| The important porcine pathogens are frequently complicated with an increased incidence of viral superinfections, which endanger respiratory system, neural system, and reproductive system in the intensive swine industry. Furthermore, the difficulty of laboratory and clinical diagnosis of porcine pathogenic infections would be increased. The rapid and exact diagnosis of epidemic disease and adopting relevant measures is a precontion for loimic prevention. Current routine techniques for identification and detection of porcine pathogenic viruses are still based on virus isolation in tissue culture and serological method, but they are laborious, time-consuming and have a relatively low sensitivity. The advent of several molecular methods are established as a specific, fast and sensitive method for virus detection, such as PCR and southern blotting, but the incidence of false positivity is high and the capacity of these assays are limited. To address some of the limitations of existing methods for pathogens detection, in this paper, a low-density oligonucleotide microarray was develpoed coulped with asymmetric PCR and indirect labeled method for the rapid, accurate and high-throughput detection of epidemic viral pathogens in the swine industry.In this study, four familiar swine virues were chosen as the investigative targets, including swine influenza virus (SIV), porcine pseudorabies virus (PRV), foot-and-mouth disease virus (FMDV) and porcine reproductive and respiratory syndrome virus (PRRSV). The specific primers were designed to amplify 100-300 bp lengh fragments based on conservative regions of each swine virus. The oligonucleotide probes immobilized on the matrix were designed based on target symmetric PCR products, at the same time ensured the same length (60-mer) as well as approximately identical GC content and Tm for each capture probe. The oligonucleotide probes immobilized on the matrix were designed using DNAstar and Oligo 6.0 software based on target symmetric PCR products, at the same time ensured the same length (60-mer) as well as approximately identical GC content and Tm for each capture probe. After the labelled samples were subjected to solid and solution phase hybridization, four swine virues could be diagnosed in one assay. In this paper we also explored optimization of the following experiment processes: spotting concentration on the microarrays, scale of of restrictive and unrestrictive primers in asymmetric PCR mixture, choice of sense stand or anti-sense probe as capture probe and hybridization condition. Finally, the sensitivity and specificity of oligonucleotides microarray were appraised using this four kinds of pathogens as the detected model.This research attains the following achievements: (1) The diagnostic oligonucleotide microanay has the virtues of low background and high probed binding efficiency; (2) Through the optimized experiments, the optimal spotting concentration is confirmed to be 25μM; (3) The hybridization results revealed when the reverse primer was ascertained to be restrictive for PRV, SIV, FMDV, while the forward primer was ascertained to be restrictive for PRRSV, at the same time with a restrictive /unrestrictive primer ratio of 1: 100, the specificity of hybridization signals was the most strongest. (4) The hybridization temperature was at 60℃, the hybridization time was for 10h, the strong and stable fluorescent signal could be gained; (5) The hybridization results showed this technique could identify and distinguish the four swine viruses, there were no false positive results and cross hybridization. The detection limit of the oligonucleotide microarray system were 1.63×10~3 gene copy numbers for SIV, 7.08×10~4 gene copy numbers for PRV, 18 gene copy numbers for FMDV, 1.52×10~3 gene copy numbers for PRRSV. |
PDF Full Text Request