Method for efficiently separating pig intestinal tract coronavirus

A coronavirus and pig intestinal technology, applied in the field of animal virology, can solve the problems of ineffective multiplication of viruses, abnormal growth of cells, lysis and death, etc.

Active Publication Date: 2018-10-19
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the high toxicity of feces and small intestinal contents to cells, large inoculum volume often leads to abnormal growth of cells, or even lysis and death, so the virus cannot effectively multiply; small inoculum volume, due to low virus content, is difficult to isolate Virus

Method used

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  • Method for efficiently separating pig intestinal tract coronavirus
  • Method for efficiently separating pig intestinal tract coronavirus
  • Method for efficiently separating pig intestinal tract coronavirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The isolation rate of porcine epidemic diarrhea virus (PEDV) is compared by adopting the method of the present invention and the traditional method (control). The same sample was treated by two methods at the same time, and then inoculated into one well of African green monkey kidney passage cells (referred to as Vero cells) cultured in a 6-well plate.

[0028] Method of the present invention:

[0029]The test samples were 43 small intestines of piglets positive for porcine epidemic diarrhea virus (PEDV) (the implementation of the present invention is not limited to this virus) detected by RT-PCR in a pig farm in Hubei Province by the applicant in 2016. The specific operation method is as follows: Aseptically collect the contents of the small intestine of piglets in an ultra-clean bench, add an equal volume of serum-free DMEM culture solution (purchased from GIBCO Company), mix well, place at -80°C and freeze-thaw repeatedly 3 times, 5000r / min Centrifuge for 15 minutes...

Embodiment 2

[0035] The isolation rate of porcine deltacoronavirus (PDCoV) was compared by adopting the method of the present invention and the traditional method (control). The same sample was treated by two methods at the same time, and then each well was inoculated into a well of passaged porcine kidney cells (LLC-PK1 cells for short) cultured in a 6-well plate.

[0036] Method of the present invention:

[0037] The experimental samples were 40 small intestines of piglets tested positive for PDCoV by RT-PCR in a pig farm in Hebei Province in 2017. The specific operation method is as follows: Aseptically collect the contents of the small intestine of piglets in an ultra-clean bench, add an equal volume of serum-free MEM culture solution (purchased from GIBCO), mix well, place at -80°C for 3 times, and centrifuge at -80°C for 3 times. After 15 minutes, take the supernatant into another sterile centrifuge tube, centrifuge at 10,000r / min for 1h, take the supernatant, add penicillin at a fi...

Embodiment 3

[0043] The method of the invention and the traditional method (control) are used to compare the isolation rate of porcine transmissible gastroenteritis virus (TGEV). The same sample was treated by two methods at the same time, and then each well was inoculated with pig testicular passage cells (ST cells) cultured in a 6-well plate.

[0044] Method of the present invention:

[0045] The test samples were 50 small intestines of piglets that were tested positive for TGEV by RT-PCR in a pig farm in Hubei Province in 2017. The specific operation method is as follows: Aseptically collect the contents of the small intestine of piglets in an ultra-clean bench, add an equal volume of serum-free DMEM culture solution (purchased from GIBCO Company), mix well, place at -80°C for three times of repeated freezing and thawing, and centrifuge at 5000r / min 15min, take the supernatant to another sterile centrifuge tube, centrifuge at 10000r / min for 1h, take the supernatant, add penicillin and ...

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Abstract

The invention belongs to the technical field of animal virology, and particularly relates to a method for efficiently separating pig intestinal tract coronavirus. The pig intestinal tract coronavirusis prepared from PEDV (Porcine Epidemic Diarrhea Virus), PDCoV(Porcine Deltacoronavirus) and TGEV (Transmissible Gastroenteritis Virus). According to the method, differential centrifugation is adoptedto remove impurities, PEG(polyethylene glycol) 6000 is used for precipitating and concentrating viruses, impurity contents in a virus sample can be lowered to a maximum degree through the steps of purification of virus by the sucrose gradient centrifugation, and viruses are concentrated. The virus sample prepared with the method is especially suitable for inoculating cells and separating viruses.Compared with the virus sample prepared in the prior art, the method is characterized in that cell toxicity is greatly reduced, a virus separation rate is obviously improved, and the separation rateis improved to be above 40% from 10% of a traditional method.

Description

technical field [0001] The invention belongs to the technical field of animal virology. Specifically relates to a method for efficiently isolating porcine enteric coronavirus. The porcine enteric coronavirus includes porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and porcine transmissible gastroenteritis virus (TGEV). Background technique [0002] Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and porcine transmissible gastroenteritis virus (TGEV) all belong to the order Neveviridae and the family Coronaviridae. The three main viruses that cause intestinal infection and diarrhea in pigs, especially PEDV and PDCoV, have been clinically infected seriously in recent years, causing huge economic losses to the pig industry. [0003] Virus isolation is an important basis for disease diagnosis, and it is also the key to vaccine development and the premise of diagnostic methods. However, porcine enteric coronavirus has the proble...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N7/02
CPCC12N7/00C12N2770/20051
Inventor 肖少波方六荣周艳荣刘康王荡刘寒
Owner HUAZHONG AGRI UNIV
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