Development of Sample Preparation Methods to Improve Multiplex PCR Performance for Detection of Escherichia coli on Leafy Vegetables

Introduction: Pathogenic Escherichia coli is one of the major cause for foodborne outbreaks in Korea, and the pathogen has been detected from leafy vegetables. To detect E. coliin leafy vegetables, a multiplex PCR method as rapid detection method is used, but its detection limit is too high.

Purpose: The purpose of this study was to find sample preparation methods to improve multiplex PCR performance to detect E. coliin leafy vegetables.

Methods: A five strain-mixture of E. coli was inoculated in leafy vegetables (cabbage, lettuce and sprouts) at 1, 2 and 3 log CFU/g. The inoculated samples were placed in sample bags containing buffered peptone water (BPW), followed by hand shaking for 30 times. The homogenates were then subjected to control, centrifugation (1912×g at 4^oC for 15 min), filtration (0.45 μm), and enrichment with Escherichia coli broth at 44.2^oC. Pellets from centrifugation and filters from filtration were resuspended in 5 ml BPW. One-milliliter aliquots from the resuspensions and enriched broth were used to extract DNA, and then multiplex PCR method was performed with the DNA.

Results: For lettuce, the filtration and enrichment method (3 h - 24 h) were positive at all inoculation levels but all negative for control and centrifugation method. For cabbage, filtration and enrichment method were positive for all inoculation levels, but centrifugation method was positive only at 3 log CFU/g, and control was negative for all inoculation levels. In sprouts, filtration and enrichment methods were positive at all inoculation level, and centrifugation method was positive at 2 and 3 log CFU/g, but control was positive only at 3 log CFU/g.

Significance: The results indicate that filtration and enrichment methods should be used as a sample preparation method to improve multiplex PCR performance for E. coli on leafy vegetables.