Targeted amplicon sequencing for enhanced detection of spiked Shiga toxin-producing Escherichia coli in ready-to-eat romaine lettuce: a proof-of-concept study

The early and accurate detection of low-level pathogenic and indicator organisms in fresh produce is critical for preventing widespread foodborne outbreaks. Contamination of leafy greens with foodborne pathogens, such as Shiga toxin-producing Escherichia coli (STEC), is a significant public health issue, making rapid and sensitive detection methods critical for mitigating outbreaks. Although next-generation sequencing (NGS) is a powerful tool for pathogen identification, challenges remain in detecting low contamination levels in food products. Here, we demonstrate the use of a custom targeted amplicon sequencing (TAS) primer panel targeting species with food safety concerns, including known human foodborne pathogens, opportunistic pathogens, and indicator organisms related to food spoilage. Using a quasi-metagenomics approach, this proof-of-concept study demonstrates that, compared to whole-metagenomic sequencing (WMS), TAS is a rapid and sensitive NGS-based method for detecting low levels of pathogens. Ready-to-eat romaine lettuce was spiked with STEC and incubated in enrichment medium. DNA was isolated at 0.5, 5, and 6 h, and libraries were prepared for both WMS and TAS. The results indicated that TAS was more sensitive than WMS not only at detecting the pathogen at the species level but also at identifying key virulence markers stx1 and stx2 . Overall, our targeted sequencing approach provides a rapid and sensitive molecular method to detect and identify foodborne pathogenic bacteria, demonstrating its potential for application in food safety.