Quantification of Escherichia coli by kinetic 5¢-nuclease polymerase chain reaction (real-time PCR) oriented to sfmD gene E. Kaclı ´kova ´ 1 , D. Pangallo 2 , K. Oravcova ´ 1 , H. Drahovska ´ 3 and T. Kuchta 1 1 Department of Microbiology and Molecular Biology, Food Research Institute and 2 Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, and 3 Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovak Republic 2004/1312: received 15 November 2004, revised 2 March 2005 and accepted 15 April 2005 ABSTRACT E. KACLI ´ KOVA ´ , D. PANGALLO, K. ORAVCOVA ´ , H. DRAHOVSKA ´ AND T. KUCHTA. 2005. Aims: A kinetic 5¢-nuclease polymerase chain reaction (real-time PCR) for the quantification of Escherichia coli was developed. Methods and Results: Specific primers and a fluorogenic probe oriented to sfmD gene, encoding a putative outer membrane export usher protein, were designed. The PCR system was highly specific and sensitive for E. coli, as determined with 37 non-E. coli strains (exclusivity, 100%) and 24 E. coli strains (inclusivity, 100%). When used in real-time PCR, linear calibration lines were obtained in the range from 10 2 to 10 8 CFU ml )1 for three E. coli strains. Salmonella Enteritidis (10 6 CFU ml )1 ) or Citrobacter freundii (10 6 CFU ml 1 ) had no effect on quantification of E. coli by the method. Conclusions: The developed real-time PCR is suitable for rapid quantification of E. coli. Significance and Impact of the Study: In connection to an appropriate sample preparation technique, the method is suitable for food safety and technological hygiene applications. Keywords: Escherichia coli, polymerase chain reaction, quantification. INTRODUCTION Escherichia coli is an indicator micro-organism that is used as a predictor of faecal contamination (Tortorello 2003). Besides classical microbiological methods for the detection and quantification of the bacterium, rapid methods have been recently developed and recognized as helpful (De Boer and Beumer 1999; Rompre ´ et al. 2002). Successful rapid detection of the entire species of E. coli has been reported by several authors using methods based upon the polymerase chain reaction (PCR). In these PCR methods, primers were oriented to specific sequences of the genes uidA encoding b-glucuronidase (Bej et al. 1991), malB promoter (Candrian et al. 1991), wecA involved in the biosynthesis of entero- bacterial common antigen (Bayardelle and Zafarullah 2002) and 16S rRNA (Nakano et al. 2003). Recently, a method for a rapid quantification of E. coli has been reported, based upon kinetic 5¢-nuclease real-time PCR. However, sequences of the oligonucleotide primers and the probe have not been disclosed (Lebuhn et al. 2003). In this study, we report on the development, composition and analytical parameters of a kinetic 5¢-nuclease real-time PCR oriented to sfmD gene encoding a putative outer membrane export usher protein (Blattner et al. 1997). MATERIALS AND METHODS Bacterial strains The bacterial strains used are listed in Tables 1 and 2. Cultures were grown in nutrient broth (Merck, Darmstadt, Correspondence to: Toma ´s ˇ Kuchta, Department of Microbiology and Molecular Biology, Food Research Institute, Priemyselna ´ 4, PO Box 25, SK-82475 Bratislava 26, Slovakia (e-mail: kuchta@vup.sk). ª 2005 The Society for Applied Microbiology Letters in Applied Microbiology 2005, 41, 132–135 doi:10.1111/j.1472-765X.2005.01736.x