Next-generation sequencing as a screening tool for foodborne pathogens in fresh produce

Next generation sequencing (NGS) approaches are increasingly applied to tracing microbial contaminants entering the food chain due to NGS' untargeted nature and ability to investigate non-culturable (and/or difficult to culture) organisms while yielding genomic information about the microbiota. So far, a plethora of microbes has been shown to be associated with fresh produce, but few studies have utilised NGS to identify contamination with human pathogens. This study aims to establish the limit of detection (LoD) for Salmonella and phage MS2 (a Norovirus surrogate) contamination of fresh produce employing NGS approaches on the Illumina MiSeq: 16S amplicon-sequencing, and RNA-seq, using ScriptSeq (Illumina) and NEBNext (New England BioLabs) kits. ScriptSeq proved the most sensitive approach; delivering an LoD of 10⁴ CFU reaction^-1 (Colony Forming Units) for Salmonella and 10⁵ PFU reaction^-1 (Plaque Forming Units) for phage MS2. Use of the NEBNext kit resulted in detection of Salmonella at 10⁶ CFU reaction^-1 and phage MS2 at 10⁷ PFU reaction^-1. 16S amplicon-sequencing yielded a similar LoD of 10⁵ CFU reaction^-1 for Salmonella but could not detect MS2. The tested NGS methodologies, in combination with bioinformatics approaches applied, proved less sensitive than conventional microbial detection approaches.