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United States Department of Agriculture

Agricultural Research Service

Research Project: EPIDEMIOLOGY, ECOLOGY, AND MOLECULAR GENETICS OF ANTIMICROBIAL RESISTANCE IN PATHOGENIC AND COMMENSAL BACTERIA FROM FOOD ANIMALS

Location: Bacterial Epidemiology and Antimicrobial Resistance

Title: Multiplex Pcr Based Method for Identification of Common Clinical Serotypes of Salmonella Enterica Subsp. Enterica

Authors
item Kim, Seonghan - STATE OF WASHINGTON
item Frye, Jonathan
item Hu, Jinxin - STATE OF WASHINGTON
item Cray, Paula
item Gautom, Romesh - STATE OF WASHINGTON
item Boyle, David - STATE OF WASHINGTON

Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 8, 2006
Publication Date: October 2, 2006
Citation: Kim, S., Frye, J.G., Hu, J., Cray, P.J., Gautom, R., Boyle, D.S. 2006. Multiplex pcr based method for identification of common clinical serotypes of salmonella enterica subsp. enterica. Journal of Clinical Microbiology. 44(10)3608-3615.

Interpretive Summary: Salmonella is an important pathogen that causes diseases ranging from gastroenteritis to enteric fever. The Salmonella are separated into over 2,500 different types by serotyping with anti-sera to specific surface structures. This typing method takes several days, requires highly trained staff and expensive reagents and often cannot type up to 10% of the Salmonella tested. A new typing technique has been developed that detects genes specific for Salmonella serotypes by multiplex PCR. Two 5-plex PCR reactions were selected for target genes in the Salmonella. This assay could identify the top 31 serotypes isolated from clinical samples. This technique can identify the top 75% of clinical Salmonella serotypes, can be completed in less than five hours, requires no specialized training, no specific anti-sera, and uses inexpensive reagents. This technique could be used to increase the speed, ease and cost effectiveness of Salmonella typing for clinical laboratories.

Technical Abstract: A multiplex PCR method has been developed to differentiate between the most common clinical serotypes of Salmonella enterica subspecies 1 encountered in Washington State. Six target regions from Salmonella enterica serovar Typhimurium and four from Salmonella enterica serovar Typhi were used to create an assay consisting of two five-plex PCR reactions. The assays gave reproducible results with 31 different serotypes which represent the most common clinical isolates of Salmonella. Of these, 21 serotypes gave unique pattern codes when compared with each other and the other 10 serotypes were grouped into five pairs which were also distinct. Of these pairs, three were composed of serotypes that have been previously noted to be serologically or genetically highly similar. The other two pairs are from less common Salmonella enterica subsp. 1 serovars. The suitability of particular gene target for amplification was assessed by simultaneously screening 253 isolates from the 31 serovars for each target using macroarray hybridization. For every serotype tested the target region was identified as being present or absent. We compared this data with both conventional serotyping and PCR serotyping data and found that PCR serotyping was nearly as discriminatory as the conventional serotyping. The results from a blind test to screen 110 isolates revealed that 93% were correctly identified using the multiplex PCR assay. The assay can be easily performed on multiple samples with final results in less than 5 hours and in conjunction with PFGE forms a very robust method for the molecular subtyping of Salmonella enterica serovar enterica.

Last Modified: 10/1/2014