Development of Microarray and Multiplex Polymerase Chain Reaction Assays for Identification of Serovars and Virulence Genes in Salmonella Enterica of Human or Animal Origin

K-246 Mosier Hall, Department of Diagnostic Medicine and Pathobiology, 1800 Denison Avenue, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66502, USA.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc (Impact Factor: 1.35). 07/2010; 22(4):559-69. DOI: 10.1177/104063871002200410
Source: PubMed


Salmonella enterica is an important enteric pathogen consisting of many serovars that can cause severe clinical diseases in animals and humans. Rapid identification of Salmonella isolates is especially important for epidemiologic monitoring and controlling outbreaks of disease. Although immunologic and DNA-based serovar identification methods are available for rapid identification of isolates, they are time consuming or costly or both. In the current study, 2 molecular methods for identification of Salmonella serovars were developed and validated. A 70-mer oligonucleotide spotted microarray was developed that consisted of probes that detected genes responsible for genetic variation among isolates of Salmonella that can be used for serotyping. A multiplex polymerase chain reaction (PCR) assay was also developed, which is capable of identifying 42 serovars, thus providing a valuable prediction of the pathogenicity of the isolates by detecting the presence of virulence genes sseL, invA, and spvC. The gene spvC was the best predictor of pathogenicity. In a blind study, traditional serologic methods were correlated at 93.3% with the microarray-based method and 100% with the multiplex PCR-based serovar determination.

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    • "However, few molecular methods are available, except those based on PCR amplification of the rfb gene clusters. These specific targets from the rfb gene clusters are not sufficient in the design of multiplex PCR assays for the identification of Salmonella serogroups and serotypes; therefore, additional targets need to be mined and applied for this purpose (Arrach et al. 2008; Cardona- Castro et al. 2009; Peterson et al. 2010). For instance, multiplex PCR assays developed using rfbJ and wzx genes by Cardona-Castro et al (2009) were capable of detecting Salmonella serogroups B, C2, D and E. In this study, 32 serogroup-specific fragments were identified using a comparative genomics method for detecting Salmonella serogroups A, B, C1, C2 and D, and a multiplex PCR was developed using 5 specific primer sets based on these fragments. "
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    ABSTRACT: Comparative genomic approaches provide abundant information to reveal the diversity among Salmonella serogroups. In a local genomic sequence database, twenty-five Salmonella whole genomic sequences were divided into 6 (A, B, C1, C2, D and others) serogroups for mining the DNA fragments specific for serogroups A through D. For each serogroup, a reference sequence was selected and split into 1000-bp fragments in silico to align against all the other genomic sequences to obtain one or more serogroup-specific fragments. As a result, 2, 6, 7, 10, and 7 specific fragments were found for A, B, C1, C2 and D serogroups, respectively. Specific primer sets targeting these DNA fragments were designed for multiplex PCR assays identifying 21 Salmonella standard strains and 86 additional food isolates. The PCR results demonstrated good agreement with those from Salmonella serotyping. This means that the PCR assay may be able to identify 5 (A, B, C1, C2 and D) Salmonella serogroups and elucidate differences among them. Based on the gene annotations, the 32 serogroup-specific fragments were divided into 3 categories (membrane protein genes, rfb gene clusters, and fimbrial genes). Each gene from these three groups was conserved within the serogroup and was closely correlated with phenotypic characterization. This finding implies that these genes, which are associated with sugar synthesis and metabolism or glycosyl and O-actetyl transfer, impart the differences among Salmonella serogroups.
    Full-text · Article · Jan 2011 · International journal of food microbiology
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    ABSTRACT: For more than 80 years, subtyping of Salmonella enterica has been routinely performed by serotyping, a method in which surface antigens are identified based on agglutination reactions with specific antibodies. The serotyping scheme, which is continuously updated as new serovars are discovered, has generated over time a data set of the utmost significance, allowing long-term epidemiological surveillance of Salmonella in the food chain and in public health control. Conceptually, serotyping provides no information regarding the phyletic relationships inside the different Salmonella enterica subspecies. In epidemiological investigations, identification and tracking of salmonellosis outbreaks require the use of methods that can fingerprint the causative strains at a taxonomic level far more specific than the one achieved by serotyping. During the last 2 decades, alternative methods that could successfully identify the serovar of a given strain by probing its DNA have emerged, and molecular biology-based methods have been made available to address phylogeny and fingerprinting issues. At the same time, accredited diagnostics have become increasingly generalized, imposing stringent methodological requirements in terms of traceability and measurability. In these new contexts, the hand-crafted character of classical serotyping is being challenged, although it is widely accepted that classification into serovars should be maintained. This review summarizes and discusses modern typing methods, with a particular focus on those having potential as alternatives for classical serotyping or for subtyping Salmonella strains at a deeper level.
    Preview · Article · Aug 2011 · Applied and Environmental Microbiology
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