A novel real-time polymerase chain reaction-based method for the detection and quantification of lactose-fermenting Enterobacteriaceae in the dairy and other food industries

Alignment of a 63-bp region of the β-galactosidase (lacZ) gene from 22 representative coliform strains (Citrobacter freundii, Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Enterobacter sakazakii, Citrobacter koseri, Citrobacter amalonaticus, and Raoultella planticola) using Clustal W software (Altschul et al., 1997). The locations of the primers are shown by arrows, an asterisk denotes identical sequences, and a dash indicates a mismatch with the consensus sequence.

Standard curves for the log number of coliform cells per reaction versus the cycle threshold (C_t) value for the fluorescent signal for A) Escherichia coli CECT 515, B) Enterobacter cloacae CECT 194, C) Citrobacter freundii CECT 401, and D) Klebsiella pneumoniae ssp. pneumoniae CECT 143. The error bars indicate standard deviations for 3 independent experiments. CECT=Colección Española de Cultivos Tipo, Burjasot, Valencia, Spain.