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.

Keywords: Adhesion; Biofilm; Digital PCR (dPCR); Food safety; Listeria monocytogenes; Real-time PCR (qPCR).