Multiplexed Single Intact Cell Droplet Digital PCR (MuSIC ddPCR) Method for Specific Detection of Enterohemorrhagic E. coli (EHEC) in Food Enrichment Cultures

Abstract

Foodborne illness attributed to enterohemorrhagic E. coli (EHEC), a highly pathogenic subset of Shiga toxin-producing E. 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 of eae-negative STEC and eae-positive E. 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 the stx and eae genes 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 (stx and eae) using standard TaqMan chemistries with ddPCR. Performance of the method was tested with panels of EHEC and non-target E. coli. By determining the linkage (i.e., the proportion of droplets in which stx and eae targets were both amplified), samples containing EHEC (typically greater than 20% linkage) could be distinguished from samples containing mixtures of eae-negative STEC and eae-positive E. 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 background E. coli and 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.

Rapid quantification of Escherichia coli O157:H7 in lettuce and beef using an on‐chip staining microfluidic device

“…High‐risk foods, such as ground beef and unpasteurized milk, are thought to become contaminated with E. coli O157:H7 via exposure to animal fecal matter, particularly from ruminant animals in which Shiga toxin‐producing E. coli (STEC) are prevalent (Duffy, Burgess, & Bolton, 2014; Gill & Gill, 2010; Karmali, Gannon, & Sargeant, 2010; Mathusa, Chen, Enache, & Hontz, 2010). The detection and identification of STEC, including E. coli O157:H7, in the screening of raw food materials and food products are usually performed by enriching the samples in a selective broth culture and screening for the presence of enterohemorrhagic E. coli using polymerase chain reaction (PCR) methods (ISO/TS TS13136:2012 Technical specification) (McMahon, Blais, Wong, & Carrillo, 2017).…”

A 4-plex Droplet Digital PCR Method for Simultaneous Quantification and Differentiation of Pathogenic and Non-pathogenic Vibrio parahaemolyticus Based on Single Intact Cells

“…However, when the intact cell was used as a template, a single cell was contained in a droplet and the linkage among four genes in one cell was presented. In the previous report, the multiplexed single intact cell droplet digital PCR (MuSIC ddPCR) method was developed for the detection of Enterohemorrhagic E. coli (EHEC;McMahon et al, 2017). However, it was only 2-plex and displayed low amplification efficiency.…”

Optimizing Droplet Digital PCR for Environmental Samples

“…First, to identify current threshold determination approaches for environmental studies using ddPCR, we searched Web of Science (Clarivate Analytics 2018). We identified 75 papers including applications in monitoring of invasive species [26,27], detection of microbes in soil, plant tissue and water [28,29,30,31] and testing food for contamination by microbes or non-declared species [32](Table S1). The most common strategy for threshold assignment was the automatic threshold set by QuantaSoft TM , the default software provided by BioRad Technologies, Inc. (2017) followed by manually set threshold, but for the majority of the studies without explained parameters, via the QuantaSoft TM software.…”