Rapid detection and simultaneous genotyping of Cronobacter spp. (formerly Enterobacter sakazakii) in powdered infant formula using real-time PCR and high resolution melting (HRM) analysis

PLoS One. 2013 Jun 25;8(6):e67082. doi: 10.1371/journal.pone.0067082. Print 2013.

Abstract

Cronobacter spp. is an emerging pathogen that causes meningitis, sepsis, bacteremia, and necrotizing enterocolitis in neonates and children. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis targeting the OmpA gene for the specific detection and rapid identification of Cronobacter spp. (formerly Enterobacter sakazakii) in powdered infant formula. Eleven Cronobacter field isolates and 25 reference strains were examined using one pair of primers, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit of 10(2) CFU/ml without pre-enrichment, and highly concordant (100%) when compared with ISO-IDF 22964 in 89 actual samples. The method performed for Cronobacter spp. detection was less than 24 h, drastically shortened, compared to several days using standard culturing method, it is probe-free and reduces a risk of PCR carryover. Moreover, all Cronobacter strains examined in this study were genotyped into two species according to their HRM profiles. The established method should provide a molecular tool for direct detection and simultaneous genotyping of Cronobacter spp. in powdered infant formula.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Outer Membrane Proteins / genetics
  • Base Sequence
  • Cronobacter / genetics*
  • Cronobacter / isolation & purification*
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics
  • Food Contamination / analysis*
  • Genotyping Techniques / methods*
  • Humans
  • Infant
  • Infant Formula*
  • Limit of Detection
  • Nucleic Acid Denaturation
  • Powders
  • Real-Time Polymerase Chain Reaction*
  • Time Factors
  • Transition Temperature*

Substances

  • Bacterial Outer Membrane Proteins
  • DNA, Bacterial
  • Powders
  • OMPA outer membrane proteins

Grants and funding

This work was supported by The Science & Technology Program of Zhongshan (Grant No. 20123A299) to XQC, The Science Fund for Creative Research Groups of Gansu Province (Grant No. 1210RJIA006) to XQZ, and the Science & Technology Program of The General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China (Grant No. 2008IK007) to HQY. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.