title: Rapid detection of Shigella and enteroinvasive Escherichia coli in produce enrichments by a conventional multiplex PCR assay.
abstract: Faster detection of contaminated foods can prevent adulterated foods from being consumed and minimize the risk of an outbreak of foodborne illness. A sensitive molecular detection method is especially important for Shigella because ingestion of as few as 10 of these bacterial pathogens can cause disease. The objectives of this study were to compare the ability of four DNA extraction methods to detect Shigella in six types of produce, post-enrichment, and to evaluate a new and rapid conventional multiplex assay that targets the Shigella ipaH, virB and mxiC virulence genes. This assay can detect less than two Shigella cells in pure culture, even when the pathogen is mixed with background microflora, and it can also differentiate natural Shigella strains from a control strain and eliminate false positive results due to accidental laboratory contamination. The four DNA extraction methods (boiling, PrepMan Ultra [Applied Biosystems], InstaGene Matrix [Bio-Rad], DNeasy Tissue kit [Qiagen]) detected 1.6 × 10(3)Shigella CFU/ml post-enrichment, requiring ∼18 doublings to one cell in 25 g of produce pre-enrichment. Lower sensitivity was obtained, depending on produce type and extraction method. The InstaGene Matrix was the most consistent and sensitive and the multiplex assay accurately detected Shigella in less than 90 min, outperforming, to the best of our knowledge, molecular assays currently in place for this pathogen.
authors.affiliation: Division of Microbiology, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, MD 20740, USA. Electronic address: rachel.binet@fda.hhs.gov.
authors.forename: Rachel
authors.initials: R
authors.lastname: Binet
authors.affiliation: Division of Microbiology, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, MD 20740, USA.
authors.forename: Deanne M
authors.initials: DM
authors.lastname: Deer
authors.affiliation: Oak Ridge Research Institute for Science and Education, PO Box 117, Oak Ridge, TN 37830, USA.
authors.forename: Samantha J
authors.initials: SJ
authors.lastname: Uhlfelder
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification*; D005506:Food Contamination / Q000032:analysis; D005516:Food Microbiology; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012760:Shigella / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification*
keywords: DNA extraction; Enteroinvasive Escherichia coli; Fresh produce; Multiplex PCR; Rapid detection; Shigella