Methods Of CDNA Chip Assay For Quantitative Detection Of Porcine Respiratory Disease Complex Related Viruses

Porcine pseudorabies virus (PRV), Porcine parvovirus (PPV), Porcine circoviruses (PCV2), Classical swine fever virus (CSFV), Porcine reproductive and respiratory syndrome virus (PRRSV) are five kinds of pathogens associated with porcine respiratory disease complex (PRDC) that cause great threat to swine industry.At present, some techniques are used to detect these pathogens, such as ELISA, PCR, immunofiuorescene, in-situ hybridization and so on. But there are some disadvantages for these techniques, such as time consuming, limited sample quantity per detection. DNA chips technique, developed in 90's age of 20 century, is a new technology for research on nucleic acid. Because of the advantage of high throughput, flexibility, sensitivity, simple performance, the technique of DNA chips has been used in many fields.In this experiment, five kinds of virus-specific nucleotide sequences as probes were fixed on the amino group modified slides. Asymmetric PCR were conducted using cDNA or DNA of viruses as templates and five pairs of oligo nucleotides as primers. The amplified products were labeled with CY3 and then hybridized with the cDNA probes on the slides. The hybridizing results were scanned by Genepix4000A scanner. The results were as follow: that the amplified product of these five kind viruses could specifically bind to the cDNA on the slide chips, which indicated that these probes could be used to detected viruses; That the sensitivity of the test was 100 times higher than the common PCR; that the results from interassay test were consistent with that from intraassay test, which suggested the technique to detect these five kinds of viruses has preferable stability. The results above showed that cDNA chips technique to detect these viruses has a characteristic of high specificity, sensitivity and stability, which will be a novel, reliable method to detect these viruses.