| Two microarray designs were developed and produced to screen for multiple bovine pathogens commonly found in the cattle industry today. The first microarray was designed, built, and processed in-house using conventional material and equipment and targeted Pasteurella multocida, Manheimia haemolytica, Histophilus somni, and Arcanobacterium pyogenes. For each pathogen, 12 perfect-match oligonucleotide probes, which were also designed in-house, targeted different sections of the respective 16S ribosomal genes, and were coupled with 12 corresponding mismatched probes for background. These arrays were able to produce distinct hybridization patterns for each pathogen that were easily visible without the need for computer analysis. However, the need for PCR amplification of the 16S gene prior to hybridization motivated us to explore more efficient array options. The second designed microarray, a custom Affymetrix GeneChip, targeted Escherichia coli, Salmonella typhimurium, and Salmonella dublin in addition to the previously mentioned pathogens and was more successful in overall performance than the "in-house" arrays. In addition to the 16S gene, oligonucleotide probes targeted other genes (from 2 to >4500, depending on whether the genome was sequenced) that were unique to each pathogen. This array also differed from the "in-house" arrays in that mismatched probes were not designed. The different probe sets performed at different detection limits as P. multocida, A. pyogenes, S. typhimurium, and S. dublin were detected with as little as 250ng of hybridized genomic DNA (gDNA), while M. haemolytica, H. somni, and E. coli required as much as 1mug gDNA. These pathogens were also spiked into bovine tissue to simulate multiorgan infections in which they were individually detected with the microarray design. |
