Design of a Targeted Amplicon Sequencing Panel for Detection of Foodborne Pathogens and its Application in Detection of Spiked Shiga Toxin-Producing Escherichia coli in Romaine Lettuce

Background: Contamination of leafy greens with foodborne pathogens like Shiga toxin–producing Escherichia coli (STEC) poses a public health concern as 40 documented outbreaks in the United States and Canada occurred between 2009–2018. Early detection and identification of foodborne pathogens helps mitigate outbreaks. One way to implement this is through the application of next-generation sequencing (NGS) methods which offer high throughput, resolution, and sensitivity for both detection and identification of foodborne pathogens. Here we demonstrate the use of a custom targeted amplicon sequencing (TAS) panel targeting 135 known human foodborne pathogens. Despite the scope and power of NGS methods, technical challenges remain in detecting low levels of pathogens in contaminated food. Using a quasimetagenomics approach, this study demonstrates that compared to whole metagenomic sequencing (WMS), targeted amplicon sequencing (TAS) is a rapid and sensitive NGS based method for detecting low levels of pathogens as demonstrated with spiked STEC in bagged chopped ready-to-eat (RTE) romaine lettuce. Results Here we evaluate the utility, specificity, and limit of detection of a targeted amplicon sequencing (TAS) approach for detection and identification of STEC in spiked RTE romaine lettuce. Romaine lettuce was inoculated with STEC at different concentrations. Post inoculation, cells were harvested using a modified version of the Bacteriological Analytical Methods (BAM), at 0.5 h, 5 h and 6 h from primary enrichments and DNA was isolated. DNA libraries were prepared for whole metagenome sequencing (WMS) as well as TAS. Data obtained indicate that TAS is more sensitive than WMS at not only detecting the pathogen at the species level, but also at detecting virulence markers such as stx1 and stx2. Conclusions The targeted sequencing approach described here provides a rapid and sensitive molecular method to detect and identify foodborne pathogenic bacteria. As proof of principle, we use STEC spiked RTE romaine lettuce to demonstrate the applicability of TAS in foodborne pathogen detection.