title: Development of digoxigenin-labeled PCR amplicon probes for use in the detection and identification of enteropathogenic Yersinia and Shiga toxin-producing Escherichia coli from foods.
abstract: By including digoxigenin-11-dUTP in a polymerase chain reaction (PCR), amplification products were produced that contained nonisotopic markers for use as DNA hybridization probes. Because these labeled amplicons encode pathogenic traits for specific foodborne bacteria, they can be used to detect the presence of potentially virulent organisms that may be present in foods. This technology allows the synthesis of a variety of shelf-stable probe reagents for detecting a number of foodborne microbes of public health concern. We used this technology to detect four genes in two potential pathogens: virF and yadA in enteropathogenic Yersinia and stx1 and stx2 in Shiga-like toxin-producing Escherichia coli. Results of DNA hybridizations of dot blots of 68 Yersinia strains and 24 of 25 E. coli strains were consistent with results of equivalent PCR analyses. DNA colony hybridization with nonisotopic virF probes of colonies arising on spread plates from artificially contaminated food homogenates was able to detect potentially pathogenic Y. enterocolitica. When compared with oligonucleotide probes, amplicon probes are much less sensitive to changes in hybridization and wash temperatures, allowing greater reproducibility. Labeled probe preparations were reused more than five times and have been stored at -20 degrees C for more than 8 months. This method conveniently generates probes that are safe, stable, inexpensive, reusable, and reliable.
authors.affiliation: Seattle District Laboratory, U.S. Food and Drug Administration, Bothell, Washington 98041, USA. sweagant@caora.fda.gov
authors.forename: S D
authors.initials: SD
authors.lastname: Weagant
authors.forename: J A
authors.initials: JA
authors.lastname: Jagow
authors.forename: K C
authors.initials: KC
authors.lastname: Jinneman
authors.forename: C J
authors.initials: CJ
authors.lastname: Omiecinski
authors.forename: C A
authors.initials: CA
authors.lastname: Kaysner
authors.forename: W E
authors.initials: WE
authors.lastname: Hill
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D001427:Bacterial Toxins / Q000235:genetics; D015342:DNA Probes; D004076:Digoxigenin; D004926:Escherichia coli / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D005516:Food Microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D022603:Shiga Toxins; D037521:Virulence Factors; D015007:Yersinia / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D015008:Yersinia enterocolitica / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D015011:Yersinia pseudotuberculosis / Q000145:classification / Q000235:genetics / Q000302:isolation & purification
chemical_list: D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D015342:DNA Probes; D022603:Shiga Toxins; D037521:Virulence Factors; C064832:YadA protein, Yersinia; D004076:Digoxigenin