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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 N, Lee HS, Jeon WJ, Choi KS, Kweon CH, Yoo HS - J. Vet. Sci. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: National Veterinary Research and Quarantine Service, Anyang 430-824, Korea.

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 log(10) CFU/ml for S. Typhimurium and S. Enteritidis for beef, 1.45 +/- 0.21 and 1.65 +/- 0.07 log(10) 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.

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Related in: MedlinePlus

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).
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Figure 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).

Mentions: The detection limits for boiling method were 3.57 ± 0.02 and 3.57 ± 0.03 log10 CFU/ml for S. Typhimurium and S. Enteritidis in beef, and 4.57 ± 0.02 and 2.26 ± 0.05 log10 CFU/ml for S. Typhimurium and S. Enteritidis in pork (Figs. 3 and 4).


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 N, Lee HS, Jeon WJ, Choi KS, Kweon CH, Yoo HS - J. Vet. Sci. (2009)

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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2801102&req=5

Figure 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).
Mentions: The detection limits for boiling method were 3.57 ± 0.02 and 3.57 ± 0.03 log10 CFU/ml for S. Typhimurium and S. Enteritidis in beef, and 4.57 ± 0.02 and 2.26 ± 0.05 log10 CFU/ml for S. Typhimurium and S. Enteritidis in pork (Figs. 3 and 4).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: National Veterinary Research and Quarantine Service, Anyang 430-824, Korea.

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 log(10) CFU/ml for S. Typhimurium and S. Enteritidis for beef, 1.45 +/- 0.21 and 1.65 +/- 0.07 log(10) 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.

Show MeSH
Related in: MedlinePlus