According to current estimates, Salmonella contamination produces illness in between two and four million people around the globe every year.1 Because this is a significant source of foodborne illness, food producers observe strict food handling controls to prevent contamination of food products and mitigate the risk to consumers. In order for these controls to make maximum impact, production facilities require access to rapid microbiological testing that is sensitive and accurate for the purpose of not only detecting contamination but also tracking and tracing outbreaks that occur, so that producers can take decisive corrective action.
In an informative poster, Ortiz et al. described a collaboration undertaken by the Custom Assay Development team at Thermo Fisher Scientific (formerly Life Technologies) to determine if repeated contamination by Salmonella at a specific production facility derived from a single strain.1 In that case, the development of a custom PCR assay was a critical component of corrective action and monitoring going forward.
The team first sequenced 18 Salmonella samples acquired from the production facility where they had been determined to be Salmonella Heidelberg strains. To do this, they used a PureLink Genomic DNA Mini Kit to obtain nucleic acid for input into an Ion Xpress Plus Fragment Library Kit. Then they quantified and enriched the resultant libraries with Ion Library TaqMan Quantitation Kit and an Ion OneTouch instrument before sequencing with the Ion PGM System (all Thermo Scientific).
Next, the team acquired an additional 108 Salmonella Heidelberg genomes from sequencing projects like Genbank and used this resource to identify common conserved regions. Ultimately, they identified three single nucleotide polymorphisms (SNPs) capable of distinguishing the genome sequences from the production facility samples from all other S.Heidelberg genomes and designed TaqMan assays for these SNPs. Finally, the researchers performed qPCR using a 7500 Fast Real-Time PCR System and Sequence Detection Software (both Thermo Scientific). In allelic discrimination mode, the assay results triggering positives can be detected on the VIC channel while negatives populate on the FAM channel.
The authors indicate that the custom assay development process generally requires four to 12 weeks, depending on sample complexity and final assay format. In the case presented here, the team was able to complete development and manufacturing of the custom assay in 10 days, including three days for sequencing and eight days for genome assembly and TaqMan primers/probes design. The resultant assay detects S. Heidelberg genomic DNA at less than 10 CFU and excludes all other serovars as well as non-Salmonella organisms, serving as a robust tool for tracing specific contamination at the production facility.
Further information on microbiology poultry solutions and custom molecular services for food applications can be found on the Thermo Fisher website; these and other food safety issues can be followed on the Accelerating Science blog.
1 Ortiz et al. (2013) ‘Custom Assay for a Sub-population of Salmonella Heidelberg.’ Life Technologies Poster, Thermo Scientific.