Infectious bovine rhinotracheitis virus strain and preparation method and application thereof
A rhinotracheitis virus and infectious technology, applied in the field of infectious bovine rhinotracheitis virus, can solve the problems of time-consuming, false positives and high sensitivity, and achieve the effects of simple operation, easy operation and high accuracy
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Examples
Embodiment 1
[0018] Example 1: Using PCR to detect and identify infectious bovine rhinotracheitis virus, add 0.01mol / L PBS to the bovine nasal cavity cotton swab, shake and wash thoroughly, take 200 μL of the flushing solution and use the Viral Nucleic AcidExtraction Kit II DNA extraction kit to extract DNA , IBRV PCR method and IBRV fluorescent PCR method for detection, PCR primers are:
[0019] gB-1: 5'-TAC GAC TCG TTC GCG CTC TC-3';
[0020] gB-2: 5'-GGT ACG TCT CCA AGC TGC CC-3';
[0021] The size of the amplified fragment is 478bp. Add 12.5 μL of 2×Master Mix (product of TAKARA company), 5 μL of sample nucleic acid, and ddH2O to 25 μL in the 25 μL PCR reaction system. The amplification conditions are: 95°C pre-denaturation for 5min, 95°C for 1min, 65°C for 45s, 72°C for 45s, 10 cycles; 95°C for 1min, 54°C for 45s, 72°C for 45s, 15 cycles; 95°C for 1min, 60°C for 45s , 72°C for 45s, 10 cycles, and finally 72°C for 10min. After the PCR product was electrophoresed on 1.6% agarose gel,...
Embodiment 2
[0022] Embodiment two: detect and identify infectious bovine rhinotracheitis virus with fluorescent PCR, fluorescent PCR primers are:
[0023] gB-F: 5'-TGT GGA CCT AAA CCT CAC GGT 3';
[0024] gB-R: 5'-GTA GTC GAG CAG ACC CGT GTC-3';
[0025] TaqMan probe: 5'-FAM-AGG ACC GCG AGT TCT TGC CGC-TAMRA-3'.
[0026] Using TaqMan fluorescent PCR kit (product of TAKARA company), add 12.5 μL of 2×Master Mix, 5 μL of sample nucleic acid, and ddH2O to 25 μL in the 25 μL reaction system. Amplification conditions were: 95°C pre-denaturation for 5 minutes, 95°C for 15s, 60°C for 45s (signal collection), 45 cycles, and observed the amplification curve and Ct value. At the same time, the TaqMan fluorescent RT-PCR kit of BVDV was used to detect BVDV, the bovine bluetongue virus (BTV) nested RT-PCR method was used for detection, and the bovine leukemia virus (BLV) nested RT-PCR method was used for detection. Foot-and-mouth disease virus (FMDV) was detected by fluorescent RT-PCR. All reaction...
Embodiment 3
[0027] Example 3: Isolation of virus, IBRV PCR and fluorescent PCR were taken positive, after the cavity cotton swab washing solution was mixed, double-resistant penicillin 1000IU / mL and streptomycin 1000μg / mL were added, and then acted at 37°C for 2h, centrifuged at 2000g for 10min, and taken The supernatant was inoculated on a 96-well plate overgrown with a single layer of MDBK cells, 100 μL / well, plus 6 wells. Adsorb at 37°C for 1 hour, discard the supernatant, add maintenance solution (MEM containing 200 IU / mL penicillin, 200 μg / mL streptomycin and 2% calf serum), culture at 5% CO2 at 37°C, and observe the cytopathic changes (CPE ), when 70%-80% of the cells appear CPE to collect the poison, if the cells do not appear CPE, after 5 days blind passage, the isolate is passed to the fifth generation. At 48h of the first generation, it was found that the dead cells increased significantly. By the 4th day, the cells basically fell off and died, while the control cells grew well;...
PUM
| Property | Measurement | Unit |
|---|---|---|
| diameter | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to view more - R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap