The Establishment Of Oligonucleotide Microarray Diagnostic Technique Of Three Porcine Viral Infections

Under typical conditions of intensive swine production it is common for swine to be simultaneously infected with two or more viral pathogens. Porcine circovirus-2 (PCV-2), porcine parvovirus (PPV) and classical swine fever virus (CSFV) are the most widespread species, which have made enormous economic loss and severely crippled a country's swine industry. Therefore, the development of rapid, high-throughput and accurate method to detect these viruses is vital for immediate treatment and prevention. Current methods for the detection of viral pathogens include virus isolation, a variety of serological methods. Some of these methods have limitations such as lack of sensitivity and long turn-around time for test results. While pestivirus PCR-based assays are rapid, sensitive, the capability of PCR assay is limited and there may be some false positive result.To address some of the limitations of existing methods for pathogens detection, in this study, we developed an oligonucleotide microarray coupled with asymmetrical PCR system. Species-specific 60-mer oligonucleotide probes (oligoprobes) acted as the capture probes were designed and printed onto the surface of aminooxyacetyl functionalized glass slides. Asymmetrical PCR was used to prepare abundant single-stranded target DNA. And during this process aa-dUTP was mixed into single-stranded-DNA targets and the targets were labeled with Cy-dye. Following purification, the labeled asymmetrical PCR products were hybridized to the microarrays. The presence of the virulence genes was established by measuring a fluorescent signal above the background noise. In the paper we also explored optimization of the following experiment processes: the sample operated method, the asymmetrical PCR scheme and hybridization condition etc. Finally, the sensitivity and specificity of the optimized oligonucleotides microarray technologic system was appraised using the three kinds of pathogens as the detected model.The research arrived the following achievements: (1) The oligonucleotides microarray has the virtues of low background and high specificity; (2) We ascertained the appropriate conditions of asymmetrical PCR to amplify target genes by our optimal experiment; (3) Using the indirect labeling technology which conducted the fluorophores into the DNA, and at the hybridization condition of 60℃for 12h we can get the strong and stable fluorescent signal; (4) The scan results showed that the spots of capturing probes corresponded with the three target viral pathogens displayed a positive result, while the negative and blank controls were found to show no signal, and there are no false positive results and cross hybridization; (5) This approach is capable of detecting as few as 10 copies of the viral genomes for PCV2 and PPV, and 100 copies of the genome for CSFV.In conclusion, compared with conventional methods, the oligonucleotides microarray detection system has high sensitivity, good specificity, stability, simplicity and speed. This technique has potential applications for diagnostic and preventive purposes.