Detection Classical Swine Fever Virus and Porcine Circovirus Type 2 of Microarray Technique
|School||Henan Agricultural University|
|Course||Preventive Veterinary Medicine|
|Keywords||Classical swine fever virus Porcine Circovirus type 2 Detection Gene chip application|
In this study using gene chip technology, using PCR and DNA markers to construct a classical swine fever virus and porcine circovirus type 2 gene chip of detection .Not only for a variety of infectious diseases in the same piece of pig Lay the foundation for testing the chip, But also for other animal diseases were detected by gene chip method provides a theoretical basis. Mainly divided into the following sections:1. CSFV-PCV2 cloning and identification of the target gene of detection gene chipCSFV and PCV2 by the pathogen and molecular biological analysis, and literature references reported in GenBank CSFV and PCV2 sequences, select the conserved sequence and using the software Primer Primier5.0 primers. By PCR or RT-PCR amplified gene, obtained two gene fragments; with pMD18-T or pUCm-T cloning vector for gene cloning and recombinant plasmid PCR or restriction endonuclease digestion, then sequenced. Multiple sequence alignments further show that: cloning the gene for the pathogen identified highly conserved genes, provides a true and reliable material for the CSFV-PCV2 detection construction of gene chip.2. CSFV-PCV2 detection Construction of gene chipPrimers were labeled by so fluorescent (Cy3) incorporation into PCR products, through to the amino-modified probe to make it better with the aldehyde of the substrate through better integration of the probe to be more well fixed on the substrate; by PCR amplification of recombinant plasmid with the appropriate hybridization temperature and hybridization time optimization, filtering out specific good hybrid gene and identified appropriate hybridization temperature and hybridization time, and finally through The results of the hybrid scanner to scan for computer software, hybrid image and analyze the results of hybridization, detection of CSFV-PCV2 complete the construction of gene chips, the results are as follows:1) Preparation of target gene: The gene recombinant plasmid DNA as template, PCR amplification, purification of the target gene isopropanol precipitation, the concentration and purity requirements are in line with chip production. 2) To determine the optimum point of the probe sample concentration 100ng/μL, strong hybridization signals were screened out, a good gene-specific hybridization to determine the CSFV-PCV2 detection of gene chip hybridization temperature is 48℃, hybridization time is 5h .3) Establishing a point-like environmental parameters: relative humidity of 55-65%, temperature 15-30℃. The probe spacing is 1.5mm.3. CSFV-PCV2 detection of gene chip technologyExperiment as negative control using the PRV strains of CSFV-PCV2 built specifically for detecting gene chips were evaluated, results showed that: PRV negative control and blank control had no hybridization signal, while the CSFV and PCV2 and the hybridization signal was visible, gene chip specificity; to the extracted plasmid by 10-fold dilution to 10-8, the use of PCR, and gene chip were different gradient of the recombinant plasmid were detected and compared to evaluate the sensitivity of the chip detection method, the results that: the sensitivity of gene chip methods are better than the PCR method; by in situ detection of suspicious samples to evaluate the application of gene chip results showed that: gene chip is fairly effective and were tested further.