A kind of isolation and cultivation method of porcine epidemic diarrhea virus

A porcine epidemic diarrhea, isolation and culture technology, applied in the field of isolation and culture of porcine epidemic diarrhea virus, can solve the problems of unfavorable PEDV timely diagnosis, cumbersome virus sample processing process, low virus purity, etc., so as to improve the separation success rate and shorten the cycle. , the effect of increasing viral content

Active Publication Date: 2021-07-20
浙江美保龙生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The traditional PEDV isolation method has a long cycle, the virus sample processing process is cumbersome, and it is easy to contaminate. The purity of the isolated virus is not high, which is not conducive to the timely diagnosis of PEDV. The rapid diagnosis and research of the disease are of great significance

Method used

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  • A kind of isolation and cultivation method of porcine epidemic diarrhea virus
  • A kind of isolation and cultivation method of porcine epidemic diarrhea virus
  • A kind of isolation and cultivation method of porcine epidemic diarrhea virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: the preparation of virus treatment solution

[0027] Sodium chloride: 8g

[0028] Potassium chloride: 1g

[0029] Potassium dihydrogen phosphate: 0.5g

[0030] Calcium chloride: 1g

[0031] Sodium citrate: 2 g (purchased from Dezhou Runxin Experimental Instrument Co., Ltd.)

[0032] Sodium thiosulfate: 10g (purchased from Sinopharm Chemical Reagent Co., Ltd.)

[0033] Penicillin: 80 mg

[0034] Streptomycin: 80 mg

[0035] After mixing the above ingredients, dissolve them in 1000 ml double distilled water, and use 0.1M NaHCO 3 Adjust the pH to 8.0, filter through a 0.22 um filter membrane, and store at 4°C for later use.

[0036] Also can adopt to get sodium chloride 5g, potassium chloride 0.01g, potassium dihydrogen phosphate 0.05g, calcium chloride 0.01g, sodium citrate 0.05g, sodium thiosulfate 5g, penicillin 60mg and streptomycin 60mg for preparation, or use 12g of sodium chloride, 2g of potassium chloride, 1g of potassium dihydrogen phosphate,...

Embodiment 2

[0037] Embodiment 2: collection and processing of disease material

[0038] Collect intestinal contents and feces from clinically suspected PED sick piglets, add physiological saline at a mass volume ratio of 1:2, and homogenize intestinal contents, shake and mix the fecal samples, and add at a volume ratio of 1:5 Virus treatment solution, mix well, let stand for 10min. Finally, centrifuge at 5000 r / min for 10 minutes, take the supernatant, filter through a 0.45 um microporous membrane, and then filter through a 0.22 um microporous membrane to obtain the PEDV virus stock solution.

[0039] In this embodiment, the intestinal contents and feces collected from clinically suspected PED sick piglets were added to normal saline at a mass volume ratio of 1:1, or the intestinal contents and feces were collected from clinically suspected PED sick piglets at a mass volume ratio of 1:1. :3 add physiological saline; add virus treatment solution by volume ratio 1:2, 1:6 or 1:10, finally a...

Embodiment 3

[0040] Example 3: Preparation of Maintenance Solution

[0041] A kind of porcine epidemic diarrhea virus separation method is characterized in that 1000ml maintenance solution is made up of following components:

[0042] Pancreatin: 3mg (purchased from: Sigma)

[0043] pAPN: 2mg (purchased from: Sigma)

[0044] Salidroside: 7g (purchased from: Shanghai Baishikai Chemical Technology Co., Ltd.)

[0045] Chlorogenic acid: 3 g (purchased from: Shanghai Aoke Chemical Co., Ltd.)

[0046] After mixing the above ingredients, dissolve in 1000 ml DMEM, filter through a 0.22 um filter membrane, and store at 4°C for later use.

[0047] In this example, 1 mg of trypsin, 1 mg of pAPN, 6 g of salidroside and 3 g of chlorogenic acid can also be used for preparation, or 5 mg of trypsin, 5 mg of pAPN, 8 g of salidroside and 5 g of chlorogenic acid can be used g is prepared, finally also can reach the same technical effect as embodiment 3.

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Abstract

A method for isolating and cultivating porcine epidemic diarrhea virus belongs to the technical field of veterinary biological products. It includes collecting intestinal contents and feces from clinically suspected PED sick piglets, adding physiological saline respectively, homogenizing the intestinal contents, vibrating and mixing the fecal samples, adding virus treatment solution, mixing evenly, standing, Centrifuge, take the supernatant, first filter through a microporous membrane, and then filter through a microporous membrane to obtain the PEDV virus stock solution; take Vero cells that have been cultured for 24 hours and grow well on the wall, discard the cell culture medium, and PEDV The virus stock solution and the maintenance solution were inoculated into the cell bottles, and the cell bottles were cultured in the incubator, and the cell lesions were observed every day, and the cell culture solution with obvious lesions were collected and stored at -20°C. The separation and cultivation method proposed by the invention has low cost and short separation period, and is beneficial to timely diagnosis and research of porcine epidemic diarrhea virus disease.

Description

technical field [0001] The invention belongs to the technical field of veterinary biological products, and in particular relates to a method for isolating and culturing porcine epidemic diarrhea virus. Background technique [0002] Porcine epidemic diarrhea (porcine epidemic diarrhea, PED) is caused by porcine epidemic diarrhea virus (porcine epidemic diarrhea virus, PEDV), a piglet with diarrhea, vomiting and dehydration as the main clinical symptoms, eventually destroying the electrolyte balance and causing the death of sick pigs an acute diarrheal disease. At present, PED affects a wide range, and cases have been reported all over the world. At the same time, the incidence of PED is high. The infection rate of young pigs is as high as 100%, and the infection mortality rate of piglets under 7 days old is 80% -100%. Pig farming causes severe economic losses. [0003] The epidemic of porcine epidemic diarrhea caused by PEDV is complex and difficult to control. Many scholar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C12R1/93
CPCC12N7/00C12N2770/24351
Inventor 李阳沈建军张秀文
Owner 浙江美保龙生物技术有限公司
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