Monopterus albus rhabdovirus Cr-ERV and RT-PCR detection primer and application thereof

A rhabdovirus, detection primer technology, applied in the application, virus, antiviral agent and other directions, can solve the problem of no detection method, and achieve the effect of ensuring high specificity, fast and simple method, and improving detection efficiency

Active Publication Date: 2019-01-11
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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  • Monopterus albus rhabdovirus Cr-ERV and RT-PCR detection primer and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] A kind of eel rhabdovirus CrERV, its isolation process is as follows:

[0027] 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.

[0028] Medium: M199, 10% fetal bovine serum, pH 7.0-7.2

[0029] 2) Collect the diseased cells, and use transmission electron microscopy for morphological identification.

[0030] 3) The virus is collected, and its whole genome is sequenced, and the sequence is shown in SEQ ID NO.1.

[0031] Morphological characteristics of eel rhabdovirus: the virus is bullet-shaped or spindle-shaped, a...

Embodiment 2

[0034] 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:

[0035]A kind of eel rhabdovirus CrERV RT-PCR detection primers, including F1: 5'-GAGATGAAGGAAGGACTGAGGAGAA-3', R1: 5'-CCCGACACCACTAAAGTATAAAA-3'; F2: 5'-GAGGAGCATTAGTTACTTAAGGGTC-3', R2: 5'-TCACAATTTGAATCGTCATGACATA -3'.

Embodiment 3

[0037] Utilize the eel rhabdovirus CrERV RT-PCR detection primers to detect the eel rhabdovirus, the method includes:

[0038] 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 sterilized double-distilled water, mix well at 65°C for 5 minutes, and immediately put it on ice; add 4 μl of 5×Reaction Buffer, 1 μl of RNase inhibitor, 2 μL of 5mM dNTPs, Add 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.

[0039] 2. Polymerase chain reaction amplification:

[0040] Use 25μl reaction system: add 10μM primers F1 and R1, add 1μL each to the PCR reaction tube, then add 2.5mMdNTPs 2μL, 25mM MgCl 2 1.5 μL, 0.5 μL of 5U Taq polymerase, 1 μl of t...

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Abstract

The invention belongs to the virus detection field, in particular, discloses a Chinese rice virus-Field eel rhabdovirus Cr-ERV and RT-PCR detection primer and an application thereof. The Monopterus albus rhabdovirus is isolated from diseased Monopterus albus seedlings, and the virus is (Chinese rice virus-Field eel rhabdovirus) CrERV, deposited as CCTCC NO: V201819, is of pioneering significance for the study of Monopterus albus rhabdovirus. Primers are designed to detect rhabdovirus of Monopterus albus. The primers have 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 rice field eel rhabdovirus CrERV and RT-PCR detection primers and applications. 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 is the most well-known, which can cause rabies in humans or other warm-blooded animals, and the fatality rate is almost 100%. 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 (ICTV) published the latest tenth report on virus classification in 2017, dividing Rhabdoviridae into 18 ge...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N15/47C12N15/11C12Q1/70C12Q1/686A61K39/205A61P31/14C12R1/93
CPCA61K39/12A61K2039/552A61P31/14C12N7/00C12N2760/20021C12N2760/20022C12N2760/20034C12Q1/686C12Q1/701C12Q2521/107
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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