P-051. Development of a Real Time PCR Method for the Detection of Yersinia enterocolitica in Green Leafy Vegetables

A. A. Rodriguez1, P. M. Regan1, A. B. Margolin2;
1U.S. FDA, Winchester, MA, 2Univ. of New Hampshire, Durham, NH.

Psychrotrophic bacteria, such as Yersinia enterocolitica, can grow at the temperatures used to maintain the post harvest quality of produce. This highlights the importance of developing a fast and sensitive method to detect this organism in ready to eat green leafy vegetables. In this study, samples of romaine lettuce, spinach and arugula in broth were inoculated with 0.01 to 105 CFU/g of Y. enterocolitica. They were then incubated at 300 C for 18 h with agitation. Sample aliquots were tested by a duplex real time PCR both before and after incubation. Two sets of probes were selected, one for the chromosomal ail gene which is unique to this species, and another for the virulence plasmid virF gene. The detection limits for the ail gene ranged from 102 CFU/g for the lettuce and arugula to 104 CFU/g for the spinach. While the detection limits for the virF gene ranged from 104 CFU/g for the lettuce and spinach to 105 CFU/g for the arugula. Following an 18 h incubation the sensitivity of the ail gene increased to 0.1 CFU/g for the lettuce and arugula and to 1 CFU/g for the spinach. While the detection limit the virF gene increased to 1 CFU/g for the lettuce and to 10 CFU/g for the spinach and arugula. The assay demonstrated high specificity to Y. enterocolitica when tested against and array of Yersinia, as well as, non Yersinia species. Additionally, it was able to differentiate virulent from non virulent strains. The assay evaluated here was specific, sensitive and fast. It can potentially be applied to the detection of this pathogen in green leafy vegetables.