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A CRERV RT-CPA detection prim for Monopterus albus rhabdovirus and application thereof

A technology of RT-CPA and rhabdovirus, which is applied in the field of virus molecular detection, can solve the problems of no detection method, etc., and achieve the effect of ensuring high specificity, improving detection efficiency, and fast and simple method

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

At present, there is no public report on the full-length sequence of Chinese rice-field eel rhabdovirus (CrERV), let alone an effective detection method.

Method used

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  • A CRERV RT-CPA detection prim for Monopterus albus rhabdovirus and application thereof
  • A CRERV RT-CPA detection prim for Monopterus albus rhabdovirus and application thereof
  • A CRERV RT-CPA detection prim for Monopterus albus rhabdovirus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] A kind of eel rhabdovirus CrERV, its isolation process is as follows:

[0020] 1) After taking out and grinding the eel spleen and kidneys carrying the virus, the culture medium was diluted 100 times, centrifuged at 2000rpm, and the supernatant was filtered, inoculated into a monolayer of crucian carp brain cell line (GibelCarpBrain, GiCB), cultured at 25°C for 7 On the next day, the cytopathic effect (CPE) occurred, and it could be seen that the GiCB 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) Collect the diseased cells, and use transmission electron microscopy for morphological identification.

[0023] 3) The virus is collected, and its whole genome is sequenced, and the sequence is shown in SEQ ID NO.1.

[0024] Morphological characteristics of eel rhabdovirus: the virus is bullet-shaped or spindle-shaped, a...

Embodiment 2

[0027] Primers designed based on the difference between the full sequence of eel rhabdovirus CrERV (shown in SEQ ID NO.1) and the full sequence of other viruses:

[0028] A primer for detecting eel rhabdovirus CrERV RT-CPA, including 1s: CAGGATATACGACCCAATC; 2a1s: TAACTCTTTGAGCAGGCCT-CAGGATATACGACCCAATC; 2a: TAACTCTTTGAG CAGGCCT; 3a: CATCAGATTCATGTGTACA; 4s: CAGAAGTCTCAGGACCACCA; 5a: AG GGATGGGCTGCCTTGGAGC.

Embodiment 3

[0030] The detection of eel rhabdovirus by using CrERV RT-CPA detection primers includes:

[0031] 1. Extraction of total RNA from eel tissue or infected cells and synthesis of cDNA: using Extract the total RNA of the sample to be tested. Configure the reverse transcription reaction system: 3 μL of total RNA, 1 μL of Oligo(dT)18 primer, 8 μL of sterile double distilled water, mix well at 65°C for 5 minutes and immediately put it on ice; mix 4 μl of 5×Reaction Buffer, 1 μl of RNase inhibitor, 5 mM dNT Ps Add 2 μL of M-MLV reverse transcriptase and 1 μL of M-MLV reverse transcriptase to the reaction solution in an ice bath, mix well, store at 42°C for 1 hour, and 95°C for 5 minutes, and store the obtained cDNA template at -20°C for later use.

[0032] 2. Polymerase chain reaction amplification:

[0033] Use 25μl reaction system: Reaction Buffer 2.5μL, Betaine 0.7mol / L, Bst DNA polymerase 8U, primer 2a1s 1.0μmol / L, primer 2a, 3a each 0.5μmol / L, primer 4s, 5a each 0.6 μmol / L, ...

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Abstract

The invention belongs to the field of virus detection, in particular discloses a rhabdovirus of Monopterus albus CrERV RT- CPA detection prim and application, inventors first isolate a kind of Monopterus albus rhabdovirus from diseased Monopterus albus seedlings, which is Monopterus albus rhabdovirus (Monopterus albus rhabdovirus- Field eel rhabdovirus) CrERV, primers designed for the virus sequence, 1s: CAGGATATACGACCCAATC; 2a1s:TAACTCTTTGAGCAGGCCTCAGGATATACGACCCAATC; 2a:TAACTCTTTGAGCAGGCCT; 3a:CATCAGATTCATGTGTACA; 4s:CAGAAGTCTCAGGACCACCA; 5a:AGGGATGGGCTGCCTTGGAGC; Monopterus albus rhabdovirus can be detected with this primer, which 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 detection primer and application of eel rhabdovirus CrERV RT-CPA. technical background [0002] Rhabdoviridae viruses are a class of negative-strand RNA viruses with a wide range of hosts, and their particles are bullet-shaped or rod-shaped. Among them, rabies virus (Rabies virus) is the most well-known, can cause rabies in humans or other warm-blooded animals, and the case fatality rate is almost 100%, which has caused widespread concern worldwide, causing society to panic about the safety of breeding pets. Therefore, it is of great significance to conduct in-depth research on rhabdoviruses. In addition, rhabdoviruses can also cause a variety of acute infectious diseases in livestock (such as bovine epidemic fever, ephemeral fever), as well as infectious diseases in fish and a variety of crops. The International Committee on Taxonomy of Viruses ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12R1/93
CPCC12Q1/6844C12Q1/701C12Q2521/107
Inventor 刘文枝曾令兵范玉顶周勇
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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