title: Development of a Droplet Digital Polymerase Chain Reaction for Rapid and Simultaneous Identification of Common Foodborne Pathogens in Soft Cheese.
abstract: Dairy products can harbor various microorganisms (e.g.,Campylobacterspp.,Salmonellaspp.,Listeria monocytogenes, verocytotoxin-producingEscherichia coli) arising from animal reservoirs, and which can become important sources of foodborne illness. Therefore, early detection of food pathogens is crucial to prevent diseases. We wished to develop an accurate quantitative protocol based on a droplet digital polymerase chain reaction (ddPCR) involving eight individual TaqMan™ reactions to detect simultaneously, without selective enrichment,Listeriaspp.,L. monocytogenes, Salmonellaspp., verocytotoxin-producingE. coliandCampylobacterspp. in cheese. ddPCR (a "third-generation PCR") provides absolute quantification of target DNAs without requirement of a standard curve, which simplifies experimentation and data comparability. The accuracy, specificity and sensitivity of the developed ddPCR system were assessed using purified DNA from 50 reference pathogenic and non-pathogenic strains from international or Italian collections and analyzing soft cheese samples artificially contaminated with serial dilutions (from 4 × 106to 4 × 101CFU/g) of pure cultures from the American Type Culture Collection. Finally, the performance of our ddPCR system was compared by parallel testing with quantitative PCR: it gave higher sensitivity (102CFU/g for theListeriaspp. assay) without the necessity of a standard curve. In conclusion, this is the first ddPCR system developed for simultaneous detection of common foodborne pathogens in cheese using a single set of amplification conditions. As such, it could become a useful strategy for high-throughput screening of microorganisms to evaluate the quality and safety of food products.
authors.affiliation: Institute of Agricultural Biology and Biotechnology, National Research Council Lodi, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Cremonesi
authors.affiliation: Institute of Agricultural Biology and Biotechnology, National Research Council Lodi, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Cortimiglia
authors.affiliation: DeFENS, University of Milan Milan, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Picozzi
authors.affiliation: PTP Science ParkLodi, Italy; DiMeVet, University of MilanMilan, Italy.
authors.forename: Giulietta
authors.initials: G
authors.lastname: Minozzi
authors.affiliation: PTP Science Park Lodi, Italy.
authors.forename: Michela
authors.initials: M
authors.lastname: Malvisi
authors.affiliation: Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna Lodi, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: Luini
authors.affiliation: Institute of Agricultural Biology and Biotechnology, National Research Council Lodi, Italy.
authors.forename: Bianca
authors.initials: B
authors.lastname: Castiglioni
keywords: cheese; ddPCR; detection; foodborne pathogens; qPCR