title: Upstream sample processing facilitates PCR detection of Listeria monocytogenes in mayonnaise-based ready-to-eat (RTE) salads.
abstract: Sample pretreatment to reduce volume and concentrate cells of the target organism(s) prior to molecular detection offers a useful supplement or alternative to cultural enrichment. The purpose of this study was to develop an upstream processing method to facilitate the detection of Listeria monocytogenes in ready-to-eat (RTE) salads by PCR. Potato salad, a model RTE commodity, was seeded with L. monocytogenes and processed by two alternative upstream sample processing methods (designated one-step and two-step centrifugation), followed by DNA extraction, PCR amplification, and Southern hybridization. The two-step method resulted in 1,000-fold improvements in the PCR detection limit, from 10(6) Cfu/g (no sample processing) to 10(3) Cfu/g. The two-step method was applied for upstream sample processing of four representative deli salad items artificially inoculated with L. monocytogenes at levels ranging from 10(1)-10(6) Cfu/g. Following DNA extraction, PCR amplification, and Southern hybridization, detection was achieved at input levels of 10(5) Cfu/g for chicken salad, 10(4) Cfu/g for macaroni salad, and 10(3) Cfu/g for potato and seafood salads. The two-step method reported here facilitates the production of a final sample concentrate of reduced volume and improved purity which was compatible with PCR amplification. This approach offers further progress in our efforts to reduce or eliminate cultural enrichment in an effort to speed time to results when applying molecular methods to the detection of pathogens in foods.
authors.affiliation: Department of Food Science, North Carolina State University, Raleigh, North Carolina, USA.
authors.forename: Jamie
authors.initials: J
authors.lastname: Isonhood
authors.forename: MaryAnne
authors.initials: M
authors.lastname: Drake
authors.forename: Lee-Ann
authors.initials: LA
authors.lastname: Jaykus
mesh_terms: D015139:Blotting, Southern; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000032:analysis*; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D005784:Gene Amplification; D008089:Listeria monocytogenes / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011198:Solanum tuberosum / Q000382:microbiology; D013997:Time Factors
chemical_list: D004269:DNA, Bacterial