A kind of cultivation method of porcine epidemic diarrhea virus stable passage
A technology for porcine epidemic diarrhea and virus passage, which is applied in the field of stable culture and passage of porcine epidemic diarrhea virus, and can solve the problems of impeding the passage of circulating virulent strains, weakening the passage of virus strains, and the loss of viruses.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0054] - culture of porcine epidemic diarrhea virus,
[0055] 1. Wash the monolayer of well-grown Vero cells (gifted by the Key Open Laboratory of Livestock and Poultry Infectious Diseases, Ministry of Agriculture, Yangzhou University) three times with PBS, and then wash once with 0.02% EDTA solution, and finally rinse with trypsin. The nutrient solution with a concentration of 60 μg / ml was treated at 37°C for 10 minutes to sensitize the cell monolayer;
[0056] 2. Warm the ZC strain (ZC Xinnuo Biotechnology Taizhou Co., Ltd. isolated from a pig farm in Taizhou, Jiangsu, named ZC strain) to room temperature, and add a-MEM nutrient solution with a final concentration of trypsin at 60 μg / ml in the Activation at 37°C for 10 minutes;
[0057] 3. Take the above-mentioned activated virus strain and inoculate it into the sensitized cell monolayer according to 10% of the final culture system;
[0058] 4. Incubation: Incubate at 37°C for 1 hour, with slight shaking twice during the p...
Embodiment 2
[0062] - Harvested virus identification;
[0063] (1) TCID 50 detection
[0064] Take the cell venom and make a 10-fold serial dilution of the virus with MEM culture solution containing 20 μg / ml trypsin -5 、10 -6 、10 -7 The three titers were respectively inoculated in 4 wells of a 96-well culture plate containing 100 μl of Vero cell solution in each well, 100ul in each well, and a control well of cells not exposed to the virus was set up at 37°C and 5% CO 2 Incubator, cultivate for 3 days, observe cell lesions, and calculate TCID 50 . porcine epidemic diarrhea virus content of 10 8.0 TCID 50 / ml.
[0065] (2) ZC method detection
[0066] RNA extraction kit (TaKaRa, AK704) was used to extract viral RNA, and one-step RT-PCR kit (TaKaRa, AK3601) was used for identification. The primers were sF and sR to amplify the target fragment. The amplification procedure was: pre-denaturation at 94°C for 4 minutes; 94°C for 30s, 60°C for 30s, 72°C for 30s, 30 cycles, and finally 72...
Embodiment 3
[0068] ——Serial passage test
[0069] Using the above method for continuous passage, from the P1 generation to the P150 generation, the PCR results of some generations are as follows: figure 2 .
[0070] In the technical scheme of the present invention, the maintenance fluid composition is (V / V): phosphotrypsin Broth (Tryptosephosphate broth, TPB) 0.1%, yeast extract (Yeast extract) 0.003%, trypsin (Trypsin) 20ug / ml, dimethylsulfoxide (DMSO) 0.1%, fetal bovine serum 1%, EDTA0.001 %, double antibody 1%, a-MEM 95.9. ;
[0071] In the technical solution of the present invention, the double antibody is a solution containing 10000 IU / ml of penicillin and 10 mg / ml of streptomycin.
[0072] In the technical scheme of the present invention, the composition and content of the PBS buffer solution are: NaCl 8g / L, KCl 0.2g / L; KH 2 PO 4 0.24g / L; Na 2 HPO 4 12H 2 O 3.65g / L, pH 7.2-7.4.
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More