A robotic DNA purification protocol and real-time PCR for the detection of Enterobacter sakazakii in powdered infant formulae.
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 102 to 103 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.


CONCLUSIONS
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.
Derzelle
Sylviane
S
Laboratoire d'Etudes et de Recherches sur la Qualité des Aliments et des Procédés agro-alimentaires, Agence française de sécurité sanitaire des aliments (AFSSA), 23 Avenue du Général de Gaulle, 94706 Maisons Alfort cedex, France. s.derzelle@afssa.fr
Dilasser
Françoise
F
D001331:Automation; D044083:Cronobacter sakazakii / Q000235:genetics / Q000302:isolation & purification*; D003470:Culture Media; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D007223:Infant; D007225:Infant Food / Q000382:microbiology*; D041943:Infant Formula; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D003470:Culture Media; D004269:DNA, Bacterial