Comparison of polymerase chain reaction methods and plating for analysis of enriched cultures of Listeria monocytogenes when using the ISO11290-1 method Marion Dalmasso a , Andrei Sorin Bolocan b , Marta Hernandez c , Anastasia E. Kapetanakou d , Tomáš Kuchta e , Stavros G. Manios d , Beatriz Melero c , Jana Minarovičová e , Meryem Muhterem f , Anca Ioana Nicolau b , Jordi Rovira c , Panagiotis N. Skandamis d , Beatrix Stessl f , Martin Wagner f , Kieran Jordan a, ⁎, David Rodríguez-Lázaro c a Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland b Faculty of Food Science and Engineering, Dunarea de Jos University of Galati, Romania c University of Burgos, Burgos, Spain d Agricultural University of Athens, Iera odos 75, 118 55 Athens, Greece e Food Research Institute, Priemyselná 4, 824 75 Bratislava, Slovakia f Institute for Milk Hygiene, Milk Technology and Food Science, Department of Veterinary Public Health and Food Science, University of Veterinary Medicine, Vienna, Veterinärplatz 1, 1210 Vienna, Austria abstract article info Article history: Received 6 September 2013 Received in revised form 18 December 2013 Accepted 20 December 2013 Available online 31 December 2013 Keywords: Listeria monocytogenes Detection Processing environment sample Food sample RTi-PCR ISO11290-1 standard Analysis for Listeria monocytogenes by ISO11290-1 is time-consuming, entailing two enrichment steps and sub- sequent plating on agar plates, taking five days without isolate confirmation. The aim of this study was to deter- mine if a polymerase chain reaction (PCR) assay could be used for analysis of the first and second enrichment broths, saving four or two days, respectively. In a comprehensive approach involving six European laboratories, PCR and traditional plating of both enrichment broths from the ISO11290-1 method were compared for the de- tection of L. monocytogenes in 872 food, raw material and processing environment samples from 13 different dairy and meat food chains. After the first and second enrichments, total DNA was extracted from the enriched cultures and analysed for the presence of L. monocytogenes DNA by PCR. DNA extraction by chaotropic solid- phase extraction (spin column-based silica) combined with real-time PCR (RTi-PCR) was required as it was shown that crude DNA extraction applying sonication lysis and boiling followed by traditional gel-based PCR re- sulted in fewer positive results than plating. The RTi-PCR results were compared to plating, as defined by the ISO11290-1 method. For first and second enrichments, 90% of the samples gave the same results by RTi-PCR and plating, whatever the RTi-PCR method used. For the samples that gave different results, plating was signifi- cantly more accurate for detection of positive samples than RTi-PCR from the first enrichment, but RTi-PCR detected a greater number of positive samples than plating from the second enrichment, regardless of the RTi-PCR method used. RTi-PCR was more accurate for non-food contact surface and food contact surface samples than for food and raw material samples especially from the first enrichment, probably because of sample matrix interference. Even though RTi-PCR analysis of the first enrichment showed less positive results than plating, in outbreak scenarios where a rapid result is required, RTi-PCR could be an efficient way to get a preliminary result to be then confirmed by plating. Using DNA extraction from the second en- richment broth followed by RTi-PCR was reliable and a confirmed result could be obtained in three days, as against seven days by ISO11290-1. © 2013 Published by Elsevier B.V. 1. Introduction The detection time of pathogen presence in foods and food processing environments is often crucial as it has a direct consequence on the reaction time for decision-making related to risk management and/or risk communication. In this context, speeding up the methods for patho- gen detection became a goal of many research teams. This especially applies to the detection of Listeria monocytogenes, a Gram-positive, rod- shaped bacterium well-known to be the causative agent of listeriosis in humans. Owing to its elaborate physiological adaptation mechanisms, L. monocytogenes can survive and even proliferate under adverse environ- mental conditions such as low pH, high salinity, low temperature and the Journal of Microbiological Methods 98 (2014) 8–14 ⁎ Corresponding author at: Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland. Tel.: +353 2542451. E-mail address: kieran.jordan@teagasc.ie (K. Jordan). 0167-7012/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.mimet.2013.12.018 Contents lists available at ScienceDirect Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth