Detection Of The Six Pathogenic Bacteria In Animal Furby DNA Chip

According to the authority of WHO and OIE released the national epidemicsituation and relevant literatures, combined with laboratory pathogen isolation andidentification results we explored and researched an DNA chip method for detectionof Brucella, E.coli O157, Staphylococcus aureus, β-Streptococcus, Erysipelothrixrhusiopathiae and Pseudomonas aeruginosa in animal fur simultaneously in this study,two pair of universal primers and the specific oligonucleotide probes of the six kindsof bacteria were designed and synthesized based on the16S rDNA and gyrB genesequence, respectively. Finally, evaluate the clinical application effect of this method.Main research contents and results are as follows:1.Using CTAB method, phenol chloroform method, NaI method and NaOH-SDSmethod to extract DNA of the bacteria in animal fur based on identifying to Brucella,E.coli O157, Staphylococcus aureus, β-Streptococcus, Erysipelothrix rhusiopathiaeand Pseudomonas aeruginosa in animal fur, and followed up to comparison ofnucleic acid content, purity and integrity. Finally, we found that phenol chloroformmethod can be used as an universal nucleic acid extraction method for DNA chipdetection.2. In this research, two pair of universal primers and the one to five specificoligonucleotide probes of the six kinds of bacteria were designed and synthesizedbased on the16S rDNA and gyrB gene sequence which had great significanceinbacteria biological, respectively. And each forward primer was markered byTAMARA fluorescent groups. The probes were diluted for30μmol/L by DNA chipspot liquid, and then DNA chips were prepared by spotting the probes onamino-modified glass slides to18x10(row/column)points each matrix of the sampleand each probe points5repeat. Followed by DNA hybridization with productsamplified from the bacteria by asymmetric PCR. Under the optimized hybridizationconditions including incubation for rotating4hours in47%formamide at42℃, theDNA chip method was no cross-reactivity with other bacteria as follows: Bacilluscereus, Bacillus thuringiensis, Escherichia coli O152, Listeria, Salmonella andEnterobacter sakazakii. The sensitivity was approximate10copies.3. We randomly selected163fur samples in DNA chip detectionand found thatthe coincidence rate of the DNA chip and the traditional methods of isolation andidentification or the PCR for clinical fur sample was100%. Our data indicated theDNA chip assay was a high throughput, sensitive and specific for rapid quarantine of the6bacteria in animal fur.