title: A Systematic Investigation of Parameters Influencing Droplet Rain in the Listeria monocytogenes prfA Assay - Reduction of Ambiguous Results in ddPCR.
abstract: The droplet digital polymerase chain reaction (ddPCR) determines DNA amounts based upon the pattern of positive and negative droplets, according to Poisson distribution, without the use of external standards. However, division into positive and negative droplets is often not clear because a part of the droplets has intermediate fluorescence values, appearing as "rain" in the plot. Despite the droplet rain, absolute quantification with ddPCR is possible, as shown previously for the prfA assay in quantifying Listeria monocytogenes. Nevertheless, reducing the rain, and thus ambiguous results, promotes the accuracy and credibility of ddPCR. In this study, we extensively investigated chemical and physical parameters for optimizing the prfA assay for ddPCR. While differences in the concentration of all chemicals and the dye, quencher and supplier of the probe did not alter the droplet pattern, changes in the PCR cycling program, such as prolonged times and increased cycle numbers, improved the assay.
authors.affiliation: Christian Doppler Laboratory for Monitoring of Microbial Contaminants, Institute of Milk Hygiene, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria.
authors.forename: Anna Kristina
authors.initials: AK
authors.lastname: Witte
authors.affiliation: Christian Doppler Laboratory for Monitoring of Microbial Contaminants, Institute of Milk Hygiene, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria.
authors.forename: Patrick
authors.initials: P
authors.lastname: Mester
authors.affiliation: Christian Doppler Laboratory for Monitoring of Microbial Contaminants, Institute of Milk Hygiene, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria.
authors.forename: Susanne
authors.initials: S
authors.lastname: Fister
authors.affiliation: Department of Psychology, University of Graz, Graz, Austria.
authors.forename: Matthias
authors.initials: M
authors.lastname: Witte
authors.affiliation: Christian Doppler Laboratory for Monitoring of Microbial Contaminants, Institute of Milk Hygiene, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria.|Institute of Milk Hygiene, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria.
authors.forename: Dagmar
authors.initials: D
authors.lastname: Schoder
authors.affiliation: Christian Doppler Laboratory for Monitoring of Microbial Contaminants, Institute of Milk Hygiene, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria.|Institute of Milk Hygiene, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria.
authors.forename: Peter
authors.initials: P
authors.lastname: Rossmanith
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis*; D004269:DNA, Bacterial / Q000032:analysis; D005453:Fluorescence; D008089:Listeria monocytogenes / Q000235:genetics*; D010454:Peptide Termination Factors / Q000032:analysis*; D016012:Poisson Distribution; D016133:Polymerase Chain Reaction / Q000379:methods*; D011891:Rain
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D010454:Peptide Termination Factors; C545356:PrfA protein, Listeria monocytogenes