Use of oligonucleotide array for identification of six foodborne pathogens and Pseudomonas aeruginosa grown on...

Identification of presumptive foodborne pathogens grown on selective media may take one to several days and requires a different battery of biochemical tests for each microorganism. A molecular identification method was developed in which universal primers were used to amplify the 16S to 23S rDNA intergenic spacer of target microorganisms, and PCR products were hybridized to a panel of species-specific oligonucleotides that were immobilized on a nylon membrane. The seven target microorganisms were Bacillus cereus, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella, Staphylococcus aureus, and Vibrio parahaemolyticus. After testing a large collection of target bacteria (29 to 51 strains) and nontarget bacteria (> 500 strains), the performances (sensitivity and specificity) of the oligonucleotide array were as follows: B. cereus (100 and 77%), E. coli (100 and 100%), L. monocytogenes (100 and 90%), P. aeruginosa (100 and 100%), Salmonella (100 and 100%), S. aureus (100 and 100%), and V. parahaemolyticus (100 and 94.2%). Other species in the B. cereus group cross-hybridized to the probes used for identification of B. cereus, and positive results should be confirmed by additional morphological observation of colonies. Listeria innocua cross-reacted with probes used to identify L. monocytogenes, but a simple hemolysis test was used to differentiate the two species. Some strains of Vibrio harveyi and Vibrio mimicus cross-hybridized with probes used for identification of V. parahaemolyticus and caused false-positive reactions. The advantage of the array is that a common protocol was used to identify the seven target microorganisms and multiple different microorganisms could be simultaneously identified on a single array.