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Title: High-throughput Molecular Determination of Salmonella enterica Serovars Use of Multiplex PCR and Capillary Electrophoresis Analysis

Author
item LEADER, BRANDON - State Of Washington
item Frye, Jonathan
item HU, JINXIN - State Of Washington
item Cray, Paula
item BOYLE, DAVID - State Of Washington

Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/8/2009
Publication Date: 5/1/2009
Citation: Leader, B.T., Frye, J.G., Hu, J., Cray, P.J., Boyle, D.S. 2009. High-throughput Molecular Determination of Salmonella enterica Serovars Use of Multiplex PCR and Capillary Electrophoresis Analysis. Journal of Clinical Microbiology. 47(5):1290-1299.

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 serotypes using a test which is specific for unique surface structures. This typing method takes several days, requires highly trained staff and expensive reagents and often cannot type up to 7% of the Salmonella tested. A new typing technique was developed that detects genes specific for Salmonella serotypes by multiplex PCR and automated capillary electrophoresis analysis. The assay assigns a unique identification number for each serotype called a SMART (Salmonella Multiplex Assay for Rapid Typing) code. SMART was used to analyze 759 blind clinical isolates. 84.2% of these isolates were successfully identified by SMART. This technique can be automated, completed in one day, and cost $1.50 per isolate as compared to ~$40.00 for traditional serotyping, thus increasing speed seed, ease and cost effectiveness of Salmonella typing for clinical and public health laboratories.

Technical Abstract: Salmonella enterica is a zoonotic pathogen and is a leading cause of food-borne illness worldwide. There are over 2,500 serotypes of Salmonella reported. Identification of the serotype is key in defining the etiological agent during an outbreak investigation. In the current study, a high-throughput molecular assay was developed to determine the most common clinical and non-human serovars of S. enterica in the United States. Sixteen genomic targets were identified based on their differential distribution among common serovars. Primers were designed to amplify regions of each of these targets in a single multiplex PCR reaction while incorporating a FAM-labeled, universal primer to fluorescently-label all amplicons. The fluorescently-labeled PCR products were separated using capillary electrophoresis and Salmonella Multiplex Assay for Rapid Typing (SMART) codes were generated for each isolate based upon the presence or absence of PCR products generated from each target gene. Seven hundred and fifty-one blind clinical isolates of Salmonella from Washington State collected in 2007 which had been previously serotyped via antiserum were screened with the assay. 85.1% of the isolates were correctly identified based on comparison to a panel of representative SMART codes previously determined for the top 50 most common serovars in the United States. Of the remaining isolates, 6.2% represented isolates that produced a new SMART code for a previously-determined serotype while the final 8.8% were from serotypes not screened in the original panel used to score isolates in the blinded study. This high-throughput multiplex PCR assay allowed simple and accurate typing of the most prevalent clinical serovars of Salmonella enterica at a level comparable to conventional serotyping, but at a fraction of both the cost and time required per test.