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5842 NORDSTROM ET AL. APPL. ENVIRON. MICROBIOL.
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FIG. 1. Standard curves of three target genes in multiplex and simplex PCR. (A) Standard curve for the multiplex assay as described in
Materials and Methods; the r2 value was 0.99 for the standard curve of each target. (B, C, and D) Standard curves for each of the targets when
run as a single target with the same reaction conditions as described in Materials and Methods. Results for the tlh, tdh, and trh targets are shown
in panels B, C, and D, with r2 values of 0.98, 0.99, and 0.99, respectively. Each standard curve was run in triplicate; each replicate is plotted. V.
parahaemolyticus with all three target genes from an exponential-phase culture was diluted in PBS, boiled, and used as a template for all
experiments, with concentrations of 0.9 ⫻ 105 to 9 ⫻ 105 CFU/reaction, except that the tlh simplex assay was tested with 0.9 ⫻ 104 to 9 ⫻ 104
CFU/reaction. rxn, reaction.
ington, 11 from Texas, 6 from New York, 5 from California, and 1 from Con- bottles. One gram of homogenate was added to 10 ml of APW in each of three
necticut), 36 environmental isolates (13 from Alabama, 12 from Alaska, 10 from separate tubes, and 1 g was added to 9 ml of PBS for dilutions. A 10-fold
Washington, and 1 from New York), 7 food isolates from the Pacific Northwest, dilution series was done in PBS, and 1 ml of each dilution was added to 10 ml
and 10 isolates of unknown source. Additionally, various concentrations of of APW in each of three tubes. All tubes were incubated overnight at 35°C.
pathogenic strains were tested in combination with higher concentrations of (iii) Culture isolation. After overnight incubation, the top 1 cm of growth
nonpathogenic strains to determine the ability of the assay to detect low copy from all APW tubes was streaked onto thiosulfate-citrate-bile salts-sucrose
numbers of the tdh and trh genes against a very high background of the tlh gene, (TCBS; Difco, Sparks, MD) plates. The TCBS plates were incubated over-
as might be expected for environmental samples. night at 35 ⫾ 2°C and suspect colonies were streaked for purification onto
Environmental studies. (i) Sample collection. Twenty-seven oyster samples nonselective media (TSA with an additional 2% NaCl added). Purified iso-
were collected at various locations in Prince William Sound and other coastal lates were subjected to further biochemical identification that included oxi-
areas of Alaska from August 14 to September 11, 2004. Oysters (10 to 12 per dase, arginine glucose slant, motility test medium (MTM), urea broth, and
sample) were collected from cages suspended from rafts and transported on salt tolerance. Suspect V. parahaemolyticus isolates were confirmed by using
bagged ice to the analyzing laboratory (Alaska Department of Environmental an API 20E assay (bioMérieux, Inc., Durham, NC) and sent to the FDA Gulf
Conservation, Palmer, AK). Analysis was conducted within 24 h of collection. Coast Seafood Laboratory for characterization by alkaline phosphatase (AP)
(ii) Sample preparation. Oysters were scrubbed and shucked according to gene probe hybridization. Each isolate was inoculated into 100 ␮l of APW in
American Public Health Association guidelines (3). The entire shell contents a 96-well plate and incubated overnight. Growth was transferred to a T1N3
(animal and liquor) were emptied into a sterile blender and homogenized at (1% tryptone, 3% sodium chloride, 2% agar) plate with a 48-prong replicator
high speed for 90 s. A three-tube MPN method was done as described in the (Boekel, Feasterville, PA). After overnight incubation, colony lifts were done
FDA Bacteriological Analytical Manual (11), with slight modifications. as previously described (11, 36) and isolates were identified using AP-tlh,
Briefly, 10 g of homogenate was added to 90 ml of alkaline peptone water AP-tdh, and AP-trh probes (11, 36). Any tube that produced at least one
(APW; 1% peptone, 1% NaCl, pH 8.5 ⫾ 0.2) in each of three separate confirmed V. parahaemolyticus (tlh-positive) isolate was scored positive for

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