| So far, PEDV, TGEV and PoRV is a major cause of diarrhea viral pathogens in our country. These diseases brought great harm to the piglets in our country.These diseases brought a high mortality rate of piglets. These disease made the prevention and diagnosis of the disease to be more difficult. The advent of these circumstances make the pig industry need much more pathogenic high-throughput screening and diagnosis technology research.It is the first time to developed PEDV-TGEV-PoRV Diagnostic DNA Microarray in this study.1. Clone, identification and analysis of Probe gene1) On the basis of the existing research in our laboratory, six pairs of specific primers were designed.Six pairs of the probe gene,inclouding PM, PS, TN, TS, NSP4 and VP7 were amplified by PCR, and they were inserted intothe T cloning vector.After that, the positioning gene was amplified by PCR in which the λ DNA was templated.The extent of probe gene was about 500bp.2) The results of the gene sequencing were used to be campared with the virus in China. The results of comparison showed that the gene order was the target gene, and the probe genes of PEDV and TGEV were highly homologous which of homology was higher than 95%.The homology of the probe genes of PoRV which was still higher than 85%and 75% was lower than them.But all of them were met the requirements.2. The construction of PEDV,TGEV and PoRV DNA diagnostic microarray.1)Three purification methods of the probe genes and locate genes were compared in this study, including anhydrous ethanol sedimentation method, Isopropanol purification methods and Law purification kit.The analysis of the results showed that the best method to directly sublime the genes from the PCR product was ethanol precipitation in which the max density was 4227.5 ng/μl and the min density was 555ng/μl, and most density of the gene higher then the orthers. Then the probe genes and locate genes was sublimed, so that they were complied with the requirements of the study.2) After this, the concentration of DNA should be controled in 200ng/μl Then, the study selected the printing buffer which was maken by Beijing CapitalBio Corporation to dilute the target genes and location genes in the condition in which the humidity was 50% -60%. The spotting parameters were spot diameter,220μm and center-to-center space 500μm. The microarray was dried at room temperature for 6h, hydrated at 60℃ water vapor for 10s, immobilized under ultraviolet for 30min,washed in 0.2% sodium dodecylsulphate(SDS)for 5min, and dried by centrifuge immediately. Then we evaluated the quality of DNA microarray. λ. DNA was chosed to cross with the DNA microarray. The positive standard formulated as SNR≥1.5 or signal intensity ≥1500. The result of the evaluation showed that the quality of the DNA microarray we made was very good.3. The application of PEDV,TGEV and PoRV DNA diagnostic microarray.1)One kind of multiple labeling method was established by dividing six pairs of primers into two groups (HY1:PM, TN, NSP4; HY2:PS, TS, VP7) and employed mixed cDNA of PEDV, TGEV and PoRV as templated successfully. The final prerequisite was Tm 54℃,dNTP 10μmol/μl) 3.5μl,Mgcl2 (25μmol/μl) 3.0μl, and Taq E (2.5U/μl) 0.55μl.2)The specificity, sensitivity and repetitive use and preservation period of PEDV-TGEV-PoRV DNA diagnostic microarray were researched. The result showed that the specificity of the microarray was good. The PEDV-TGEV-PoRV DNA diagnostic microarray could simultaneoussly detect PEDV, TGEV, PoRV and had no cross hybridization among PPV, PRV, PRRSV, CSFV and JEV. The result of sensitivity tests showed it had high sensitivity,and the minimum detectable concentration was 20pg/μl. It can be reused at least 7 times.The microarray had finely detection effect in 150 days storage. |
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