Detection of Shigella spp. in food with a nested PCR method-sensitivity and performance compared with a conventional...

A nested PCR method was developed and its performance evaluated by detection of Shigella flexneri in food. The nested PCR amplifies sequences within an invasion-associated locus (ial) on the invasion plasmid specific for Shigella and enteroinvasive Eschrichia coli (EIEC). The nested PCR detected Sh. flexneri in lettuce inoculated with 2, 20 and 200 cfu g-1 after 1, 7 and 18 d of storage, respectively. In comparison, a culture method (NMKL no. 151) detected 10(5) cfu g-1 after 1 but not after 7 d of storage. The presence of inhibitors in blue cheese and shrimps reduced the sensitivity of the PCR assay. To overcome this inhibition, a sample preparation step based on buoyant density centrifugation was developed. This treatment resulted in a successful recovery of Sh. flexneri in lettuce, milk, shrimp and blue cheese inoculated with 10 cfu g-1. The proposed method, which includes a combination of enrichment, buoyant density centrifugation and a nested PCR, can be completed in less than two working days.