Quantification of Escherichia coli by kinetic 5'-nuclease polymerase chain reaction (real-time PCR) oriented to sfmD gene.
OBJECTIVE
A kinetic 5'-nuclease polymerase chain reaction (real-time PCR) for the quantification of Escherichia coli was developed.


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.


CONCLUSIONS
In connection to an appropriate sample preparation technique, the method is suitable for food safety and technological hygiene applications.
Kaclíková
E
E
Department of Microbiology and Molecular Biology, Food Research Institute, Bratislava, Slovak Republic.
Pangallo
D
D
Oravcová
K
K
Drahovská
H
H
Kuchta
T
T
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis; D003851:Deoxyribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification*; D029968:Escherichia coli Proteins / Q000235:genetics*; D005516:Food Microbiology; D007700:Kinetics; D016133:Polymerase Chain Reaction / Q000379:methods*
D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C505399:sfmD protein, E coli; D003851:Deoxyribonucleases