Virus isolation method for low-content sore mouth virus sample

A canker sore virus and virus separation technology, which is applied in the field of virus isolation of samples with low content of canker sore virus, can solve the problems of time-consuming, inability to isolate cane canker sore virus, and restricting researchers' access to wild canker sore virus strains, etc., and achieves the separation cycle. Short, maintain integrity and activity, good integrity and activity

Active Publication Date: 2015-07-01
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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  • Virus isolation method for low-content sore mouth virus sample
  • Virus isolation method for low-content sore mouth virus sample
  • Virus isolation method for low-content sore mouth virus sample

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 until homogenized with a homogenizer, freeze and thaw repeatedly 3 times, centrifuge at 5000r / min for 5min, take the supernatant, filter 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;

[0057] Add 10mL...

Embodiment 2

[0067] Step 1: Handling of materials:

[0068] Processing method of saliva and milk samples:

[0069] After centrifugation 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 three 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 solut...

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 filter membrane, and store the filtrate at -20℃ for later use;

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

[0088] Take 0.5g sample, wash it 3 times 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 3000r / min for 10min, take the supernatant, filter it 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 hour...

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Abstract

The invention relates to a virus isolation method for a low-content sore mouth virus sample. The traditional sore mouth virus is separated from incrustation and oral lip scurf of an ill goat, the isolation period is long, lesion is caused after 5 generations of blind passage, the pathological sample source range is narrow, and the research work of the sore mouth virus is limited. The method disclosed by the invention comprises the following steps: performing centrifugal filtration treatment on samples such as saliva, breast milk and internal organs by using a special Virus holder solution, inoculating the cattle primary testis cells with the samples for culturing, and discarding the venom; adding cell maintenance medium for culturing, and collecting the virus; and repeatedly freezing-thawing and collecting the cell culture, inoculating the cattle testis cells from the third generation within 72 hours, and observing the cytopathic effect. The samples are treated by using the special Virus holder solution, the sore mouth virus can be isolated from a sample with extremely low virus content, the material taking range is wide, the virus isolation period is short, the stability is high, and the integrity and activity of the virus can be well maintained.

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 Applications(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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