Detection Of Six Animal Original Zoonosis Pathogens By Microarray

Avian influenza virus serotype H5(AIV-H5), Rabies virus (RV), Streptococcussuis2(S.suis2), Bacillus anthracis (B.anthracis), Salmonella, Escherichia coli O157(E.coli O157) were the six animal original zoonosis pathogens which closely relatedto humans lives and occurred frequently recently, it can cause serious results not onlyto cultivation industries but also to humans safety when these pathogens happened. Akind of microarray method which according to the principle of nucleic acidhybridization for simultaneously detection of Avian influenza virus serotype H5,Rabies virus, Streptococcus suis2, Bacillus anthracis, Salmonella, Escherichia coliO157was established on account of the shortcomings of conventional methods andthe quarantine requirement of local port.Downloaded many gene sequences of AIV-H5, RV, S.suis2, B.anthracis,Salmonella, E.coli O157that published on GenBank, and then designed specificprimers and probes by Primer Express2.0in the region of the hemagglutinin (HA)gene of AIV-H5, the nucleoprotein (N) gene of RV, the capsular polysaccharide (CPS2J) gene of S.suis2, the coating protein (CapB) gene of plasmid pXO2of B.anthracis,the invasion protein (invA) gene of Salmonella, the rfb E gene of E.coli O157aftermultiple alignment by Clustal X, joined Biotin in5，end of every forward primer andlinked15-28Poly（T）in5，end of every probe when synthesis.The microarray was prepared by spotting probes on matrix, and then dried atroom temperature for5-10minutes, immobilized for7-13minutes under UVcrosslinker (254nm,0.6-1.2J), washed with Milli-Q water, washed with95%alcohol,dried at room temperature for10minutes. The temperature optimization test showsthat47℃was the most suitable temperature of microarray among45℃、47℃、49℃、51℃. Extracted the nucleic acid of the six pathogens by magic beads kit, and then diluted to seven concentration gradient by10fold serial dilution afterquantitation. The specificity test and the sensitivity test shows that no nonspecificreaction existed and the limited detections were1.38×10-5pg/μL to151pg/μLnucleotide of the pathogens. A total of930clinical samples were tested by themicroarray, the results showed that the established assay was in accordancecompletely with real-time PCR detection.Establishment of the method for detection of six animal zoonosis pathogens byTaqMan probe real-time PCR, and discussed in detail about real-time PCR by rabiesvirus as a representative.