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 x 102 and 4 x 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. 'a9Springer Science+Business Media, LLC 2007.