Establishment and application of multiplex fluorescent RT-PCR detection method for foot-and-mouth disease, vesicular stomatitis and porcine vesicular disease
A technique for vesicular stomatitis and swine vesicular disease
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
preparation example Construction
[0065] 2.3.1.3 Preparation of standards for quantification
[0066] Using specific primers for the 3D protein encoding gene of foot-and-mouth disease virus, the viral RNA was amplified by PCR in a 50 μL system, and the PCR product obtained was purified and cloned into the pGEM-T easy vector and transformed into E. coli TOP10 competent cells. The plasmid was extracted from the purified and amplified recombinant bacteria, purified by the kit, and the plasmid concentration was measured by OD260 with a UV reflectometer, and the concentration was converted into gene copy number according to Avogadro constant. The formula for calculating plasmid copy number is as follows:
[0067] Copy number (copy / μL)=(plasmid concentration (g / μL)×6.02×10 23 ) / [Base Pair (bp)×660(g / mol] (Formula 1-1).
[0068] According to formula 1-1, the copy number of recombinant plasmid pGEM T-3D is calculated to be 1.21×10 10 Copy / μL.
[0069] The extraction process of recombinant plasmid DNA was performed according ...
Embodiment 1
[0136] Example 1 Establishment of a fluorescent RT-PCR detection method for foot-and-mouth disease virus
[0137] 1.1 Method establishment and specificity
[0138] Synthesize primers and probes for the 3D protein coding gene sequence of foot-and-mouth disease virus. After exploring the reaction conditions, a reaction system was established with a total of 50 μL. Take foot-and-mouth disease virus (FMDV) RNA as a positive control, take genomic RNA extracted from cells that are not infected with foot-and-mouth disease virus as a negative control, and use water as a blank control. Other viruses such as Akabane virus RNA, bovine leukemia virus cDNA, and bovine viral diarrhea virus RNA , Vesicular stomatitis virus RNA, swine vesicular disease virus RNA and Mycobacterium paratuberculosis DNA to identify the specificity of the fluorescent quantitative RT-PCR detection method. The test results are attached figure 1 .
[0139] 1.2 Establishment of standard curve
[0140] According to serial ...
Embodiment 2
[0145] Example 2 Establishment of a fluorescent RT-PCR detection method for vesicular stomatitis virus
[0146] 2.1 Method establishment and specificity
[0147] Design and synthesis of primers and probes according to the coding gene sequence of N protein of vesicular stomatitis virus. After exploring the reaction conditions, a reaction system was established with a total of 50 μL. Vesicular stomatitis virus (VSV) RNA was used as a positive control, genomic RNA extracted from cells not infected with vesicular stomatitis virus was used as a negative control, and water was used as a blank control. Other viruses such as Akabane virus RNA, bovine leukemia virus eDNA, Bovine viral diarrhea virus RNA, foot-and-mouth disease virus RNA, swine vesicular disease virus RNA and Mycobacterium paratuberculosis DNA were used to identify the specificity of the fluorescent quantitative RT-PCR detection method. The test results are attached Figure 5 .
[0148] 2.2 Establishment of standard curve
...
PUM
Abstract
Description
Claims
Application Information
- 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, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com