title: Evaluation of the performance of quantitative detection of the Listeria monocytogenes prfA locus with droplet digital PCR.
abstract: Fast and reliable pathogen detection is an important issue for human health. Since conventional microbiological methods are rather slow, there is growing interest in detection and quantification using molecular methods. The droplet digital polymerase chain reaction (ddPCR) is a relatively new PCR method for absolute and accurate quantification without external standards. Using the Listeria monocytogenes specific prfA assay, we focused on the questions of whether the assay was directly transferable to ddPCR and whether ddPCR was suitable for samples derived from heterogeneous matrices, such as foodstuffs that often included inhibitors and a non-target bacterial background flora. Although the prfA assay showed suboptimal cluster formation, use of ddPCR for quantification of L. monocytogenes from pure bacterial cultures, artificially contaminated cheese, and naturally contaminated foodstuff was satisfactory over a relatively broad dynamic range. Moreover, results demonstrated the outstanding detection limit of one copy. However, while poorer DNA quality, such as resulting from longer storage, can impair ddPCR, internal amplification control (IAC) of prfA by ddPCR, that is integrated in the genome of L. monocytogenes ΔprfA, showed even slightly better quantification over a broader dynamic range. Graphical Abstract Evaluating the absolute quantification potential of ddPCR targeting Listeria monocytogenes prfA.
authors: Christian Doppler Laboratory for Monitoring of Microbial Contaminants, Institute of Milk Hygiene, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Veterinärplatz 1, 1210, Vienna, Austria.
authors: Anna Kristina
authors: AK
authors: Witte
authors: Christian Doppler Laboratory for Monitoring of Microbial Contaminants, Institute of Milk Hygiene, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Veterinärplatz 1, 1210, Vienna, Austria.
authors: Susanne
authors: S
authors: Fister
authors: Christian Doppler Laboratory for Monitoring of Microbial Contaminants, Institute of Milk Hygiene, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Veterinärplatz 1, 1210, Vienna, Austria.
authors: Patrick
authors: P
authors: Mester
authors: Christian Doppler Laboratory for Monitoring of Microbial Contaminants, Institute of Milk Hygiene, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Veterinärplatz 1, 1210, Vienna, Austria.|Institute of Milk Hygiene, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Veterinärplatz 1, 1210, Vienna, Austria.
authors: Dagmar
authors: D
authors: Schoder
authors: Christian Doppler Laboratory for Monitoring of Microbial Contaminants, Institute of Milk Hygiene, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Veterinärplatz 1, 1210, Vienna, Austria. peter.rossmanith@vetmeduni.ac.at.|Institute of Milk Hygiene, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Veterinärplatz 1, 1210, Vienna, Austria. peter.rossmanith@vetmeduni.ac.at.
authors: Peter
authors: P
authors: Rossmanith
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D002611:Cheese / Q000382:microbiology; D004269:DNA, Bacterial / Q000235:genetics*; D005506:Food Contamination / Q000032:analysis*; D015870:Gene Expression; D056426:Genetic Loci; D006801:Humans; D057230:Limit of Detection; D008089:Listeria monocytogenes / Q000235:genetics* / Q000302:isolation & purification; D010454:Peptide Termination Factors / Q000235:genetics*; D016012:Poisson Distribution; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D010454:Peptide Termination Factors; C545356:PrfA protein, Listeria monocytogenes
keywords: Heterogeneous matrix; Listeria monocytogenes; Poisson distribution; ddPCR; prfA; qPCR