Development of quantitative real-time PCR for detection and enumeration of Enterobacteriaceae

Int J Food Microbiol. 2017 Apr 4;246:92-97. doi: 10.1016/j.ijfoodmicro.2016.12.015. Epub 2017 Jan 4.

Abstract

The family Enterobacteriaceae, members of which are widely distributed in the environment, includes many important human pathogens. In this study, a rapid real-time PCR method targeting rplP, coding for L16 protein, a component of the ribosome large subunit, was developed for enumerating Enterobacteriaceae strains, and its efficiency was evaluated using naturally contaminated food products. The rplP-targeted real-time PCR amplified Enterobacteriaceae species with Ct values of 14.0-22.8, whereas the Ct values for non-Enterobacteriaceae species were >30, indicating the specificity of this method for the Enterobacteriaceae. Using a calibration curve of Ct=-3.025 (log CFU/g)+37.35, which was calculated from individual plots of the cell numbers in different concentrations of 5 Enterobacteriaceae species, the rplP-targeted real-time PCR was applied to 51 food samples. A <1log difference between the real-time PCR and culture methods was obtained in a majority of the food samples (81.8%), with good correlation (r2=0.8285). This study demonstrated that the rplP-targeted real-time PCR method could detect and enumerate Enterobacteriaceae species in foods rapidly and accurately, and therefore, it can be used for the microbiological risk analysis of foods.

Keywords: Enterobacteriaceae; Hygiene indicator; Real-time PCR; rplP.

MeSH terms

  • Colony Count, Microbial
  • DNA Primers
  • Enterobacteriaceae / isolation & purification*
  • Food Microbiology / methods*
  • Hygiene
  • Linear Models
  • Real-Time Polymerase Chain Reaction / methods*
  • Reproducibility of Results
  • Sensitivity and Specificity

Substances

  • DNA Primers