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Double-RNA virus, method for quickly identifying double-RNA virus, and application

An RNA virus and segment technology, applied in double-stranded RNA viruses, DNA/RNA fragments, viruses, etc., can solve the problem that the virus cannot be effectively detected, and achieve the effect of good immune protection.

Active Publication Date: 2021-12-03
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the limitation of these methods is that the gene sequence or protein sequence information of the virus must be predicted in advance, and it cannot be effectively detected for unknown new viruses

Method used

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  • Double-RNA virus, method for quickly identifying double-RNA virus, and application
  • Double-RNA virus, method for quickly identifying double-RNA virus, and application
  • Double-RNA virus, method for quickly identifying double-RNA virus, and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, viral metagenomic sequencing

[0035] 1. Collection of diseased samples: In 2018, an unknown pathogenic disease broke out in a largemouth bass farm in Sanshui, Guangdong. Researchers in our laboratory went to the diseased place to collect tissue samples of the diseased largemouth bass and preserved them.

[0036] Such as figure 1 As shown in the table, the clinical symptoms of the largemouth bass with the disease were mainly diarrhea and enteritis, and the clinical signs of inflammation such as congestion spots in the liver and yellow mucus in the intestinal tract were found in the autopsy.

[0037] 2. Virus metagenomic sequencing

[0038] In order to determine the specific cause of the disease outbreak, the applicant sent samples of the diseased largemouth bass to Guangzhou Meige Gene Technology Co., Ltd. for viral metagenomic sequencing.

[0039] The DNA and RNA of the diseased samples were extracted separately, and the DNA and RNA were divided into ...

Embodiment 2

[0046] Embodiment 2, virus isolation and observation

[0047] Take 100 mg of liver, spleen and kidney mixed tissues of diseased largemouth bass and put them in a 1.5 mL centrifuge tube, add 1 mL of PBS to it, homogenize for 3 min, add 10 μL of double antibodies (penicillin and streptomycin) to each tube, and incubate at 4°C for 2 After ~4h, centrifuge at 4°C, 8000r / min for 20min, filter with a 0.22μm filter, and dilute the filtered virus solution by 100 times, inoculate the monolayer PSF, CCO, FHM, CIK, EPC, CPB cells cultured for 16~24h respectively , as a control, use L-15 medium instead of virus liquid, 28°C without CO 2 After incubation in the incubator for 1 hour, add L-15 medium with a final concentration of 2% FBS, culture in the incubator, and observe every day. If there is no obvious CPE within 7 days, the cells will be blindly passed on the 7th day (at most 5 passages), when obvious CPE appeared, repeated freezing and thawing twice, collected by filtration and sto...

Embodiment 3

[0052] Embodiment 3, viral genome nucleic acid type determination

[0053] Nucleotype determination: The DNA biosynthesis inhibitor 5-bromo-2-deoxyuracil (BUDR) was used to identify the nucleic acid type of the virus. Largemouth bass ranavirus (LBRV), a DNA virus, was used as a positive control. Take 4 flasks of cells, inoculate LBBV and LBRV respectively, and inoculate 2 flasks of cells (treatment and control) with each virus. After incubation for 1 h, one of the flasks is supplemented with 2% FBS L-15 medium containing BUDR (the final concentration of BUDR is 500μg / mL), another bottle of L-15 medium supplemented with 2% FBS without BUDR, placed in 28℃ without CO 2 Cultivate in an incubator. After 80% of the control cells have lesions, freeze and thaw twice, collect the supernatant, and measure the virus TCID 50 .

[0054] Virus RNA electrophoresis: collect the virus supernatant, centrifuge at 4000rpm / min for 10min, filter with a 0.22μm filter, and collect the virus liqu...

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Abstract

The invention relates to the technical field of biology, and particularly discloses a double-RNA virus, a method for quickly identifying the double-RNA virus, and application. The double-RNA virus comprises a genome A segment and a genome B segment, the whole genome sequence of the double-RNA virus comprises four ORFs, namely VP1, VP2, VP3 and VP4, the genome A segment contains two gene spacer regions, the gene spacer region of the VP2 and the VP4 is 1458bp-1949bp, and the gene spacer region of the VP3 and the VP4 is 2445bp-2663bp. According to the double-RNA virus, the method for quickly identifying the double-RNA virus, and the application, the virus pathogen causing symptoms of morbidity of largemouth bass of defecation and enteritis is identified as the novel double-RNA virus, the whole genome sequence of the virus is obtained, and the range of unknown pathogen is narrowed through a virus metagenome technology, so that the working efficiency of determining the unknown pathogen is greatly improved, and a novel rapid and effective technical method is provided for determining other unknown pathogens.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a biRNA virus and a method and application for rapid identification of the biRNA virus. Background technique [0002] There are a large number of unknown new virus types in nature, but the viruses recognized by humans only account for 0.1% of all potential virus types. Therefore, the rapid identification technology of emerging viral pathogens is very important for clinical treatment and epidemic prevention and control. [0003] The largemouth bass (Micropterus salmoides) is a premium freshwater fish. In recent years, the average survival rate of largemouth bass seedlings is only about 5%, mainly due to diseases such as fry defecation, enteritis, spinning, and ripening caused by unknown pathogens, resulting in nearly 100% fry mortality, which has seriously restricted largemouth bass. The healthy development of the black bass aquaculture industry has become a bottleneck for the devel...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12Q1/70C12Q1/6869C12N15/11A61K39/12A61P31/14
CPCC12N7/00C12Q1/701C12Q1/6869A61K39/12A61P31/14C12N2720/10021C12N2720/10034A61K2039/552A61K2039/5252C12Q2531/113
Inventor 李宁求付小哲罗明菊林强梁红茹刘礼辉牛银杰罗霞
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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