Purpose: To determine if two different but complementary molecular methods could be designed to work efficiently together and act as confirmation of one another for the detection of foodborne pathogens.
Methods: PCR assays were designed and the best assays were selected based on inclusion/exclusion testing and efficiency studies. Two independent PCR assays were designed for Salmonella species, a real-time PCR assay against apeE and an end-point PCR assay against invA. Initially, real-time PCR was performed on the 7500Fast instrument and end-point amplicons were hybridized to an array and detected using standard colorimetric methods. Food samples were enriched according to standard protocols and DNA was isolated using the PrepSEQ™ Nucleic Acid Extraction Kit. Subsequently, DNA extraction, real-time PCR and endpoint PCR was automated using a prototype sample-to-answer system.
Results: Real-time and end-point PCR were coupled in a single reaction tube and tested for Salmonella species. Each method included a separate internal positive control. The real-time PCR assay demonstrated detection of 100 inclusion strains and no detection of 30 exclusion strains, whereas the end-point PCR assay demonstrated detection of 30 inclusion strains and no detection of 20 exclusion strains. The level of detection was less than 100 genomic equivalents. The results show proof of principle for a novel sample-to-answer system that includes sample prep, real-time PCR and end point PCR detection steps.
Significance: Performing screening and confirmation together in an automated workflow provides greater ease of use and confidence to food safety labs.