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

Author
item PETERSON, GREG - Kansas State University
item GERDES, BRYAN - Kansas State University
item BERGES, JAMI - Kansas State University
item NAGARAJA, T - Kansas State University
item Frye, Jonathan
item BOYLE, DAVID - Path
item NARAYANAN, SANJEEV - Kansas State University

Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/10/2010
Publication Date: 7/1/2010
Citation: Peterson, G., Gerdes, B., Berges, J., Nagaraja, T.G., Frye, J.G., Boyle, D.S., Narayanan, S. 2010. Development of Microarray and Multiplex Polymerase Chain Reaction Assays for Identification of Serovars and Virulence Genes in Salmonella enterica of Human or Animal Origin. Journal of Veterinary Diagnostic Investigation. 22(4):559-569.

Interpretive Summary: Salmonella is an important pathogen often associated with gastroenteritis in humans. The Salmonella are separated into over 2,500 different serotypes using a test which is specific for unique surface structures on the bacteria. Rapid identification of Salmonella isolates is especially important for epidemiological monitoring and controlling outbreaks of disease. Although current serotyping methods are available for identification of isolates, they are time-consuming and/or costly. A previous study reported the development of a molecular test (polymerase chain reaction, PCR) that could rapidly identify 30 common serotypes of Salmonella associated with clinical disease by combining several PCR tests into a multiplex PCR (M-PCR). In this study, additional PCR tests were added to the M-PCR assay that not only increased the number of identifiable serotypes to 41 but also provided valuable information on the pathogenicity of the isolates by amplifying virulence genes which can be predictive of the ability to cause disease. Of these virulence genes two were present in all isolates, while one gene was more selective and was the best predictor of disease. Further, a specialized DNA test called a microarray was also developed that consisted of DNA probes which detected the genes used for serotyping by the M-PCR assay. In a blind study, traditional tests correlated 100% with the M-PCR and microarray serotype determination. This data is important for public health and diagnostic laboratories to determine the best means of treatment and control.

Technical Abstract: Salmonella enterica is an important enteric pathogen consisting of many serotypes that can cause severe clinical diseases in animals and humans. Rapid identification of Salmonella isolates is especially important for epidemiological monitoring and controlling outbreaks of disease. Although immunological and DNA-based serotyping methods are available for identification of isolates, they are time-consuming and/or costly. A previous study (30) reported the development of a multiplex PCR assay that could rapidly identify 30 common serotypes of Salmonella associated with clinical disease. In this study, an additional multiplex PCR assay was added that not only increased the number of identifiable serotypes to 41 but also provided a valuable prediction of the pathogenicity of the isolates by amplifying virulence genes sseL, invA, and spvC. Of these virulence genes, sseL and invA were present in all isolates, while the gene spvC being more selective was the best predictor of pathogenicity. A 70mer oligonucleotide spotted microarray was also developed that consisted of probes which detected genes responsible for antigenic variation and genotype that can be used for serotyping by multiplex PCR assay. In a blind study, traditional serotyping correlated 100% with the multiplex PCR- based (111 isolates) and the microarray-based (35 isolates) based serotype determination.