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Real‐time PCR for the detection of Escherichia coli O157:H7 in dairy and cattle wastewater
Author(s) -
Spano G.,
Beneduce L.,
Terzi V.,
Stanca A.M.,
Massa S.
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.2004.01634.x
Subject(s) - stx2 , wastewater , polymerase chain reaction , escherichia coli , biology , microbiology and biotechnology , real time polymerase chain reaction , manure , food science , dna extraction , veterinary medicine , gene , shiga toxin , environmental engineering , environmental science , medicine , ecology , biochemistry
Aims: Developing and evaluating a rapid real‐time polymerase chain reaction (PCR) method for the identification of Escherichia coli O157:H7 in cattle and dairy wastewater samples produced from mozzarella cheese factories, without pre‐enrichment step before DNA extraction. Methods and Results: Wastewater samples were collected from a dairy farm producing mozzarella cheese and located in Puglia (south of Italy). Plate count and other microbial assays were performed 1 h after sampling. Wastewater samples were artificially inoculated with 10 4 , 10 7 and 10 8 cells ml −1 of E. coli O157:H7, strain EDL933. PCR protocols for stx1 , stx2 and eae genes were first tested on pure DNA extracted from type strains, in order to optimize the amplification conditions and reagent concentration before real‐time PCR experiments. Three specific fragments of ca 106, 150 and 200 bp corresponding to genes eae , stx1 and stx2 , respectively, were obtained. Real‐time PCR experiments were performed with DNA extracted from dairy and manure wastewater samples inoculated with 10 4 , 10 7 and 10 8 colony‐forming units (CFU) ml −1 of E. coli O157:H7 strain EDL 933. The sensitivity limit of the assay was 10 −1 pg μ l −1 for eae , stx2 and 16SrRNA , and 1 pg μ l −1 for stx1 gene respectively. Conclusions: A real‐time PCR protocol has been developed and used in order to identify potential pathogens in dairy wastewater, in which previous methods (including standard PCR) failed to work. Significance and Impact of the Study: Cattle and dairy wastewater samples produced from mozzarella cheese factories may harbour verocytotoxin‐producing E. coli . The availability of rapid and sensitive molecular methods may be useful to monitor the persistence of verocytotoxin‐producing E. coli in general and to assess the effectiveness of wastewater treatments.