IAlignments of target (A gene segments) and probe sequences are shown on the left-hand side, and the respective hybridization signals (including internal staining marker) are given on the right-hand side

Upper part: The signal generated by duplex formation at genotype E-specific probe VD2-04 is reduced to approx. 20% when the target has a single mismatch, such as genotype B. This applies also to genotype B-specific probe VD2-03, when reacting with genotype E. Lower part: Signal is reduced to less than 10% in the case of two mismatches on the target sequence.<p><b>Copyright information:</b></p><p>Taken from "Genotyping of using a new DNA microarray assay based on sequence analysis of A genes"</p><p>http://www.biomedcentral.com/1471-2180/8/63</p><p>BMC Microbiology 2008;8():63-63.</p><p>Published online 17 Apr 2008</p><p>PMCID:PMC2362127.</p><p></p

Genes associated with antimicrobial resistance.

<p>Genes associated with antimicrobial resistance.</p

The leftmost bar in each plot represents the signal of the internal staining control (biotinylated oligonucleotide probe)

<p><b>Copyright information:</b></p><p>Taken from "Genotyping of using a new DNA microarray assay based on sequence analysis of A genes"</p><p>http://www.biomedcentral.com/1471-2180/8/63</p><p>BMC Microbiology 2008;8():63-63.</p><p>Published online 17 Apr 2008</p><p>PMCID:PMC2362127.</p><p></p

SplitsTree graph visualising strain assignments and similarities between isolates.

<p>Isolates were numbered as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089120#pone.0089120.s001" target="_blank">file S1</a>. <i>mecA</i> positives are indicated with squares, PVL-positives with crosses.</p

Genes associated with virulence.

<p>Genes associated with virulence.</p

Typing data.

<p>Typing data.</p

<i>Salmonella</i> strains used to validate the <i>Salmonella</i> serogenotyping array.

a<p><i>invA</i>, <i>galF</i> and <i>manC</i> are species marker for <i>Salmonella</i>.</p>b<p>generous gift of Paul Barrow, University of Nottingham Sutton Bonington Campus, UK.</p>c<p>only genomic DNA of <i>Salmonella</i> Typhi, generous gift of Rene S. Hendrickson, DTU Food, Denmark.</p><p>Strains were classically serotyped by the CDC (Centers of Disease Control and Prevention, Atlanta, USA), DSMZ (German Collection of Microorganism and Cell Cultures, Brunswick, Germany), SGSC (Salmonella Genetic Stock Center, Calgary, Canada) and FLI (National Reference Laboratory for Salmonellosis in cattle at the Friedrich-Loeffler-Institute, Jena, Germany).</p

Fast DNA Serotyping and Antimicrobial Resistance Gene Determination of <em>Salmonella enterica</em> with an Oligonucleotide Microarray-Based Assay

<div><p>Salmonellosis caused by <em>Salmonella</em> (<em>S.</em>) belongs to the most prevalent food-borne zoonotic diseases throughout the world. Therefore, serotype identification for all culture-confirmed cases of <em>Salmonella</em> infection is important for epidemiological purposes. As a standard, the traditional culture method (ISO 6579:2002) is used to identify <em>Salmonella</em>. Classical serotyping takes 4–5 days to be completed, it is labor-intensive, expensive and more than 250 non-standardized sera are necessary to characterize more than 2,500 <em>Salmonella</em> serovars currently known. These technical difficulties could be overcome with modern molecular methods. We developed a microarray based serogenotyping assay for the most prevalent <em>Salmonella</em> serovars in Europe and North America. The current assay version could theoretically discriminate 28 O-antigens and 86 H-antigens. Additionally, we included 77 targets analyzing antimicrobial resistance genes. The <em>Salmonella</em> assay was evaluated with a set of 168 reference strains representing 132 serovars previously serotyped by conventional agglutination through various reference centers. 117 of 132 (81%) tested serovars showed an unique microarray pattern. 15 of 132 serovars generated a pattern which was shared by multiple serovars (<em>e.g.</em>, <em>S.</em> ser. Enteritidis and <em>S.</em> ser. Nitra). These shared patterns mainly resulted from the high similarity of the genotypes of serogroup A and D1. Using patterns of the known reference strains, a database was build which represents the basis of a new PatternMatch software that can serotype unknown <em>Salmonella</em> isolates automatically. After assay verification, the <em>Salmonella</em> serogenotyping assay was used to identify a field panel of 105 <em>Salmonella</em> isolates. All were identified as <em>Salmonella</em> and 93 of 105 isolates (88.6%) were typed in full concordance with conventional serotyping. This microarray based assay is a powerful tool for serogenotyping.</p> </div

Summary of probes detecting antibiotic resistance genes and virulence factors.

<p>Summary of probes detecting antibiotic resistance genes and virulence factors.</p

Multiplex linear DNA amplification, labeling and hybridization of the ArrayStrips.

<p>(a) Linear Multiplex Amplification starting from clonal RNA free genomic DNA, extracted DNA is internally labeled with biotin (Label [L]) and amplified in a linear multiplex PCR reaction; (b) Hybridization: the biotin labeled, single-stranded DNA product hybridizes specifically under stringent conditions to the corresponding probes. The resulting duplex is detected using a horse-radish peroxidase (Enzyme [E]) – streptavidin conjugate, which converts the substrate (Seramun green [S]) into a colored local precipitate. (c) Detection: the ArrayMate™ Reader (or ArrayTube™ Reader ATR 03) enables the visualization and subsequent automated analysis of the array image. The presence of a dark precipitated spot indicates successful hybridization; (d) Analysis: the assay specific software analysis script, supplied with the ArrayMate™ Reader (or ArrayTube™ Reader ATR 03), measures the signal intensity of each probe and determines with an assay specific algorithm which genes/alleles are present in the sample. (e) Genotype analysis: the PatternMatching software supplied with the ArrayMate™ Reader (or ArrayTube™ Reader ATR 03) is comparing the resulting pattern with a local database including 132 reference serovars previously sero- and genotyped, finally a report is given to which serovar the sample strain belongs with regard to the Kauffman-White Scheme.</p