PCR-HRM primer and detection method for rapid identification of PRRSV gene subtype and application of PCR-HRM primer
A technology of PCR-HRM and PRRSV-F, which is applied in the field of PCR-HRM primers for rapid identification of PRRSV genotypes, can solve the problems of inability to quickly and accurately identify PRRSV genotypes, inability to distinguish vaccine strains from wild strains, and high cost. Reliable identification, shortened time required and high repeatability are achieved
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0038] Embodiment 1 PCR-HRM primer design
[0039] The inventors of the present invention used PCR amplification and sequencing of the PRRSV GP5 gene in clinical samples of more than 100 large-scale pig farms in Guangdong Province, and used the sequence analysis software MEGA7 to draw the genetic evolution tree of PRRSV. The results are shown in figure 1. The characteristic sequences of the four genotype subtypes of PRRSV were found through the genotyping software, and multiple pairs of primers were designed and screened repeatedly to obtain primers that could efficiently amplify all strains of PRRSV. The length of the fragment amplified by the primers is 117 bp, which contains the differential gene sites of the four genotype subtypes of PRRSV, and this region is also the main antigenic site of neutralizing antibodies produced after PRRSV vaccine immunization. The nucleotide sequences of the primers are as follows:
[0040] PRRSV-F: 5'-TTGTGGTGTATCGTGCCRT-3' (SEQ ID NO: 1); ...
Embodiment 2
[0043] Preparation and PCR-HRM analysis of embodiment 2 standard samples
[0044] (1) Preparation of positive standard samples:
[0045] In order to verify the feasibility and reliability of the method of the present invention, the standard strains of 4 gene subtypes PCR-HRM analysis of PRRSV are established simultaneously, and subtype I and subtype II select the widely used vaccine strain JXA1-R strain ( (purchased from Guangdong Animal Vaccine Supply Station) and RespPRRS MLV strain (purchased from Guangdong Animal Vaccine Supply Station) were used as the standard strains of these two genotype subtypes. Two strains, GD-GM strain (published on GenBank, gene bank accession number: KX429681.1) and GD-HH strain (already published on GenBank) of the same genotype as NADC30 strain and GM2 strain, were isolated from The gene bank accession number is: KX429682.1) as the standard strain-positive samples of these two genotypes, providing a typing reference for PCR-HRM analysis for su...
Embodiment 3
[0056] The PCR-HRM detection of embodiment 3 clinical sample
[0057] (1) Extracting viral RNA from samples suspected of being infected with PRRSV, the method is the same as the RNA extraction method in Example 2.
[0058] (2) Using the extracted RNA as a template, perform reverse transcription and PCR-HRM amplification (one-step amplification). The amplification reaction system is: 2 μL of extracted RNA, 2 times the concentration of one-step buffer (2 × Step Buffer) 10 μL, primer PRRSV-F (10 μM) 0.5 μL, primer PRRSV-R (10 μM) 0.5 μL, PrimeScript 1 Step Enzyme Mix 1 μL, saturated fluorescent dye LC Green solution (diluted 20 times according to the instructions) 1 μL, ddH 2 O to make up to 20 μL.
[0059] The amplification reaction program was: reverse transcription at 50°C for 5min; pre-denaturation at 95°C for 2min; denaturation at 95°C for 10s, extension at 72°C for 35s, 45 cycles. The heating steps of the HRM program were 92°C for 1 min, 40°C for 2 min; from 60°C to 90°C,...
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