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

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 Escherichia 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.