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

다국어 초록 (Multilingual 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.

참고문헌 (Reference)

• 1 Pang T, "Typhoid fever and other salmonellosis: a continuing challenge" 3 : 253-255, 1995
• 2 Gillespie BE, "Simultaneous detection of mastitis pathogens, Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae by multiplex real-time polymerase chain reaction" 88 : 3510-3518, 2005
• 3 Barrow PA, "Serological diagnosis of Salmonella serotype Enteritidis infections in poultry by ELISA and other tests" 21 : 55-68, 1994
• 4 Kent PT, "Salmonellae in Foods and Feeds" Department of Health and Human Services 29-, 1981
• 5 Andrews WH, "Salmonella. in: Bacteriological Analytical Manual. 8th ed. Chapter 5" AOAC International 5.01-5.20, 1998
• 6 Hein I, "Real-time PCR for the detection of Salmonella spp. in food: An alternative approach to a conventional PCR system suggested by the FOOD-PCR project" 66 : 538-547, 2006
• 7 Seo KH, "Rapid, specific detection of Salmonella Enteritidis in pooled eggs by real-time PCR" 67 : 864-869, 2004
• 8 Chiu CH, "Rapid identification of Salmonella serovars in feces by specific detection of virulence genes, invA and spvC, by an enrichment broth culture-multiplex PCR combination assay" 34 : 2619-2622, 1996
• 9 Bennett AR, "Rapid and definitive detection of Salmonella in foods by PCR" 26 : 437-441, 1998
• 10 Grant MA, "Multiplex real-time PCR detection of heat-labile and heat-stable toxin genes in enterotoxigenic Escherichia coli" 69 : 412-416, 2006
• 11 International Organization for Standardization (ISO), "Microbiology of food and animal feeding stuffs. Horizontal method for the detection of Salmonella (ISO 6579:2003)" ISO 2003
• 12 Szmolka A, "Improved diagnostic and real-time PCR in rapid screening for Salmonella in the poultry food chain" 54 : 297-312, 2006
• 13 Soumet C, "Identification by a multiplex PCR-based assay of Salmonella Typhimurium and Salmonella Enteritidis strains from environmental swabs of poultry houses" 29 : 1-6, 1999
• 14 Mead PS, "Food-related illness and death in the United States" 5 : 607-625, 1999
• 15 Korea Food & Drug Administration (KFDA), "Food & Drug Statistical Yearbook" KFDA 142-, 2008
• 16 De Medici D, "Evaluation of DNA extraction methods for use in combination with SYBR green I real-time PCR to detect Salmonella enterica serotype Enteritidis in poultry" 69 : 3456-3461, 2003
• 17 Fey A, "Establishment of a real-time PCR-based approach for accurate quantification of bacterial RNA targets in water, using Salmonella as a model organism" 70 : 3618-3623, 2004
• 18 Malorny B, "Diagnostic real-time PCR for detection of Salmonella in food" 70 : 7046-7052, 2004
• 19 Imre A, "Development of a PCR system forthe characterisation of Salmonella flagellin genes" 53 : 163-172, 2005
• 20 Rossmanith P, "Detection of Listeria monocytogenes in food using a combined enrichment/real-time PCR method targeting the prfA gene" 157 : 763-771, 2006
• 21 Seo KH, "Detection and enumeration of Salmonella Enteritidis in homemade ice cream associated with an outbreak: comparison of conventional and real-time PCR methods" 69 : 639-643, 2006
• 22 Wilson MA, "Comparison of DNA fingerprints and somatic serotypes of serogroup B and E Pasteurella multocida isolates" 30 : 1518-1524, 1992
• 23 Clouthier SC, "Characterization of three fimbrial genes, sefABC, of Salmonella enteritidis" 175 : 2523-2533, 1993
• 24 Tan W, "Automated detection of Salmonella spp. in foods" 37 : 87-91, 1999
• 25 Miyamoto T, "Application of random amplified polymorphic DNA analysis for detection of Salmonella spp. in foods" 61 : 785-791, 1998
• 26 Lin CK, "An improved PCR primer pair based on 16S rDNA for the specific detection of Salmonella serovars in food samples" 67 : 1335-1343, 2004
• 27 Trkov M, "An improved 16S rRNA based PCR method for the specific detection of Salmonella enterica" 80 : 67-75, 2003
• 28 Baay MF, "Alternative antigens reduce cross-reactions in an ELISA for the detection of Salmonella enteritidis in poultry" 74 : 243-247, 1993
• 29 Mackinnon A, "A spreadsheet for the calculation of comprehensive statistics for the assessment of diagnostic tests and inter-rater agreement" 30 : 127-134, 2000
• 30 Sails AD, "A real-time PCR assay for the detection of Campylobacter jejuni in foods after enrichment culture" 69 : 1383-1390, 2003
• 31 Holicka J, "A rapid (one day), sensitive real-time polymerase chain reaction assay for detecting Escherichia coli O157:H7 in ground beef" 52 : 992-998, 2006
• 32 Quinn C, "A comparison of conventional culture and three rapid methods for the detection of Salmonella in poultry feeds and environmental samples" 20 : 89-91, 1995