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 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.
Lindqvist
R
R
National Food Administration, Uppsala, Sweden. roli@slv.se
D002499:Centrifugation, Density Gradient; D003470:Culture Media; D005511:Food Handling; D005516:Food Microbiology; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012763:Shigella flexneri / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*
D003470:Culture Media