Quantification of Escherichia coli by kinetic 5'-nuclease polymerase chain reaction (real-time PCR) oriented to sfmD gene

• DOI: 10.1111/j.1472-765x.2005.01736.x
• Publication Date: 2005-07-20T01:32:46Z
• Authors: E. Kaclikova; D. Pangallo; K. Oravcova; H. Drahovska; T. Kuchta
• Keywords: DNA, Bacterial; 0301 basic medicine; Kinetics; 03 medical and health sciences; Deoxyribonucleases; Escherichia coli Proteins; Escherichia coli; Food Microbiology; Polymerase Chain Reaction; Bacterial Outer Membrane Proteins; 3. Good health

A kinetic 5'-nuclease polymerase chain reaction (real-time PCR) for the quantification of Escherichia coli was developed.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.The developed real-time PCR is suitable for rapid quantification of E. coli.In connection to an appropriate sample preparation technique, the method is suitable for food safety and technological hygiene applications.