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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: Rapid Molecular Determination of Serotype from Clinical Isolates of Salmonella Enterica

Authors
item Boyle, David - WASHINGTON DEPT OF HEALTH
item Leader, Brandon - WASHINGTON DEPT OF HEALTH
item Frye, Jonathan
item Russell, Denny - WASHINGTON DEPT OF HEALTH
item Cray, Paula
item Hu, Jinxin - WASHINGTON DEPT OF HEALTH

Submitted to: Proceedings of the International Conference on Emerging Infectious Diseases
Publication Type: Abstract Only
Publication Acceptance Date: March 6, 2008
Publication Date: March 16, 2008
Citation: Boyle, D.S., Leader, B.T., Frye, J.G., Russell, D., Cray, P.J., Hu, J. 2008. Rapid Molecular Determination of Serotype from Clinical Isolates of Salmonella Enterica. Proceedings of the International Conference on Emerging Infectious Diseases.(325)184-185.

Technical Abstract: Background: The conventional serotyping of Salmonella Enterica is time consuming, costly, and requires highly skilled staff. In the present study, we report a multiplex PCR typing method using capillary electrophoresis for fragment analysis that allows for the identification of the 30 most common human clinical serotypes of S. enterica. The use of capillary electrophoresis dramatically improves the throughput, accuracy, discrimination, and sensitivity of this technique compared to conventional agarose gel electrophoresis. This new method of Salmonella typing has been used to test clinical isolates of S. enterica from Washington State and offers an alternative to conventional serotyping. Methods: Fifteen genes were identified based on their distribution’s ability to discriminate different serotypes of S. enterica. Regions of these genes were targeted for amplification in a single multiplex PCR reaction. All forward primers incorporated a 5’ universal sequence complementary to a fluorescently-label complementary universal probe containing FAM. Hot Start PCR was performed with template DNA extracted from either boiled PFGE plugs or from boiled colonies. Samples were diluted 1:100 (v/v) in formamide containing CHIMERx GENEFLO 625 DNA Ladder and separated in an ABI 3100 Avant gene analyzer. Data was analyzed using ABI GeneMapper v3.5. Results: Multiple isolates representing the thirty most common serotypes were analyzed by the multiplex PCR assay to determine representative amplicon codes for each serotype. Using the representative codes, we correctly typed 83% of 400 previously serotyped clinical isolates. Eight percent of the 17% of isolates that we were unable to type with our assay represented rare serotypes that we had not initially screened with our assay and can be defined in future assays. Conclusions: Our fragment analysis-based multiplex PCR typing method is comparable to conventional serotyping in its accuracy, especially when used in conjunction with PFGE. Most importantly it is faster, more cost effective, and avoids the current QC issues arising from the availability, storage, and validation of the numerous reagents required for serotyping. We propose that this method will allow for the rapid and highly accurate typing of extremely large collections of specimens, thus providing an effective alternative to conventional serotyping.

Last Modified: 7/22/2014
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