J Vet Sci.  2009 Mar;10(1):43-51. 10.4142/jvs.2009.10.1.43.

A multiplex real-time PCR for differential detection and quantification of Salmonella spp., Salmonella enterica serovar Typhimurium and Enteritidis in meats

• Lee SH
• Jung BY
• Rayamahji
• Lee HS
• Jeon WJ
• Choi KS
• Kweon CH
• Yoo HS

• KMID: 1089345
• DOI: http://doi.org/10.4142/jvs.2009.10.1.43

Abstract

Salmonella (S.) Typhimurium and S. Enteritidis are the major causative agents of food-borne illnesses worldwide. Currently, a rapid detection system using multiplex real-time polymerase chain reaction (PCR) has been applied for other food-borne pathogens such as Escherichia coli, Staphylococcus aureus and Streptococcus spp. A multiplex real-time PCR was developed for the simultaneous detection of Salmonella spp., especially S. Typhimurium and S. Enteritidis, in beef and pork. For the specific and sensitive multiplex real-time PCR, three representative primers and probes were designed based on sequence data from Genbank. Among the three DNA extraction methods (boiling, alkaline lysis, and QIAamp DNA Mini Kit), the QIAamp DNA Mini Kit was the most sensitive in this study. The optimized multiplex real-time PCR was applied to artificially inoculated beef or pork. The detection sensitivity of the multiplex real-time PCR was increased. The specificity of the multiplex real-time PCR assay, using 128 pure-cultured bacteria including 110 Salmonella isolates and 18 non-Salmonella isolates, was 100%, 100% and 99.1% for Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. The sensitivity was 100%, 100% and 91.7% for Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. The multiplex real-time PCR assay developed in this study could detect up to 0.54 +/- 0.09 and 0.65 +/- 0.07 log10 CFU/ml for S. Typhimurium and S. Enteritidis for beef, 1.45 +/- 0.21 and 1.65 +/- 0.07 log10 CFU/ml for S. Typhimurium and S. Enteritidis for pork, respectively, with all conditions optimized. Our results indicated that the multiplex real-time PCR assay developed in this study could sensitively detect Salmonella spp. and specifically differentiate S. Typhimurium from S. Enteritidis in meats.

Keyword

multiplex real time-PCR; Salmonella Enteritidis; Salmonella spp.; Salmonella Typhimurium

MeSH Terms

Animals
Cattle
DNA, Bacterial
*Food Microbiology
Meat/*microbiology
Polymerase Chain Reaction/*veterinary
Salmonella/*isolation & purification
Sensitivity and Specificity
Swine

Figure

• 

  Fig. 1 Standard curves for the multiplex real-time PCR for Salmonella (S.) Typhimurium. The results of the multiplex real-time PCR were determined using decimal dilution of S. Typhimurium ATCC 14028 DNA. The PCR reaction contained primers and probes for all Salmonella spp., S. Typhimurium and S. Enteritidis. Vertical (y) axis, fluorescence intensity; horizontal (x) axis, PCR cycle numbers. Standard curves for the multiplex real-time PCR of S. Typhimurium. The reactions of S. Typhimurium were always positive at 555 nm (JOE) and 510 nm (FAM). The threshold values (CT) were plotted against the corresponding bacterial cell number (log10 CFU/ml).

• 

  Fig. 2 Standard curves of the multiplex real-time PCR for Salmonella (S.) Enteritidis. The results of the multiplex real-time PCR were determined using decimal dilution of S. Enteritidis ATCC 13076 DNA. The PCR reaction contained primers and probes for all Salmonella spp., S. Typhimurium and S. Enteritidis. Vertical (y) axis, fluorescence intensity; horizontal (x) axis, PCR cycle numbers. Standard curves for the multiplex real-time PCR of S. Enteritidis. The reactions of S. Enteritidis were always positive at 665 nm (Cy5) and 510 nm (FAM). The threshold values (CT) were plotted against the corresponding bacterial cell number (log10 CFU/ml).

• 

  Fig. 3 Comparison of sensitivity of the multiplex real-time PCR on Salmonella Typhimurium ATCC 14028 using the three DNA extraction methods. (A) The results at 555 nm (JOE). (B) The results at 510 nm (FAM).

• 

  Fig. 4 Comparison of sensitivity of the multiplex real-time PCR on Salmonella Enteritidis ATCC 13076 using the three DNA extraction methods. (A) The results at 555 nm (JOE). (B) The results at 510 nm (FAM).

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