A multiplex ligation detection assay for the characterization of Salmonella enterica strains Henk J.M. Aarts a, ⁎ ,1 , Pieter Vos b , Jonas T. Larsson c , Angela H.A.M. van Hoek a,1 , Stephan Huehn d,2 , Thijs Weijers b , Hugo Ahlm Grønlund e , Burkhard Malorny d a RIKILT-Institute of Food Safety, Wageningen UR, Akkermaalsbos 2, 6708 WB, Wageningen, the Netherlands b Check-Points B.V., Binnenhaven 5, 6709 PD, Wageningen, the Netherlands c Statens Serum Institut, Department of Bacteriology, Mycology and Parasitology, Artillerivej 5, 2300 Copenhagen S, Denmark d Federal Institute for Risk Assessment (BFR), National Salmonella Reference Laboratory, 12277 Berlin, Germany e National Food Institute, Technical University of Denmark (DTU), Mørkhøj Bygade 19, 2860 Søborg, Denmark abstract article info Article history: Received 25 March 2010 Received in revised form 28 September 2010 Accepted 17 October 2010 Keywords: Bio-traceability LDR Microarray Pathogenicity Salmonella A proof of principle of a multi-target assay for genotyping Salmonella has been developed targeting 62 genomic marker sequences of Salmonella related to pathogenicity. The assay is based on multiplex ligation detection reaction (LDR) followed by customized ArrayTube® microarray detection. The feasibility of the developed assay was verified in a method comparison study with conventional PCR using 16 Salmonella ‘test’ strains comprising eight serovars. Subsequently, the feasibility of the LDR microarray assay was also tested by analyzing 41 strains belonging to 23 serovars. With the exception of four serovars each serovar was characterized by a unique virulence associated gene repertoire. The LDR microarray platform proved to be a convenient, rapid and easy to use tool with potential in tracing a Salmonella contamination in the food chain, for outbreak studies, and to provide data for risk assessors that support bio-traceability models. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Salmonellosis is still an important food-borne infective disease worldwide. In the EU for instance 151,995 confirmed cases of human salmonellosis were reported in 2007 (Anonymous, 2009). The Salmonella strains isolated from humans and warm blooded animals usually belong to S. enterica subsp. enterica. This subspecies is further divided into more than 1531 serovars according to the White–Kauffmann–Le Minor scheme (Grimont and Weill, 2007). The incidence of the serovars causing disease with symptoms like nausea, vomiting and abdominal pain varies significantly. For example the serovars Enteritidis and Typhimurium are responsible for at least 80% of all human infections (Anonymous, 2007), whereas other serovars are found in frequencies below 3%. Food-producing animals and related products are the main source of salmonellosis (Callaway et al., 2008). Consequently, to ensure food safety it is very important to control the microbial hazards at the various stages of the food and feed chain. Subtyping food-borne pathogens is an approach often applied to facilitate the epidemiological investigation of outbreaks of gastroin- testinal disease and to identify the source of entry into the food chain. Several molecular based tools have been developed to characterize bacteria genotypically. Pulsed-field gel electrophoresis (PFGE) is currently the method of choice for molecular subtyping Salmonella serovars. It has been proven to be a useful discriminatory method which was standardized by the PulseNet Consortium (Gerner-Smidt et al., 2006). Although this approach is certainly valuable it, however, does not reveal any data on the gene repertoire and biological properties of a strain in order to estimate the risk for humans by consumption. To tackle this weakness, various Salmonella associated DNA microarrays have been developed successfully, specifically targeting genes involved in antibiotic resistance profiles (van Hoek et al., 2005; Majtan et al., 2007; Batchelor et al., 2008; van Hoek and Aarts, 2008), phage types (Pelludat et al., 2005) or serovars (Yoshida et al., 2007; Wattiau et al., 2008). A condensed selection of various Salmonella genetic markers comprising the detection of flagellar and somatic antigens, important virulence genes, phage-associated ele- ments and antibiotic resistance determinants have been used to show the usefulness of DNA microarrays for the discriminative character- ization of Salmonella serovars (Malorny et al., 2007; Huehn et al., 2009). Here we describe a feasibility study of a novel microarray platform that is able to detect various informative genotypic properties to characterize Salmonella strains isolated from a pig slaughterhouse. The novel designed probes target mainly genes which are known to International Journal of Food Microbiology 145 (2011) S68–S78 ⁎ Corresponding author. Present address: Laboratory for Zoonoses and Environmen- tal Microbiology (LZO), National Institute for Public Health and the Environment (RIVM)–Centre for Infectious Disease Control (CIb), P.O. Box 1, 3720 BA, Bilthoven, the Netherlands. Tel.: +31 30 2743008; fax: +31 30 2744434. E-mail address: henk.aarts@rivm.nl (H.J.M. Aarts). 1 Current address: National Institute for Public Health and the Environment (RIVM), Antonie van Leeuwenhoeklaan 9, 3721 MA Bilthoven, the Netherlands. 2 Current address: Free University Berlin, Institute of Food Hygiene, Königsweg 69, 14163 Berlin, Germany. 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.10.010 Contents lists available at ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro