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Title: Rapid multiplex PCR and Real-Time TaqMan PCR assays for detection of Salmonella enterica and the highly virulent serovars Choleraesuis and Paratyphi C

Author
item WOODS, DAVID - UCC NATL UNIV OF IRELAND
item REEN, F. JERRY - UCC NATL UNIV OF IRELAND
item GILROY, DEIRDRE - CORK INST OF TECHNOLOGY
item BUCKLEY, JIM - CORK COUNTY COUNCIL
item Frye, Jonathan
item BOYD, E. FIDELMA - UNIV OF DELAWARE

Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/17/2008
Publication Date: 12/1/2008
Citation: Woods, D.F., Reen, F., Gilroy, D., Buckley, J., Frye, J.G., Boyd, E. 2008. Rapid multiplex PCR and Real-Time TaqMan PCR assays for detection of Salmonella enterica and the highly virulent serovars Choleraesuis and Paratyphi C. Journal of Clinical Microbiology. 46(12):4018-4022.

Interpretive Summary: Salmonella enterica serovars Choleraesuis (Cs) and Paratyphi C (Pc) are food borne pathogens that cause highly invasive infections in humans world wide. To prevent their transmission to humans, a rapid technique is needed to detect them in contaminated foods. A multiplex polymerase chain reaction (mPCR) assay specific for Cs and Pc including a Salmonella species internal control was determined to be specific for detection of these serovars when 152 Salmonella strains were tested. The sensitivity of the technique for detection of Salmonella, Cs and Pc in ground turkey and milk was also determined and when the assay was adapted to a real-time test as few as 3 bacterial cells in 10 grams of food could be detected in less than two days. This method can rapidly screen food for contamination with Salmonella, Cs and Pc to prevent transmission to humans resulting in improved food safety and consumer confidence.

Technical Abstract: Salmonella enterica is a human pathogen with over 2,500 serovars characterized. S. enterica serovars Choleraesuis (Cs) and Paratyphi C (Pc) are two globally distributed serovars. We have developed a rapid molecular typing method to detect Cs and Pc in food samples by using a comparative genomics approach to identify regions unique to each serovar from the sequenced genomes. A Salmonella specific primer pair based on the oriC was designed as an internal amplified control to establish accuracy, sensitivity, and reproducibility. Serovar specific primer sets based on regions of difference between Cs and Pc were also designed for use in the real time PCR assays. The optimized multiplex PCR (mPCR) assay employed these three primer sets and was used to screen a collection of over 152 Salmonella strains to validate the specificity of the PCR assay. In this mPCR assay, Cs and Pc gave unique amplification patterns. To develop the technique for practical use, its sensitivity for detection of Salmonella in a food matrix was determined by spiking experiments. The technique was also adapted to a Real-Time (RT) PCR rapid detection assay for both Cs and Pc. This assay utilizes bioinformatics and molecular epidemiological approaches to develop a practical application that eliminates the current lengthy procedures for Salmonella isolation and serotyping of these important serovars.