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Real-time PCR for the detection of Escherichia coli O157:H7 in dairy and cattle wastewater.
Lett Appl Microbiol. 2005; 40(3):164-71.LA

Abstract

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 microl(-1) for eae, stx2 and 16SrRNA, and 1 pg microl(-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.

Authors+Show Affiliations

Spano G
Department of Food Science, Foggia University, Foggia, Italy.
Beneduce L
No affiliation info available
Terzi V
No affiliation info available
Stanca AM
No affiliation info available
Massa S
No affiliation info available

MeSH

Adhesins, BacterialAnimalsCattleCheeseDNA, BacterialDairyingEscherichia coli O157Escherichia coli ProteinsPolymerase Chain ReactionRNA, Ribosomal, 16SShiga ToxinsWaste Disposal, FluidWater MicrobiologyWater Pollutants

Pub Type(s)

Evaluation Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15715639

Citation

Spano, G, et al. "Real-time PCR for the Detection of Escherichia Coli O157:H7 in Dairy and Cattle Wastewater." Letters in Applied Microbiology, vol. 40, no. 3, 2005, pp. 164-71.
Spano G, Beneduce L, Terzi V, et al. Real-time PCR for the detection of Escherichia coli O157:H7 in dairy and cattle wastewater. Lett Appl Microbiol. 2005;40(3):164-71.
Spano, G., Beneduce, L., Terzi, V., Stanca, A. M., & Massa, S. (2005). Real-time PCR for the detection of Escherichia coli O157:H7 in dairy and cattle wastewater. Letters in Applied Microbiology, 40(3), 164-71.
Spano G, et al. Real-time PCR for the Detection of Escherichia Coli O157:H7 in Dairy and Cattle Wastewater. Lett Appl Microbiol. 2005;40(3):164-71. PubMed PMID: 15715639.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Real-time PCR for the detection of Escherichia coli O157:H7 in dairy and cattle wastewater. AU - Spano,G, AU - Beneduce,L, AU - Terzi,V, AU - Stanca,A M, AU - Massa,S, PY - 2005/2/18/pubmed PY - 2005/5/25/medline PY - 2005/2/18/entrez SP - 164 EP - 71 JF - Letters in applied microbiology JO - Lett Appl Microbiol VL - 40 IS - 3 N2 - 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 microl(-1) for eae, stx2 and 16SrRNA, and 1 pg microl(-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. SN - 0266-8254 UR - https://www.unboundmedicine.com/medline/citation/15715639/Real_time_PCR_for_the_detection_of_Escherichia_coli_O157:H7_in_dairy_and_cattle_wastewater_ DB - PRIME DP - Unbound Medicine ER -