title: Multiplexed Single Intact Cell Droplet Digital PCR (MuSIC ddPCR) Method for Specific Detection of EnterohemorrhagicE. coli(EHEC) in Food Enrichment Cultures.
abstract: Foodborne illness attributed to enterohemorrhagicE. coli(EHEC), a highly pathogenic subset of Shiga toxin-producingE. coli(STEC), is increasingly recognized as a significant public health issue. Current microbiological methods for identification of EHEC in foods often use PCR-based approaches to screen enrichment broth cultures for characteristic gene markers [i.e., Shiga toxin (stx) and intimin (eae)]. However, false positives arise when complex food matrices, such as beef, contain mixtures ofeae-negative STEC andeae-positiveE. coli, but no EHEC with both markers in a single cell. To reduce false-positive detection of EHEC in food enrichment samples, a Multiplexed, Single Intact Cell droplet digital PCR (MuSIC ddPCR) assay capable of detecting the co-occurrence of thestxandeaegenes in a single bacterial cell was developed. This method requires: (1) dispersal of intact bacteria into droplets; (2) release of genomic DNA (gDNA) by heat lysis; and (3) amplification and detection of genetic targets (stxandeae) using standard TaqMan chemistries with ddPCR. Performance of the method was tested with panels of EHEC and non-targetE. coli. By determining the linkage (i.e., the proportion of droplets in whichstxandeaetargets were both amplified), samples containing EHEC (typically greater than 20% linkage) could be distinguished from samples containing mixtures ofeae-negative STEC andeae-positiveE. coli(0-2% linkage). The use of intact cells was necessary as this linkage was not observed with gDNA extracts. EHEC could be accurately identified in enrichment broth cultures containing excess amounts of backgroundE. coliand in enrichment cultures derived from ground beef/pork and leafy-green produce samples. To our knowledge, this is the first report of dual-target detection in single bacterial cells using ddPCR. The application of MuSIC ddPCR to enrichment-culture screening would reduce false-positives, thereby improving the cost, speed, and accuracy of current methods for EHEC detection in foods.
authors.affiliation: Research and Development, Ottawa Laboratory (Carling), Ontario Laboratory Network, Canadian Food Inspection Agency, OttawaON, Canada; Department of Biology, Carleton University, OttawaON, Canada.
authors.forename: Tanis C
authors.initials: TC
authors.lastname: McMahon
authors.affiliation: Research and Development, Ottawa Laboratory (Carling), Ontario Laboratory Network, Canadian Food Inspection Agency, Ottawa ON, Canada.
authors.forename: Burton W
authors.initials: BW
authors.lastname: Blais
authors.affiliation: Department of Biology, Carleton University, Ottawa ON, Canada.
authors.forename: Alex
authors.initials: A
authors.lastname: Wong
authors.affiliation: Research and Development, Ottawa Laboratory (Carling), Ontario Laboratory Network, Canadian Food Inspection Agency, Ottawa ON, Canada.
authors.forename: Catherine D
authors.initials: CD
authors.lastname: Carrillo
keywords: EHEC; STEC; Shiga toxin; droplet digital PCR; intimin; quantitative PCR