Droplet Digital PCR Method for Multiple Gene Marker Determination in Single Cells Enabling Accurate Detection of Priority STEC in Food Enrichment Cultures

Introduction: The identification of priority Shiga toxin-producing E. coli (STEC) requires detection of characteristic gene markers (i.e., Shiga toxin (stx) and intimin (eae)). Microbiological methods for detection of STEC in foods often use PCR-based approaches to screen enrichment broths for these markers. However, false positives arise when complex food matrices such as beef contain both eae-negative STEC and eae-positive E. coli, and no STEC with both markers in a single cell.  

Purpose: To reduce false positive detection of priority STEC, we have developed a multiplexed droplet digital PCR (ddPCR) assay capable of detecting stx and eae genes in a single bacterial cell. 

Methods: A novel method involving: (1) dispersal of intact bacteria into droplets; (2) release of gDNA by heat lysis; and (3) amplification and detection of genetic targets (stx and eae) using standard TaqMan chemistries and the BioRad QX200^TM ddPCR system was tested with panels of target STEC and non-target E. coli.  

Results: By determining the proportion of droplets positive for both targets relative to all positive droplets, samples containing priority STEC (typically 20-40% double-positive) could be distinguished from samples containing mixtures of eae-negative STEC and eae-positive E. coli (0-2% double-positive). The use of intact cells was necessary as this linkage was not observed with gDNA extracts.  In simulated enrichment broths, STEC could be accurately identified in backgrounds with up to 1,000 x generic E. coli relative to target organisms. Similar sensitivity was achieved in spiked enrichment broths from ground beef and produce samples. 

Significance: To our knowledge, this is the first report of dual-target detection in single bacterial cells using ddPCR. The application of this assay to enrichment-culture screening would reduce false-positives, thereby improving the cost, speed and accuracy of current methods for STEC detection in foods.