Cloning and Characterisation of a Δ-prfA Listeria monocytogenes Strain Containing an Artificial Single Copy Genomic Internal Amplification Control (IAC) for Use as Internal Sample Process Control (ISPC)

Abstract

Conventional internal amplification controls (IAC) are DNA-based controls which monitor the amplification reaction of real-time PCR in food pathogen detection. Food pathogen detection using real-time PCR, however, includes necessarily sample preparation and DNA isolation/purification. This modular structure leads to an analytical chain. To cover the whole analytical chain, the concept of the IAC has to be extended to internal sample process controls (ISPCs) which include supporting pre-analytical steps. One concept for such ISPCs is the use of recombinant bacterial cells comprising a deleted target and an artificial competitive target instead, which are derived from the actual target strain. In this work, we present an ISPC for the molecular detection of Listeria monocytogenes. A Δ-prfA L. monocytogenes EGDe strain was cloned with a pPL2 phage insertion vector to include a single copy artificial DNA target, resulting in a fluorescence signal not interfering with the respective signal of the L. monocytogenes EGDe wild-type strain during realtime PCR. The recombinant strain was confirmed and characterized with conventional and real-time PCR including sequencing. Microbiological examination revealed a distinct phenotype pattern on selective plate media which enables discrimination of Δ-prfA L. monocytogenes EGDe from wild-type L. monocytogenes EGDe and Listeria innocua. The ISPC was applied in an examination of artificially contaminated ultra high temperature-treated milk to demonstrate its analytical suitability. The resulting corrected recovery values of the ISPC as obtained by the whole molecular quantification procedure correspond to the respective values determined for the actual target strain (P≤ 0.05).

Screening and characterisation of bacteriophage P100 insensitive Listeria monocytogenes isolates in Austrian dairy plants

“…monocytogenes reference strains ATCC BAA-679 (EGD-e) and D-prfA, þIAC L. monocytogenes EGDe (DMSZ) (Fruhwirth, Fuchs, Mester, Wagner, & Rossmanith, 2012) were used in this study as phage susceptible controls. All bacteria strains were grown overnight in tryptone soya broth (TSB) with 0.6% (w/v) yeast extract (Oxoid Ldt., Hampshire) at 37 C. Overnight cultures were diluted ten-fold in fresh medium and incubated at 37 C for 3e4 h to obtain a maximum number of viable cells in the logarithmic growth phase (log phase).…”

Sample Preparation for qPCR Detection of Listeria from Food

“…An ISPC provides the potential for quantitative monitoring of the efficiency of the individual steps of the protocol. In case of L. monocytogenes, an available ISPC, which confirms the previously stated parameters, is the L. monocytogenes Δ-prfA/+IAC strain (DSMZ; DSM 23639) that can be used ideally together with qPCR assays targeting prfA [2,15].…”

Essential role of polymerases for assay performance – Impact of polymerase replacement in a well-established assay

“…One milliliter of a L. monocytogenes (strain EGDe 1/2a) or Δ prfA L. monocytogenes (strain EGDe 1/2a [ 11 ], both part of the collection of bacterial strains at the Institute of Milk Hygiene, Milk Technology and Food Science, University of Veterinary Medicine, Vienna, Austria) overnight culture (grown in tryptone soya broth with 0.6% (w/v) yeast extract (TSB-Y; Oxoid, Hampshire, UK) at 37 °C) was used for DNA isolation. The DNA concentration was measured with the Qubit dsDNA Broad Range Kit (Fisher Scientific, Vienna, Austria).…”

“…In addition to classic microbiological detection methods, the prfA qPCR has been developed for detection of L. monocytogenes [ 10 ]. Besides its high sensitivity and reliability, the prfA assay is advantaged by its excellent internal amplification control (IAC) using the same primers but different probe from the original assay [ 11 ]. Moreover, the prfA assay has been tested and optimized for droplet digital PCR (ddPCR).…”