| Porcine reproductive and respiratory syndrome, Classical swine fever, Japanese encephalitis are the most common porcine reproductive disturbance diseases in clinical, they are harmful to swine industry extremely. The three diseases have the similar symptoms and mixed infection, therefore, fast inspection of these diseases is of great significance to the prevention and control of porcine reproductive disturbance diseases. Gene chip has the advantages of specificity, sensitivity, fast and accurate, high throughput. But the traditional labeled with fluorescent need expensive copolymerization laser scanner to scan and analysis results. To make the results intuitive and visual on the application of gene chip has a strong role in promoting.1. The establishment of the PRRSV-CSFV-JEV visual microarrayTake Y11 gene from PRRSV, PS8 gene from CSFV and C gene from JEV as probes. The positive quality and probes would be purified with the method of ethanol sodium acetate, and diluted to 400 ng/uL, again all the probes diluted to 200 ng/uL with Shanghai Baio company point sample buffer. The positive quality and probes sprayed to amino substrate according to a array designed, and repeat 2 times. Finally, the microarray would be baked for 4h in 80℃, purple foreign 30 min, sealed and saved in 4℃.The hybridization process of the PRRSV-CSFV-JEV visual microarray include the amplification of tags, pre-hybrid, hybrid, Nanogold-Streptavidin incubation and silver determination. The microarray’s hybrid temperature, hybrid time, Nanogold-Streptavidin dilution ratio, silver time were be explored and optimized.The specificity, sensitivity, stability and shelf life of the PRRSV-CSFV-JEV visual microarray were be validated. The PRRSV-CSFV-JEV visual microarray hybrid 60 min in 48 ℃, Nanogold Streptavidin 40 times dilution, silver stain 8-10 min the effect is the best. This study appraised the specificity, sensitivity, reproducibility and storage life with standard substance, the specificity of the PRRSV-CSFV-JEV visual microarray was good, no cross reaction between probe, the sensitivity could be up to 16.2 pg/uL, the reproducibility was well and could be sealing stored at least 180 d at 4℃.2. The preliminary application of the PRRSV-CSFV-JEV visual microarray102 clinical samples collected from Sichuan province between January 2014 and January 2015 detected by the PRRSV-CSFV-JEV visual microarray and conventional RT-PCR, the detection rate of PRRSV was 46%, the coincidence rate of the microarray and RT-RCR was 100%; the detection rate of JEV was 3%, the coincidence rate of the microarray and RT-RCR was 100%; the detection rate of CSFV was 39%, the coincidence rate of the microarray and RT-RCR was 97.5%. The phenomenon of mixed infection is common, including PRRSV and CSFV mixed infection detection rate was 6.8%, PRRSV and JEV mixed infection detection rate was 1%, the coincidence rate of the microarray and RT-RCR was 100%. The results demonstrated that the PRRSV-CSFV-JEV visual microarray can detect three porcine reproductive disturbance diseases at the same time, and have advantages of strong specificity, high sensitivity, high throughput and suitable for clinical application, laid the foundation method for the promotion of visual microarray. |
