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

Sylviane Derzelle1, Françoise Dilasser

  • 1Laboratoire 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

BMC Microbiology
|December 15, 2006
PubMed

Insights

A new real-time PCR assay accurately detects Enterobacter sakazakii in infant formula and environmental samples. This method offers rapid screening for this foodborne pathogen, enhancing infant safety.

Area of Science:

  • Food safety
  • Microbiology
  • Molecular diagnostics

Background:

  • Enterobacter sakazakii causes severe infant infections like meningitis and sepsis.
  • Contaminated infant formula is a significant source of E. sakazakii outbreaks.
  • Rapid and specific detection methods are crucial for foodborne pathogen control.

Purpose of the Study:

  • Develop a real-time PCR assay for specific Enterobacter sakazakii detection in food.
  • Evaluate the assay's performance with automated DNA extraction and standard enrichment procedures.
  • Assess the method's utility for screening infant formulae and environmental samples.

Main Methods:

  • Developed a real-time fluorescence resonance energy transfer (FRET) PCR assay with an internal amplification control (IAC).
  • Integrated the assay with an automated DNA extraction system and the E. sakazakii ISO-IDF enrichment procedure.
  • Validated the method on infant formulae and environmental production samples.

Main Results:

  • The real-time PCR assay demonstrated 100% specificity against 35 E. sakazakii and 184 non-E. sakazakii strains.
  • Detection limits ranged from 5 to 25 genomic copies of E. sakazakii.
  • Achieved 97.5% concordance with the ISO-IDF reference method when analyzing 41 naturally contaminated samples.

Conclusions:

  • The combined method of specific real-time PCR, automated DNA extraction, and ISO-IDF enrichment is effective.
  • This approach provides a valuable tool for the rapid screening of Enterobacter sakazakii in food and environmental matrices.
  • The developed assay enhances the ability to ensure the safety of infant nutrition products.
Abstract

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