Skip to main content
ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Publications at this Location » Publication #158140

Title: COMPARISON OF PRIMERS FOR THE DETECTION OF SALMONELLA ENTERICA SEROVARS USING REAL-TIME PCR.

Author
item CSORDAS, ANDREW
item Barak Cunningham, Jeri
item DELWICHE, MICHAEL

Submitted to: Journal of Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/18/2004
Publication Date: N/A
Citation: N/A

Interpretive Summary: We report the evaluation of the specificity and sensitivity of polymerase chain reaction (PCR) primers for the detection of Salmonella enterica in a real-time PCR assay. Serial dilutions of S. enterica strains were made in sterile water and unenriched whole cells were used as template for each PCR. Twenty microliters of sample were tested in a total PCR volume of 50 'l. SYBR Green dye was used for the non-specific detection of double stranded DNA (dsDNA) and amplicon size was verified with both dissociation analysis and agarose gel electrophoresis. The real-time PCR detection limits of two primer sets, INVA and Sal, with S. enterica Newport as the target organism were 3 x 104 and 3 x 103 CFU reaction-1 (rxn), respectively. A new primer set, Sen, was designed, also targeting the invA gene of S. enterica. The Sen PCR primer set detected S. enterica Newport down to 6 CFU rxn-1 in one case, with an average detection limit of 35 CFU rxn-1 over three separate runs. It was possible to significantly improve the sensitivity of the real-time PCR assay by designing a primer set that minimized double stranded DNA formation between PCR primers. Further, it was possible to achieve a sensitive assay for the detection of S. enterica Newport whole cells in sterile water without the need for DNA extraction or target bacteria enrichment.

Technical Abstract: Aims: To evaluate the specificity and sensitivity of PCR primers for the detection of Salmonella enterica (S. enterica) in a real-time PCR assay. Methods and Results: Serial dilutions of S. enterica serovars were made in sterile water and unenriched whole cells were used as template for each PCR. Twenty microliters of sample were tested in a total PCR volume of 50 'l. SYBR Green dye was used for the non-specific detection of double stranded DNA (dsDNA) and amplicon size was verified with both dissociation analysis and agarose gel electrophoresis. The real-time PCR detection limits of two primer sets, INVA (Chiu and Ou, 1996) and Sal (Wang et al., 1997), with S. enterica Newport as the target organism were 3 x 104 and 3 x 103 CFU reaction-1 (rxn), respectively. A new primer set, Sen, was designed, also targeting the invA gene of S. enterica. The Sen PCR primer set detected S. enterica Newport down to 6 CFU rxn-1 in one case, with an average detection limit of 35 CFU rxn-1 over three separate runs. Conclusions: It was possible to significantly improve the sensitivity of the real-time PCR assay by designing a primer set that minimized primer dimer formation. Further, it was possible to achieve a sensitive assay for the detection of S. enterica Newport whole cells in sterile water without the need for DNA extraction or target bacteria enrichment.