Abstract

Salmonella, widely distributed in nature, is a great human and animal health hazard of a class of pathogens. Culture-based methods may require many days to detect Salmonella. Traditional microbiology could advantageously be replaced by DNA microarray technology. We described an improved 3-D polyacrylamide gel-based DNA microarray assay based on gyrB gene (DNA gyrase B subunit gene) sequences that can be used for the identification of Salmonella species. Primers specific for a gyrB gene region common to all 13 samples were synthesized and used for PCR amplification of purified DNA. An oligonucleotide probe for specific gyrB gene regions was developed for the identification of 7 Salmonella species. Acrylamide-modified oligonucleotides solutions containing acrylamide monomer, glycerol, APS and probe were prepared at the desired concentration. The solutions were spotted on the modified glass slide by ink jet using a microarrayer and then the slide was transferred to a vacuum chamber with TEMED, after that the slide was used for hybridization with fluorescently labeled ssDNA derived from amplified sample DNA to yield a pattern of positive spots. This microarray produced unique hybridization patterns for species of Salmonella and could differentiate closely related bacterial species. The sample preparation and microarray method used in this study increased sensitivity and reduces time-to-result for detection of Salmonella. The described method allowed microarray monitoring for Salmonella contamination of food and manure for aquaculture.

Key words: Salmonella, gyrB gene, PCR, DNA microarray, TEMED.