Development of
16S rRNA targeted PCR methods for the detection and differentiation of
Vibrio vulnificus in marine environments
Myoung Sug Kim, Hyun Do Jeong-2001
Aquaculture, 193 (3-4): 199-211
Abstract:
A PCR method for the detection and differentiation of
Vibrio vulnificus strains was developed as an alternative to culture methods
by using combined primers directed against the variable regions of 16S rRNA.
Primers designed from two variable regions of the Vibrionaceae 16S rRNA
(corresponding to nucleotide numbers 1006 to 1023 and 1278 to 1258 in
Escherichia coli 16S rRNA) was found to be species-specific for V.
vulnificus by PCR. Additionally, tri-primer PCR of 16S rRNA was evaluated
for the differentiation of V. vulnificus strains. Although the third primer,
which was derived from the variable region, positions 454 to 473, cannot
discriminate V. vulnificus from other bacteria, it was used to avoid the
detection of type B 16S rRNA of this organism in PCR. The resulting 825 bp
fragment in the presence of the 273 bp fragment, which is specific to V.
vulnificus, in tri-primer PCR clearly differentiated type A 16S rRNA strains
from type B. Enumeration of V. vulnificus in the samples of oyster and
environmental samples was done by most probable numbers' (MPN) method of
five preenrichment tubes of alkaline peptone water supplemented with
polymyxin B following up the confirmation of positive tubes by streaking the
samples onto mCPC agar or by 16S rRNA gene amplification. Higher numbers of
presumptive V. vulnificus confirmed by selective media compared with those
confirmed by PCR method in MPN method suggested that there would be some
bacteria that cannot be discriminated from V. vulnificus on mCPC agar in
environmental samples. In the biotyping of the V. vulnificus isolates in
oyster samples, the majority of the strains (92.5%) belonged to biotype 1,
and 7.5% of the strains belonged to biotype 2. However, strains of 16S rRNA
of V. vulnificus isolates in the marine environment determined by tri-primer
PCR appeared to be 35% type A and 65% type B. These results implied that the
marine environment can serve as reservoir of both V. vulnificus biotypes 1
and 2, and strains of 16S rRNA type B were more frequent than strains of
type A in that environment.
(Department of Aquatic Life Medicine, Pukyong
National University, 599-1 Dae Yeon Dong, Nam Ku, Pusan 608-737, South
Korea)
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