Study On Diagnostic DNA Macroarray For Porcine Viral Disease

Multiple infections have become widespread in swinery, and have made the conditions more complex. This case increased the difficulty of clinical and laboratory diagnosis. Therefore, it's necessary to establish a pluralistic method to diagnosis the complex infective diseases. In this study, the diagnostic DNA macroarray for porcine viral disease has been constructed.Major study reads as follows:Part 1: Clone and identification of probe The primers were designed based on the published nucleotide sequences of PRRSV, CSFV, PCV-2, PPV, PRV, JEV and influenza A virus in Genbank. The virus DNA or cDNA was amplified by using high-fidelity DNA polymerase, then ligated with pMD 18-T Simple Vectors to construct recombinant plasmids, respectively. The recombinant plasmids were identified by PCR and sequencing. Aliment reports showed that the sequence had 89.1 to100 percent identities with relevant virus strains, respectively.Part 2: Construction and preliminary application of diagnostic DNA macroarray in porcine viral respiratory diseaseUsed the recombinant plasmids constructed in part 1 to prepare PRRSV, PRV, PCV-2 and SIV probes, and then to prepare diagnostic DNA macroarray for porcine viral respiratory disease.Biotin-11-dUTP concentration options: The target of PRRSV, PRV, PCV-2 and SIV were labeled by Biotin-11-dUTP in PCR. The final concentrations of Biotin-11-dUTP in PCR system were 40μM,50μM,70μM and 100μM, respectively. It was showed that all the products of four PCR systems could get positive hybridization signals. And the signals were increased with the concentration. The result indicated that 70μM concentration (at 35% level of substitution) could meet the requirements of signal analysis.Hybridization temperature options: The target of PRRSV, PRV, PCV-2 and SIV were...