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Background   Enterobacter sakazakii  is the causative agent of rare but severe food-borne infections associated with meningitis, necrotizing enterocolitis and sepsis in infants. Rehydrated powdered infant formulae have been implicated as the source of infection in several outbreaks and sporadic cases. In this work, a real time fluorescence resonance energy transfer PCR assay incorporating an internal amplification control (IAC) was developed for the specific detection of  E. sakazakii  in foods. Performance of the assay, coupled to an automated DNA extraction system and the  E. sakazakii  ISO-IDF (TS 22964/RM 210) enrichment procedure, was evaluated on infant formulae and samples from production environment.    Results  The real-time PCR assay had 100% specificity as assessed using 35  E. sakazakii  and 184 non- E. sakazakii  strains. According to the  E. sakazakii  strains tested, the detection limits ranged from 5 to 25 genomic copies. Assays on pure cultures (including real-time PCR and DNA extraction) gave a sensitivity of about 10 2  to 10 3  CFU/ml. Out of 41 naturally contaminated infant formulae and environmental samples analysed for the presence of  E. sakazakii , 23 were positive by real-time PCR and 22 by the conventional culture method, giving 97.5% concordance with the ISO-IDF reference method.    Conclusion  This method, combining specific real-time PCR, automated DNA extraction and ISO-IDF standard enrichments, provides a useful tool for rapid screening of  E. sakazakii  in food and environmental matrices.

A robotic DNA purification protocol and real-time PCR for the detection of Enterobacter sakazakii in powdered infant formulae.