title: Development of a multiplex PCR for rapid detection of verocytotoxin-producing Escherichia coli O26 in raw milk and ground beef.
abstract: Verocytotoxin-producing Escherichia coli (VTEC) O26 is an emergent pathotype that has caused an increasing number of sporadic cases and outbreaks of gastroenteritis, hemorrhagic colitis, and hemolytic uremic syndrome in the United States and Europe. Many cases are associated with the consumption of milk and undercooked or fermented meats. The stx(2) strains of VTEC O26 seem to be more likely to cause human infections than isolates expressing only stx(1). The isolation and identification of VTEC O26 from foods is labor intensive and time-consuming. We developed a multiplex PCR (M-PCR) assay for the identification and characterization of E. coli O26 VTEC and its detection in raw milk and ground beef. The method is based on the amplification of the wzx, stx(1), and stx(2) genes for the simultaneous detection of the O26 antigen and verocytotoxin types 1 and 2. This M-PCR assay had a sensitivity of 10(8) CFU/ml when applied to a bacterial suspension and of 10(6) CFU/ml or g when applied to both inoculated milk and minced beef samples. This M-PCR assay also was highly specific, and results were consistently negative for negative controls (nonpathogenic E. coli strains, uninoculated milk and beef samples, and samples inoculated with the nontarget microorganisms). This method could be used for the rapid detection of E. coli O26 VTEC from foods and for the rapid identification and characterization of clinical and environmental isolates.
authors.affiliation: Department of Health and Animal Welfare, Faculty of Veterinary Medicine, Str. Prov. per Casamassima Km 3, 70010 Valenzano, Bari, Italy.
authors.forename: V
authors.initials: V
authors.lastname: Lorusso
authors.forename: A
authors.initials: A
authors.lastname: Dambrosio
authors.forename: N C
authors.initials: NC
authors.lastname: Quaglia
authors.forename: A
authors.initials: A
authors.lastname: Parisi
authors.forename: G
authors.initials: G
authors.lastname: Lasalandra
authors.forename: G
authors.initials: G
authors.lastname: Mula
authors.forename: S
authors.initials: S
authors.lastname: Virgilio
authors.forename: G
authors.initials: G
authors.lastname: Lucifora
authors.forename: M
authors.initials: M
authors.lastname: Dario
authors.forename: G
authors.initials: G
authors.lastname: Normanno
mesh_terms: D000818:Animals; D002417:Cattle; D015169:Colony Count, Microbial; D003257:Consumer Product Safety; D004269:DNA, Bacterial / Q000032:analysis; D004196:Disease Outbreaks; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D006801:Humans; D008461:Meat Products / Q000382:microbiology*; D008892:Milk / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D054323:Shiga-Toxigenic Escherichia coli / Q000302:isolation & purification*
chemical_list: D004269:DNA, Bacterial