapia technology, applied in DNA/RNA fragments, microorganisms, recombinant DNA technology, etc., can solve the problems of tilapia breeding and export economic losses, tilapia death, etc., to ensure high specificity, The method is fast and simple, and the effect of improving the detection efficiency

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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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] A kind of tilapia parvovirus TiPV, its isolation process is as follows:

[0019] 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.

[0020] Medium: M199, 10% fetal bovine serum, pH 7.0-7.2

[0021] 2) Collect the diseased cells, and use transmission electron microscopy for morphological identification.

[0022] 3) The virus is collected and sequenced to obtain the specific gene of tilapia parvovirus TiPV, which is shown in SEQ ID NO.1.

[0023] Morphological characteristics of tilapia parvovirus: the virus is spherical, and the virus has ...

Embodiment 2

[0025] Primers designed based on the difference between the specific gene of tilapia parvovirus TiPV (shown in SEQ ID NO.1) and other viral genes:

[0026] A detection primer for tilapia parvovirus TiPVLAMP, including F3: 5'-CAACTAAAGACCCGGTTCC-3', B3: 5'-GCCTTGGTAGCGTAAGTTCA-3'; FIP: 5'-GCTATCTCCTCGTTGCTCGGTG-ATCCAGCAATTCCGGAGACA-3', BIP: 5'-AGGACGGCCCGGAAGTACTG -TCAAATCCGAGCTGTGTAGC-3'.

Embodiment 3

[0028] Utilize tilapia parvovirus TiPV LAMP to detect primers to the detection of tilapia parvovirus, the method comprises:

[0029] 1. Extraction of total DNA from tilapia tissues or infected cells: Omega (USA) DNA extraction kit was used to extract tissue DNA.

[0030] 2. LAMP reaction amplification:

[0031] Use 25μl reaction system: including: inner primers FIP and BIP each 0.8μM, outer primers F3 and B3 each 0.1μM, dNTPs1mM, Betaine 0.5M, DTT 4mM, MgCl 2 8mM, Bst DNA polymerase 8U, AMV reverse transcriptase 5U, template RNA 5μl, 1×ThermoPol Reaction Buffer. After the reaction tube was incubated at 65°C for 60 minutes, it was inactivated at 80°C for 2 minutes.

[0032] F3: 5'-CAACTAAAGACCCGGTTCC-3',

[0033] B3: 5'-GCCTTGGTAGCGTAAGTTCA-3';

[0034] FIP: 5'–GCTATCTCCTCGTTGCTCGGTG-ATCCAGCAATTCCGGAGACA-3',

[0035] BIP: 5'-AGGACGGCCCGGAAGTACTG-TCAAATCCGAGCTGTGTAGC-3'.

[0036] LAMP reaction amplification product analysis:

[0037] 1. Take 5 μL of the amplified product,...

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Abstract

The invention belongs to the field of virus detection, and specifically discloses a LAMP primer for detecting tilapia parvovirus and application thereof, 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 TiPV LAMP detection primer for tilapia parvovirus and its application. technical background [0002] Parvovirus (Parvovirus) is the smallest single-stranded DNA virus found so far. The diameter of the virus particle is icosahedron with a diameter of 18-30nm, 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, par...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/701C12Q2531/119
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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