Evaluation of the performance of quantitative detection of the Listeria monocytogenes prfA locus with droplet digital PCR

• Published in: Analytical and bioanalytical chemistry 2016-11, Vol.408 (27), p.7583-7593
• Main Authors: Witte, Anna Kristina, Fister, Susanne, Mester, Patrick, Schoder, Dagmar, Rossmanith, Peter
• Format: Article
• Language: English
• Subjects: Amplification; Analytical Chemistry; Assaying; Bacteria; Bacterial Proteins - genetics; Biochemistry; Characterization and Evaluation of Materials; Cheese - microbiology; Chemistry; Chemistry and Materials Science; Contamination; Digital; DNA, Bacterial - genetics; Droplets; Dynamic range; Flora; Food; Food Contamination - analysis; Food Science; Gene Expression; Genetic Loci; Humans; Laboratories; Laboratory Medicine; Limit of Detection; Listeria; Listeria monocytogenes; Listeria monocytogenes - genetics; Listeria monocytogenes - isolation & purification; Monitoring/Environmental Analysis; Organisms; Paper in Forefront; Pathogens; Peptide Termination Factors - genetics; Poisson Distribution; Polymerase chain reaction; Polymerase Chain Reaction - methods; Public health; Staphylococcus infections; Veterinary medicine
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-016-9861-9

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 Δ prfA , showed even slightly better quantification over a broader dynamic range. Graphical Abstract Evaluating the absolute quantification potential of ddPCR targeting Listeria monocytogenes prfA