Abstract
Enterobacter sakazakii is an emerging food-borne pathogen causing invasive infection with high mortality rates in neonates and infants. The aim of this study was to develop, optimize, and evaluate real-time 5′-nuclease polymerase chain reaction (PCR) for the specific detection and quantification of E. sakazakii. Original primers and TaqMan probe targeting a sequence of E. sakazakii palE gene were designed. The developed real-time PCR system was highly specific for E. sakazakii with 100% inclusivity determined using 54 E. sakazakii strains and 100% exclusivity determined using 99 other strains. Detection limits of 4 × 102 and 4 × 101 CFU ml−1 were determined with 100% and 90% probability, respectively. The response of the 5′-nuclease PCR system was linear (correlation coefficient ≥ 0.997) in the range of 101 to 108 CFU ml−1. Five methods of DNA sample preparation were compared. The methods of DNA preparation using the InstaGene Matrix and the simple lysis by boiling with the Triton X-100 were the most sensitive with calibration lines applicable for quantification. The developed real-time PCR targeted to the palE gene provides an alternative possibility for the detection and quantification of E. sakazakii after the suitable sample preparation.
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References
Abolmaaty A, El-Shemy MG, Khallaf MF, Levin RE (1998) J Microbiol Methods 34:133
Börnke F, Hajirezaei M, Sonnewald U (2001) J Bacteriol 183:2425
Commission Regulation (EC) No. 2073/2005, Official Journal of the European Union, L 338:22.12.2005, 1
Derzelle S, Dilasser F (2006) BMC Microbiol 6:100
Dorak MT (2007) Glossary of real-time PCR terms. Available at http://www.dorak.info/genetics/glosrt.html
Drudy D, O’Rourke M, Murphy M et al (2006) Int J Food Microbiol 110:127
Friedemann M (2007) Int J Food Microbiol 116:1
FDA Food and Drug Administration, Center for Food Safety and Applied Nutrition (2002) Isolation and enumeration of Enterobacter sakazakii from dehydrated powdered infant formula. Available at http://www.cfsan.fda.gov/~comm/mmesakaz.html
Guillaume-Gentil O, Sonnard V, Kandhai MC, Marugg JD, Joosten H (2005) J Food Prot 68:64
Gurtler JB, Kornacki JL, Beuchat LR (2005) Int J Food Microbiol 104:1
International Standard Organization (2006) ISO/TS 22964 milk and milk products—detection of Enterobacter sakazakii. International Standard Organization, Geneva
Iversen C, Forsythe S (2004) Food Microbiol 21:771
Iversen C, Forsythe SJ (2007) Appl Environ Microbiol 73:48
Iversen C, Druggan P, Forsythe S (2004) Int J Food Microbiol 96:133
Kandhai MC, Reij MW, van Puyvelde K, Guillaume-Gentil O, Beumer RR, van Schothorst M (2004) Lancet 363:39
Keyser M, Witthuhn RC, Ronquest LC, Britz TJ (2003) Biotechnol Lett 25:1893
Lai KK (2001) Medicine 80:113
Lehner A, Tasara T, Stephan R (2004) BMC Microbiol 25:43
Lehner A, Nitzsch S, Breeuwer P, Diep B, Thelen K, Stephan R (2006a) BMC Microbiol 6:15
Lehner A, Riedel K, Rattei T et al (2006b) Syst Appl Microbiol 29:609
Liu Y, Gao Q, Zhang X, Hou Y, Yang J, Huang X (2006a) Mol Cell Probes 20:11
Liu Y, Cai X, Zhang X, Gao Q, Yang X, Zheng Z, Luo M, Huang X (2006b) J Microbiol Methods 65:21
Malorny B, Wagner M (2005) J Food Prot 68:1623
Mohan Nair KM, Venkitanarayanan KS (2006) Appl Environ Microbiol 72:2539
Nazarowec-White M, Farber JM (1997) J Food Prot 60:226
Oravcová K, Kuchta T, Kaclíková E (2007) Lett Appl Microbiol 45:568
Seo KH, Brackett RE (2005) J Food Prot 68:59
Van Acker J, de Smet F, Muyldermans G, Bougatef A, Naessens A (2001) J Clin Microbiol 39:293
Acknowledgements
This work was supported by the Slovak Research and Development Agency under the contract no. APVV-27-009705. We are grateful to Ing. Katarína Fašiangová from the State Veterinary and Food Institute in Bratislava for the provision of enriched food samples for E. sakazakii isolation.
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Krascsenicsová, K., Trnčíková, T. & Kaclíková, E. Detection and Quantification of Enterobacter sakazakii by Real-time 5′-Nuclease Polymerase Chain Reaction Targeting the palE Gene. Food Anal. Methods 1, 85–94 (2008). https://doi.org/10.1007/s12161-007-9013-0
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DOI: https://doi.org/10.1007/s12161-007-9013-0