title: Quantification of Listeria monocytogenes cells with digital PCR and their biofilm cells with real-time PCR.
abstract: The purpose of this study was to develop a PCR-based method for quantification of Listeria monocytogenes adhesion in microtitre plates. We optimized isolation of DNA in the microtitre plates using cell lysis, ultrasound treatment, heating, and centrifugation of the lysate. Digital PCR was applied for quantification of L. monocytogenes DNA that was used for construction of the standard curve, and real-time PCR was used for quantification of the attached L. monocytogenes cells. This PCR-based method was applied to quantify different strains of L. monocytogenes at different times of biofilm formation, and to study the anti-adhesive actions of natural bioactive substances (epigallocatechin gallate, (-)-α-pinene). The results show that the PCR-based method developed here can be widely used as a novel approach for adhesion assays and biofilm research.
authors: Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia.
authors: Anja
authors: A
authors: Klančnik
authors: Omega d.o.o., Dolinškova 8, 1000 Ljubljana, Slovenia.
authors: Nataša
authors: N
authors: Toplak
authors: Omega d.o.o., Dolinškova 8, 1000 Ljubljana, Slovenia.
authors: Minka
authors: M
authors: Kovač
authors: Department of Microbiology and Immunology, Veterinary Medical Center, Room C4-183, Cornell University, Ithaca, NY 14853, United States.
authors: Hélène
authors: H
authors: Marquis
authors: Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia. Electronic address: barbka.jersek@bf.uni-lj.si.
authors: Barbara
authors: B
authors: Jeršek
mesh_terms: D001422:Bacterial Adhesion / Q000187:drug effects; D058491:Bacterial Load / Q000379:methods*; D018441:Biofilms / Q000254:growth & development*; D004202:Disinfectants / Q000494:pharmacology; D008089:Listeria monocytogenes / Q000187:drug effects / Q000302:isolation & purification* / Q000502:physiology*; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004202:Disinfectants
keywords: Adhesion; Biofilm; Digital PCR (dPCR); Food safety; Listeria monocytogenes; Real-time PCR (qPCR)