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A Virus Isolation Method for Low-Content Samples of Aphthus Virus

A technology for isolating oral ulcer virus and virus, which is applied in the field of virus isolation of low-content samples of oral ulcer virus, can solve the problems of time-consuming, inability to isolate oral ulcer virus, and restrict scientific research personnel from obtaining wild strains of oral ulcer virus, and achieve the separation cycle Short, maintain integrity and activity, good integrity and activity effect

Active Publication Date: 2018-02-06
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional isolation method cannot isolate the oropharynx virus from the tissue samples of this kind of virus-infected sheep, which greatly limits the rapid, timely and extensive acquisition of wild strains of the oropharyngeus virus from various places; Lesions will appear in the 5th generation, which is time-consuming

Method used

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  • A Virus Isolation Method for Low-Content Samples of Aphthus Virus
  • A Virus Isolation Method for Low-Content Samples of Aphthus Virus
  • A Virus Isolation Method for Low-Content Samples of Aphthus Virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Step 1: Handling of materials:

[0051] Processing method of saliva and milk samples:

[0052] After centrifugation at 12000r / min for 10min, take 200μL supernatant, add 800μL Virus holder solution, freeze and thaw 3 times, filter with 0.22μm filter membrane, and store the filtrate at -20℃ for later use;

[0053] Liver, kidney and other visceral sample processing methods:

[0054]Take 0.5g sample, wash it twice with sterile PBS, cut it into pieces, add 1mL Virus holder solution, grind it with a homogenizer until it is homogenized, freeze and thaw it repeatedly 3 times, centrifuge at 5000r / min for 5min, take the supernatant, and filter it at 0.22μm Membrane filtration, the filtrate was stored at -20°C for later use;

[0055] Step 2: Virus isolation:

[0056] Take 1mL of the filtrate and inoculate the bovine testicular primary cells that have covered 90% of the bottom of the culture flask, at 37°C, 5% CO 2 Discard the venom after incubating in the incubator for 1 hour;...

Embodiment 2

[0067] Step 1: Handling of materials:

[0068] Processing method of saliva and milk samples:

[0069] After centrifuging at 14000r / min for 7min, take 200μL supernatant, add 800μL Virus holder solution, freeze and thaw 3 times, filter with 0.22μm filter membrane, and store the filtrate at -20℃ for later use;

[0070] Liver, kidney and other visceral sample processing methods:

[0071] Take 0.5g sample, wash it twice with sterile PBS, cut it into pieces, add 1mL Virus holder solution, grind until homogenized with a homogenizer, freeze and thaw repeatedly 3 times, centrifuge at 4000r / min for 7min, take the supernatant, filter at 0.22µm Membrane filtration, the filtrate was stored at -20°C for later use;

[0072] Step 2: Virus isolation:

[0073] Take 1mL of the filtrate and inoculate 85% of the bovine testicular primary cells at the bottom of the culture flask, at 37°C, 5% CO 2 Discard the venom after incubating in the incubator for 1 hour;

[0074] Add 7mL cell maintenance ...

Embodiment 3

[0084] Step 1: Handling of materials:

[0085] Processing method of saliva and milk samples:

[0086] After centrifugation at 16000r / min for 5min, take 200μL supernatant, add 800μL Virus holder solution, freeze and thaw 3 times, filter with 0.22μm membrane filter, and store the filtrate at -20℃ for later use;

[0087] Liver, kidney and other visceral sample processing methods:

[0088] Take 0.5g sample, wash 3 times with sterile PBS, cut into pieces, add 1mL Virus holder solution, grind until homogenized with a homogenizer, freeze and thaw repeatedly 3 times, centrifuge at 3000r / min for 10min, take the supernatant, filter at 0.22µm Membrane filtration, the filtrate was stored at -20°C for later use;

[0089] Step 2: Virus isolation:

[0090] Take 1mL of the filtrate and inoculate the bovine testicular primary cells that have covered 80% of the bottom of the culture flask, at 37°C, 5% CO 2 Discard the venom after incubating in the incubator for 1.5 h;

[0091] Add 5mL cell...

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Abstract

The invention relates to a virus isolation method of a low-content sample of aphthus virus. Traditional aphthus virus is isolated from scabs and lip dander of diseased sheep. The separation cycle is long, and it takes 5 generations of blind transmission before lesions appear. research work. The present invention uses a unique Virus holder solution to centrifugally filter samples such as saliva, milk, and viscera, inoculate them in the primary cells of the bovine testis and discard the venom after culturing; add the cell maintenance solution to collect the poison after culturing; freeze and thaw repeatedly to collect the cells In the culture, cytopathic changes can be observed 72 hours after inoculation with bovine testis cells in the third passage. The invention uses a unique Virus holder solution to process samples, can isolate aphthus virus from samples with extremely low virus content, has a wide range of materials, short virus isolation period, high stability, and can well maintain the integrity and activity of the virus.

Description

technical field [0001] The invention relates to a virus isolation method, in particular to a virus isolation method for a low-content sample of aphthous ulcer virus. Background technique [0002] Orf contagious impetigo, commonly known as Orf in sheep, is a zoonotic infectious disease caused by aphthous sore virus. It occurs frequently in sheep and goats. The World Health Organization (IOE) lists this disease as an animal disease that needs to be declared , my country lists it as a first-class animal disease. The disease is distributed in almost all sheep-raising countries in the world. It mainly infects goats and sheep under natural conditions, and goats are more susceptible. The disease is often epidemic in groups. Lambs aged 3-6 months are most susceptible, and adult sheep are less prone to disease. Sick sheep and infected sheep are the main sources of infection for this disease. The virus can be discharged with the saliva, pustules and blister secretions of sick sheep, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C12R1/93
Inventor 陈德坤周铭刘方刘鹤媛高洋赵燕青
Owner NORTHWEST A & F UNIV
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