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Quantification of Escherichia coli by kinetic 5′‐nuclease polymerase chain reaction (real‐time PCR) oriented to sfmD gene
Author(s) -
Kaclíková E.,
Pangallo D.,
Oravcová K.,
Drahovská H.,
Kuchta T.
Publication year - 2005
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/j.1472-765x.2005.01736.x
Subject(s) - escherichia coli , citrobacter freundii , polymerase chain reaction , real time polymerase chain reaction , salmonella , biology , nuclease , microbiology and biotechnology , enterobacteriaceae , multiplex polymerase chain reaction , salmonella enteritidis , gene , chemistry , bacteria , biochemistry , genetics
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.