Porcine epidemic diarrhea virus isolated culture method

A porcine epidemic diarrhea and virus isolation technology, applied in the direction of virus/bacteriophage, microorganism-based methods, biochemical equipment and methods, etc., to achieve high success rate, strong virus virulence, and high virus purity

Inactive Publication Date: 2014-05-28
上海佳牧生物制品有限公司 +1
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  • Summary
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AI Technical Summary

Problems solved by technology

At present, there is no report on the isolation of porcine epidemic diarrhea virus by primary culture of porcine small intestinal epithelial cells (IEC) at home and abroad.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0021] A method for isolating and culturing porcine epidemic diarrhea virus, comprising the following steps:

[0022] (1) The piglets were bled to death by arterial bleeding, the small intestine was removed by aseptic operation, the mesentery was removed, and placed in a plate of DMEM-F12 incomplete medium preheated at 37°C for standby;

[0023] (2) Cut the small intestine into 8-10 cm, use a syringe to absorb PBS solution and rinse slowly from one end to the other end, and rinse repeatedly until all the intestinal contents are washed out and the liquid is clear. The second time, take out the small intestine, use DMEM-F12 complete medium to separate the IEC cells, gently scrape the intestinal mucosal tissue with a glass slide, blow it evenly with a pipette, transfer it to a cell culture bottle, and add sterile type II collagenase to 0.1 wt% final concentration; place at 4°C and 37°C for 12h and 2h respectively, add an equal amount of DMEM-F12 complete medium to stop the digest...

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Abstract

The invention relates to a porcine epidemic diarrhea virus isolated culture method which comprises the following steps: thoroughly rinsing small intestine of a piglet, culturing the small intestine in a culture medium, inoculating, diluting a PEDV (Porcine Epidemic Diarrhea Virus) sample which is positive through detection into a suspension with the volume ratio of 1:1 by using normal saline, culturing and harvesting cells with the cytopathic effect (CPE), continuously freeze-thawing and inoculating single-layer vero cells, culturing by using the culture medium to obtain the cells with the cytopathic effect (CPE), namely, separating to obtain the porcine epidemic diarrhea virus. Compared with the prior art, the method is high in separation success rate and the separated porcine epidemic diarrhea virus is high in virulence and high in purity.

Description

technical field [0001] The invention relates to a virus isolation and cultivation method, in particular to a porcine epidemic diarrhea virus isolation and cultivation method. Background technique [0002] Porcine epidemic diarrhea virus (PEDV) is a contagious enteric disease mainly caused by pigs, characterized by vomiting, diarrhea, and dehydration. It first appeared in the UK in 1971, and the disease occurred successively in my country in the early 1980s. The virus mainly infects pigs through the intestinal tract, and has obvious characteristics of intestinal tissue tropism. The virus infection route is mainly the digestive tract. After the virus is infected through the mouth and nose, it enters the small intestine, and then the virus exists in the intestinal villi epithelial cells and mesenteric lymph nodes. . Virus replication occurs in the cytoplasm of the villous epithelium of the small intestine and colon. Because the virus multiplies firstly to cause organelle dam...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12R1/93
Inventor 张春玲倪建平訾玉华何锡忠李春华蒋凤英张婉华周宗清彭丽英
Owner 上海佳牧生物制品有限公司
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