Preliminary Construction Of DNA Microarray For Detection Of Porcine Reproductive And Respiratory Syndrome、Classical Swine Fever And Japanese Encephalitis

Porcine reproductive and respiratory syndrome, swine fever, Japanese encephalitis which belong to porcine reproductive disturbance diseases are relatively common and complicated diseases in domestic swine industry. One integrated DNA microarray that can detect the three diseases of porcine reproductive and respiratory syndrome virus, swine fever virus. Japanese encephalitis virus is very necessary.On the basis of our laboratory research, this study selected PRRSV:T/PRRS-PS9, T/Yll; CSFV:T/CSF1, T/PS8; JEV:T/PRM/M, T/JEV-C recombinant plasmid bacteria from the target gene library. Six pairs of specific primers were designed and synthesized for the six kinds of recombinant plasmid bacteria, then six kinds of target gene fragment were amplified by PCR. Location gene was amplified using λDNA as template. The target genes and location gene were diluted into200ng/ul with printing buffer, and print on the amino-modified slides; then the slides were processed to attach the target genes and location gene, prepared a PRRSV-CSFV-JEV microarray for detecting porcine reproductive and respiratory syndrome virus, swine fever virus and Japanese encephalitis virus. This study optimized several factors that effect target gene amplification productivity, compared several purification methods to control the quality of target genes and location genes. The optimal amplification conditions are:the final concentration of Mg2+is2.0mmol/L, the final concentration of dNTP is200umol/L, the number of amplification cycles is35. The best purification method is using the OMEGA kits directly purification from the PCR product. Before preparing the gene chip, we optimized the spot pin, printing buffer and printing humidity to control the gene chip conditions the process of preparation. We selected BaiO(?) printing buffer to dilute the target genes and location gene under the humidity of50%-60%. The printed gene chip must be processed to attach the target gene, we optimized the baking time as8hours, hydration time10sec, UV irradiation time as25min in post-process.In this study,5pieces of PRRSV-CSFV-JEV microarray selected randomly were hybridized by probes to assay the repeatability of PRRSV-CSFV-JEV microarray. Intensity values with higher than1000, SNR>2from the results indicated good repeatability.The specificity and sensitivity of PRRSV-CSFV-JEV microarray was appraised in the study by testing PRRSV、CSFV、 JEV、PPV、PRV、TGEV and PEDV using the gene chip. The results showed no cross hybridization among PRRSV、CSFV、JEV、and no hybridization reaction between PRRSV-CSFV-JEV microarray and PPV、PRV、TGEV PEDV. Sensitivity test showed the fluorescent intensity is testable when probes DNA concentration was0.295pg/ul, which showed high specificity and sensitivity of the DNA microarray. In the preservation time experiment of PRRSV-CSFV-JEV microarray, different storage time(30d,60d,90d,120d) with sealed dry condition in4℃. The results showed that the DNA microarray has fine detection effect after120days storage.A sample multiple labeling method was established by dividing six pairs of primers into two groups (HY1:PRM/M, PS8, PS9; HY2:JEC-C, Y11, CSF1) and employed mixed cDNA of PRRS-C14, CSFV, JEV-NJ1as template,31samples were labeled using this method with the following detection by PRRSV-CSFV-JEV microarray. The DNA microarray test showed positive rate of PRRSV12.9%, CSFV12.9%, JEV9.68%, co-infection rate9.68%of PRRSV and CSFV, co-infection rate3.22%CSFV and JEV, which suggests it can be used to detect porcine reproductive and respiratory syndrome, swine fever, Japanese encephalitis.