Detection and Quantification of Enterobacter sakazakii by Real-time 5′-Nuclease Polymerase Chain Reaction Targeting the palE Gene

摘要

Enterobacter sakazakii is an emerging food-borne pathogen causing invasive infection with high mortality rates in neonates and infants. The aim of this study was to develop, optimize, and evaluate real-time 5′-nuclease polymerase chain reaction (PCR) for the specific detection and quantification of E. sakazakii. Original primers and TaqMan probe targeting a sequence of E. sakazakii palE gene were designed. The developed real-time PCR system was highly specific for E. sakazakii with 100% inclusivity determined using 54 E. sakazakii strains and 100% exclusivity determined using 99 other strains. Detection limits of 4 × 102 and 4 × 101 CFU ml?1 were determined with 100% and 90% probability, respectively. The response of the 5′-nuclease PCR system was linear (correlation coefficient ≥ 0.997) in the range of 101 to 108 CFU ml?1. Five methods of DNA sample preparation were compared. The methods of DNA preparation using the InstaGene Matrix and the simple lysis by boiling with the Triton X-100 were the most sensitive with calibration lines applicable for quantification. The developed real-time PCR targeted to the palE gene provides an alternative possibility for the detection and quantification of E. sakazakii after the suitable sample preparation.