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., Campylobacter spp., Salmonella spp., Listeria monocytogenes, verocytotoxin-producing Escherichia 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, Listeria spp., L. monocytogenes, Salmonella spp., verocytotoxin-producing E. coli and Campylobacter spp. 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 × 106 to 4 × 101 CFU/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 (102 CFU/g for the Listeria spp. 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.

New Approaches on Quantification of Campylobacter jejuni in Poultry Samples: The Use of Digital PCR and Real-time PCR against the ISO Standard Plate Count Method

“…has been quantified in the poultry slaughterhouse water samples by the plate count method, droplet dPCR (ddPCR), and qPCR; both molecular-based methods outperformed the culture-based method commonly used in the testing of poultry processing water samples (Rothrock et al, 2013 ). Cremonesi et al ( 2016 ) quantified the common foodborne pathogens including Campylobacter spp. in the soft cheese using ddPCR and qPCR, discovering higher sensitivity of ddPCR.…”

Comparison of the intensity of biofilm formation by Listeria monocytogenes using classical culture-based method and digital droplet PCR

“…The qPCR technique has been used to determine the level of expression of genes involved in biofilm formation of L. monocytogenes (agr and prfA) (Gandra et al 2019). Third-generation PCR, known as a droplet digital PCR (ddPCR), allows fast and quantitative detection (Cremonesi et al 2016). Droplet digital PCR, based on the amplification of a single target DNA molecule in a plurality of separate droplets, enables an exact quantification of DNA copy numbers (Witte et al 2016b).…”

Waterborne pathogen monitoring in Jaipur, India reveals potential microbial risks of urban groundwater supply

“…Another promising development is the use of ddPCR for the absolute quantification of nucleic acid templates 59,60 . Droplet digital PCR has been used to quantify Salmonella in river sediments, Shiga-toxin-producing E. coli (STEC) in bovine feces, foodborne pathogens in soft cheeses, and fecal indicators in environmental water [61][62][63] . The method shows great promise for the detection of waterborne pathogens in drinking water due to its reported sensitivity and resilience to inhibitory substances [64][65][66] .…”