A New Validated Real-Time PCR-Based Method for the Specific and Fast Detection of Cronobacter spp. in Infant Formula
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A New Validated Real-Time PCR-Based Method for the Specific and Fast Detection of Cronobacter spp. in Infant Formula Marçal Soler & Olaya Ruiz-Rueda & Mireia Lopez-Siles & Laia Calvó & Eva Kaclíková & Jesús L. García-Gil
Received: 22 March 2011 / Accepted: 16 May 2011 / Published online: 27 May 2011 # Springer Science+Business Media, LLC 2011
Abstract In this study, a real-time polymerase chain reaction (PCR)-based method was designed for the fast detection of Cronobacter spp. (a newly proposed genus formerly known as Enterobacter sakazakii) in infant formula. The real-time PCR was positively tested with 70 Cronobacter strains, including members of all the species of this genus, and 88 non-Cronobacter strains. This new PCR-based system was validated against the reference standard ISO/TS 22964: 2006 (ISO International Organization for Standardization 2006) using 70 food matrices including powdered infant formula, follow-up formula, and hydrolyzed cereals for infants. The detection limit of the technique was found to be of 1 cfu in 10 g of food, fulfilling the requirements of the European Commission. The time of analysis, which comprises around 3–6 days using the reference method, is considerably reduced to less than 24 h using the real-time PCR-based system hereby described, allowing food industry a faster release of the stocks for commercialization. Moreover, this method includes an internal amplification control, co-amplified during each PCR run to verify the results. M. Soler : O. Ruiz-Rueda : M. Lopez-Siles : L. Calvó (*) Microbial. Sistemes i Aplicacions Analítiques, S.L. Parc Científic i Tecnològic de la Universitat de Girona. Ed. Jaume Casademont, E C/Pic de Peguera, 15 E-17003 Girona, Spain e-mail: [email protected] E. Kaclíková Department of Microbiology and Molecular Biology, Food Research Institute, Priemyselná 4, PO Box 25, SK-82475 Bratislava 26, Slovakia J. L. García-Gil Department of Biology, Universitat de Girona, Campus de Montilivi s/n, E-17071 Girona, Spain
Keywords Cronobacter spp. . Enterobacter sakazakii . Powdered infant formula (PIF) . Real-time PCR . Validation
Introduction Formerly known as Enterobacter sakazakii (Farmer et al. 1980), Cronobacter spp. is a new genus within the Enterobacteriaceae family recently proposed by Iversen et al. (2007a, 2008a) based on a polyphasic approach. The new genus comprises six species: Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter muytjensii, Cronobacter dublinensis (which in turn contains three subspecies: C. dublinensis subsp. dublinensis, C. dublinensis subsp. lactaridi and C. dublinensis subsp. lausannensis) and Cronobacter genomospecies 1. All species are considered pathogenic as they have been linked to clinical cases of infection in infants or adults (FAO/WHOM 2008). However, given that most physiological and epidemiological studies were carried on using the taxonomy proposed by Farmer et al. (1980), the name E. sakazakii is still commonly used. To avoid confusion due to the new classification,
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