Typing Vibrio parahaemolyticus without a culture

Blackstone GM; Depaola A
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Detection of foodborne pathogens can be greatly facilitated by nucleic acid assays but their usefulness for epidemiology and regulatory purposes has been limited unless a culture has also been isolated. In 1998, the largest US outbreak of Vibrio parahaemolyticus occurred involving oysters harvested from Galveston Bay, Texas that contained the O3:K6 serogroup, a new clone with an unusually high attack rate. During an environmental investigation of these oysters, colony lifts from two samples contained colonies that hybridized with a DNA probe for the thermostable direct hemolysin gene (tdh) which is a pathogenicity marker. Cultures from these colonies were not isolated and it was impossible to determine if the signals were actually V. parahaemolyticus, or from the O3:K6 outbreak strain. A PCR method was developed to amplify nylon or Whatman 541 filter-bound DNA previously hybridized with digoxigenin or alkaline phosphatase probes. Signal positive colonies were cut from the filter and template was prepared by boiling filter cut outs in water or by inserting a portion of the filter into the PCR reaction tube. The PCR products from the filter-bound DNA were the same as those from broth cultures using primers for tdh, the species specific thermolabile direct hemolysin (tlh), and the O3:K6 serogroup specific tox RS or ORF8. This method was applied to filter-bound DNA from the 1998 Texas oyster samples. All colonies yielded appropriate PCR amplicons for tlh and tdh but there was no evidence of amplicons indicative of tox RS or ORF8. The ability to confirm tdh signals by a second method and to subtype using filter-bound DNA in the absence of a viable culture can provide additional information vital to determining the public health significance of colony hybridization results

DNA; hemolysin;Foods; Infection; Methods and Techniques;Meeting Abstract;oyster: host [Pelecypoda]; Vibrio parahaemolyticus: pathogen [Vibrionaceae];Pelecypoda: Animals,Invertebrates,Mollusks; Vibrionaceae: Bacteria,Eubacteria,Microorganisms;[00520] General biology - Symposia,transactions and proceedings;[06704] Vibrionaceae;[10062] Biochemistry studies - Nucleic acids,purines and pyrimidines;[13502] Food technology - General and methods;[31000] Physiology and biochemistry of bacteria;[61500] Pelecypoda;[61500] Pelecypoda,Mollusca,Invertebrata,Animalia [06704] Vibrionaceae,Facultatively Anaerobic Gram-Negative Rods,Eubacteria,Bacteria,Microorganisms;[64026] Invertebrata: comparative,experimental morphology,physiology and pathology - Mollusca;