the 30 cycles of denaturation at 958C for 30 s, annealing at 658C for 30 s and extension at 728C for 30 s.
2.3. Collection and treatment of samples Coastal sea water, sediment, and cultured oyster samples were obtained at the
Hadong area, located at the southern coast of Korea, in late August, 1999. The temperature of the water was 228C and the salinity, as determined from the refractive
index, was 32 ppt. To enumerate the V. Õulnificus, samples were analyzed by using a five-tube MPN method with alkaline peptone water preenrichment supplemented with
Ž .
polymyxin B APWP
followed by the identification of presumptive V. Õulnificus positive tubes using mCPC agar plating or PCR gene amplification with specific primers
Ž .
against V. Õulnificus. Top-layer sediment approximately 500 g and oysters obtained Ž
. from local farmers 10 oysters, average body weight 15 g were transported in a sealed
Ž sterile plastic bag to the laboratory and homogenized with an equal volume of PBS pH
. Ž
. 7.5 in a stainless steel commercial blender Sam Sung at high speed for 60 s. Water
Ž samples were collected along with oyster samples. Homogenized samples 0.01, 0.1, and
. Ž
. 1 g were inoculated into 10 ml of APWP five tubes for each weight of sample and
incubated at 378C for 24 h. For the determination of the positive tubes of V. Õulnificus by the MPN method, samples showing turbidity were analyzed by PCR gene amplifica-
tion with the primers Vib 2 and Vib 3R or by the appearance of yellow colonies after streaking onto mCPC agar plates with incubation at 408C for 24 h. MPN tables were
Ž used to estimate the number of V. Õulnificus cells originally present in a sample US
. Food and Drug Administration, 1984 .
2.4. Identification and characterization of V. Õulnificus isolates From the colonies on mCPC agar plates of different samples, 111 V. Õulnificus-like
yellow colonies were picked randomly and used to isolate total nucleic acid. PCR gene Ž
. amplification was performed with two primers Vib 2 and Vib 3R to confirm that the
isolated colonies were V. Õulnificus. All verified isolates as V. Õulnificus were further Ž
. analyzed to determine the biotypes by indole production in tryptone broth Difco
supplemented with Kovacs indole reagent and the 16S rRNA types were determined by Ž
. PCR gene amplification using tri-primers Vib 1,Vib 2, and Vib 3R .
3. Results
3.1. DeÕelopment of the PCR method Amplification of genomic DNA isolated from three different strains of V. Õulnificus
with primers Vib 2 and Vib 3R resulted in a product with the predicted length of 273 bp, while no products were obtained from those of other species of bacteria including six
Ž .
different species of Vibrionaceae Fig. 1 . For verification, the PCR product derived from the amplification of the total nucleic acid of V. Õulnificus, CJVVO4 was sequenced
Ž .
Fig. 1. Agarose 2 gel electrophoresis analysis of the PCR products from nucleic acids of bacteria with V. Õ
ulnificus specific primers, Vib 2 and Vib 3R. Lane 1: V. Õulnificus CJVVO4; Lane 2: V. Õulnificus HUFP 5002; Lane 3: V. Õulnificus ATCC 27562; Lane 4: V. alginolyticus HUFP 9701; Lane 5: V. cholerae POI
9001; Lane 6: V. fluÕialis; Lane 7: V. mimicus; Lane 8: V. parahaemolyticus HUFP 9114; Lane 9: A. hydrophila ATCC7966; Lane 10: E. coli HB101; Lane 11: Edw. tarda EDK-2; Lane 12: Streptococcus sp.;
Lane M: 100 bp DNA ladder.
and showed no differences compared with the same target region in the 16S rRNA gene Ž
. of V. Õulnificus biotype 1 strain ATCC 27562 data not shown . The PCR amplification
of a 10-fold serial dilution of V. Õulnificus DNA indicated that at least 5 pg of nucleic Ž
. acid was required to yield a visible fragment on agarose gel electrophoresis Fig. 2 . The
Fig. 2. Detection limit of the total nucleic acids isolated from V. Õulnificus CJVVO4 by PCR and RT-PCR Ž .
Ž . with V. Õulnificus specific primers. A Product of PCR gene amplification with primers Cyt1 and Cyt2; B
Ž . product of PCR gene amplification with primers Vib 2 and Vib 3R; C product of RT-PCR gene amplification
with primers Vib 2 and Vib 3R; Lane 1 through 4; 50 pg, 5 pg, 500 fg, 50 fg of V. Õulnificus CJVVO4 nucleic acids; Lane M: 100 bp DNA ladder.
sensitivity was close to that of PCR with Cyt1 and Cyt2 primers. With RT-PCR amplification to increase the sensitivity, it was found that 500 fg of the nucleic acids,
which was 10-fold lower than the threshold sensitivity of PCR amplification, was Ž
. sufficient to yield a visible fragment by agarose gel electrophoresis Fig. 2 .
3.2. Determination of rRNA types of V. Õulnificus V. Õulnificus could be separated into the following two groups corresponding to the
two different 16S rRNA sequences: type A, 16S rRNA corresponding to V. Õulnificus ATCC 27562; and type B, 16S rRNA corresponding to V. Õulnificus C7184 or TW1
Ž .
Aznar et al., 1994 . The complete 16S rRNA sequences of type A strains were slightly Ž
. Ž
different 17 bases from the corresponding sequences of the type B strains Aznar et al., .
1994 . Primers Vib 2 and Vib 3R used in the experiment above were targeted to the variable regions of 16S rDNA of Vibrio species and were specific for both rRNA types
of V. Õulnificus. Another PCR sense primer Vib 1 is complementary to position 454 to 473 in 16 rDNA of V. Õulnificus ATCC 27562 known to be rRNA type A strain.
Although it is not complementary to the target region of the 16S rRNA type B strain of V. Õulnificus, it can hybridize to the 16S rDNA of other species of bacteria depending
upon the phylogenetic relatedness of the 16S rRNA. Upon PCR amplification with
Ž .
Fig. 3. Agarose 2 gel electrophoresis of amplicons generated by tri-primers PCR with the total nucleic acid isolated from V. Õulnificus strains of different 16S rRNA types and other eubacteria. Lane 1: V. Õulnificus
Ž .
Ž .
ATCC 27562 type A 16S rRNA ; Lane 2: V. Õulnificus CJVVO4 type B 16S rRNA ; Lane 3: mixture of the Ž
isolated nucleic acids from five different strains of Vibrionaceae V. alginolyticus HUFP 9701, V. cholerae .
POI 9001, V. fluÕialis, V. mimicus, V. parahaemolyticus HUFP 9114 ; Lane 4: mixture of three different Ž
. Ž
. strains of G y bacteria
A. hydrophila ATCC 7966, E. tarda EDK-2, E. coli HB101 ; Lane M: 100 bp DNA ladder.
Ž .
tri-primers Vib 1, Vib 2, and Vib 3R , the presence of two amplified products, the 273 and 825 bp fragments, discriminated the nucleic acid of rRNA type A V. Õulnificus from
Ž that of rRNA type B V. Õulnificus, which produced a single fragment 273 bp long Fig.
. 3 . The sequence of the 825 bp fragment was exactly the same as that of V. Õulnificus
Ž .
Ž C7184 data not shown , which is reported to be a type B 16S rRNA strain Aznar et al.,
. 1994 .
Tri-primer PCR amplification using nucleic acid isolated from other strains of bacteria as a template yielded no fragments or only a single fragment 825 bp long.
3.3. Characteristics of V. Õulnificus occurred in oyster and enÕironmental samples For counting of V. Õulnfificus by using a five-tube MPN method, two different
methods were employed to determine the presence of V. Õulnificus in the tubes with turbidity. One was the subculturing of the tube samples preenriched in APWP followed
by observation of the yellow colonies that appeared on mCPC agar as presumptive V. Õ
ulnificus. The other one was PCR analysis with the primers Vib 2 and Vib 3R against the total nucleic acids isolated from the bacteria of the cultured tube samples. In a
comparison of these two methods, the number of V. Õulnificus counted when samples from oyster, coastal mud, and sea water was analyzed by PCR appeared to be 49,
13, and 12 of those counted with subculturing on mCPC agar plate, respectively Ž
. Table 3 .
Furthermore, the proportion of V. Õulnificus isolates confirmed by PCR analysis among the presumptive colonies of V. Õulnificus on mCPC agar plates also showed
similar to that of the compared result of MPN method described above and appeared to be 47, 25, and 15 with the colonies of the tube samples of oyster, coastal mud, and
Ž .
sea water, respectively Table 4 . For the classification of the isolated V. Õulnificus Ž
. depending upon the biochemical characteristics biotype and the sequence of rRNA
Ž .
Ž .
Ž . rRNA type , a total of 40 V. Õulnificus isolates from oyster 35 , coastal mud 3 , and
Ž . sea water 2 were tested for indole production and the presence of the 825 fragment on
Ž .
tri-primer PCR amplification Table 4 . The prevalence of V. Õulnificus biotype 2 in the samples of oyster, coastal mud, and sea water was 2 of 33, 1 of 2, and 0 of 2,
respectively. However, the rRNA types of the isolated V. Õulnificus did not show such a
Table 3 Characteristics and confirmation of the identities of V. Õulnificus isolates
Ž .
Samples Total number of
Number of V. Õulnificus cellsr100 ml or g Ž
. bacteria g or ml
identified by mCPC plating
PCR
a 3
3
Oyster ND
7.0=10 2.4=10
5 4
3
Coastal mud 2.1=10
1.6=10 2.1=10
2 2
Sea water 1.2=10
1.3=10 1.5=10
a
Not determined.
Table 4 Comparison of the number of V. Õulnificus identified by PCR and mCPC agar plating in five-tube MPN
method
a b
Samples Number of colonies
Biotypes of the 16S rRNA types of
verified isolates the verified isolates
Examined on Verified by
c
1 2
A B
mCPC PCR
Oyster 74
35 33
2 11
24 Coastal mud
12 3
2 1
2 1
Sea water 13
2 2
1 1
Total 111
40 37
3 14
26
a
Determined by indole test in tryptone broth.
b
Determined by tri-primer PCR gene amplification.
c
Examined yellow colonies on the mCPC agar plate were verified by PCR.
biased result as the biotype, and appeared to be 35 type A and 65 type B in the marine environmental samples.
4. Discussion
