Biology Reference
In-Depth Information
Vickery et al. (1998) made use of a random primer designated R-PSE420
( Table 5.2 ) for generating RAPD profi les of V. vulnifi cus strains. The primer
yielded 15 different DNA banding profi les with 16 strains. A great deal of
genomic heterogeneity was observed with strains derived from different
oyster samples and even from strains derived from the same patient with
wound infections.
Warner and Oliver (1999) developed an RAPD protocol for detecting
V. vulnifi cus and for distinguishing this organism from other members
of the genus Vibrio. A 10-mer primer ( Table 5.2 ) previously described
by Warner and Oliver (1998) was used. Each of 70 V. vulnifi cus strains
examined produced a unique banding pattern, indicating that members
of this species are highly heterogeneous. All of the clinical isolates
yielded a unique band (200 to 178-bp) that was only occasionally found
with environmental strains. The authors concluded that this band may
be correlated with human pathogenicity. Subsequent observations by
DePaola et al. (2003a) with this primer indicated that only 70% of clinical
isolates possessed this amplicon and that a band of ca. 460-bp was present
in 86% of these same strains.
Arias et al . (1998) determined the genetic relationships among 132 strains
of V. vulnifi cus d erived from human infections, diseased eels, seawater, and
shellfi sh with the use of ribotyping and RAPD. For ribotyping, genomic
DNA was digested with Kpn I and hybridized with the 18-mer universal
digoxigenin-labeled 1038 olignucleotide probe ( Table 5.2 ) complementary
to a highly conserved sequence in the 23S rRNA gene. RAPD was performed
with the universal primers M13 and T3 ( Table 5.2 ) . Both ribotyping and
RAPD revealed a high level of homogeneity of diseased-eel isolates in
contrast to the genetic heterogeneity of seawater-shellfi sh isolates of the
Mediterranean. Although differentiation within diseased-eel isolates was
only possible by ribotyping, the authors proposed that RAPD is a better
technique than ribotyping for less laborious and rapid typing of new V.
vulnifi cus isolates.
Oligonucleotide Probe
Wright et al. (1993) developed an oligonucleotide probe designated VVAP
( Table 5.2 ) constructed from a portion of the V. vulnifi cus cytolysin gene vvhA
and labeled with alkaline phosphatase. Naturally occurring V. vulnifi cus
were detected without enrichment or selective media by plating dilutions
of oyster homogenates and seawater directly onto Luria agar followed
by incubation overnight at 35°C or at room temperature for 72 h. Plates
with colonies were overlayed with membrane fi lters or fi lter paper discs
which were microwaved and then treated with proteinase K to remove
background alkaline phosphatase activity prior to hybridization. The
VVAP probe was then added and hybridization was allowed to occur for
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