Materials and methods Directory UMM :Data Elmu:jurnal:A:Aquaculture:Vol193.Issue3-4.2001:

Ž . In the present study, a most probable number MPN procedure was used to enumerate V. Õulnificus with the determination and comparison of the number of presumptive positive tubes using selective media and PCR 16S rRNA gene amplifica- tion. The tri-primer PCR method for the differentiation of the confirmed isolates of V. Õ ulnificus depending upon the types of 16S rRNA was also evaluated.

2. Materials and methods

2.1. Bacterial cultures and DNA extraction Ž . A total of 12 species of bacteria, 11 species of G y bacteria including three V. Ž Õ ulnificus strains, five Vibrio species, and Streptococcus sp. were used in this study. V. Õ ulnificus CJVV04 from R D Center of Cheiljedang, Korea; V. Õulnificus HUFP 5002, V. alginolyticus HUFP 9701, V. parahemolyticus HUFP 9114 and V. cholerae POI 9001 . Ž . from Dr. K. Muroga, Hiroshima University, Japan Table 1 . All of the Vibrio species Ž . were grown aerobically on brain heart infusion broth Difco, Detroit, MI, USA Ž . supplemented with 1 wrv NaCl at 258C for 18 h. Cultured cells were harvested by centrifugation at 8000 = g for 10 min and lysed with 5.5 SDSr0.125 mgrml proteinase K solution. Bacterial nucleic acids were extracted by a phenol–chloroform– Ž . Ž . isoamyl alcohol 25:24:1 vrvrv mixture and chloroform–isoamyl alcohol 24:1 vrv mixture. The nucleic acids were precipitated by adding the two volumes of ethanol in the presence of 0.3-M sodium acetate. 2.2. Primers’ design Ž . Ž . Two senses Vib 1, Vib 2 and one antisense Vib 3R primers were constructed for Ž . V. Õulnificus 16S rRNA sequence Table 2 . These are based upon a gene alignment Table 1 Bacterial strains used in this study a Organism Source and strain Origin V. Õulnificus CJVVO4 Human HUFP5002 Hiroshima University ATCC27562 V. alginolyticus HUFP9701 Hiroshima University V. cholerae POI9001 Human V. fluÕialis Isolate in lab. Sea water V. mimicus Isolate in lab. Sea water V. parahaemolyticus HUFP9114 Hiroshima University Aeromonas hydrophila ATCC7966 Edwardsiella tarda EDK-2 Eel E. coli HB101 Commercial Streptococcus sp. Isolate in lab. Flounder a Abbreviation: ATCC, American type culture collection, Rockville, MD. Table 2 Primers used in PCR a Primer Target gene Sequence Direction Position X X Cyt1 cytolysin 5 -ACAAAGACGGCCGCAAAGTGG-3 sense 1277–1297 X X Cyt2 cytolysin 5 -AGCCCGCAGAGCCGTAAACC-3 antisense 1684–1665 X X Vib 1 16S rDNA 5 -GTGGTAGTGTTAATAGCACT-3 sense 454–473 X X Vib 2 16S rDNA 5 -TCTAGCGGAGACGCTGGA-3 sense 1006–1023 X X Vib 3R 16S rDNA 5 -GCTCACTTTCGCAAGTTGGCC-3 antisense 1278–1258 a Sequence positions for Vib 1, Vib 2, and Vib 3R are given according to the E. coli 16S rDNA numbering system. Ž using MACAW Version 2.0.5, National Center for Biotechnology Information, Na- . tional Institutes of Health, USA program for 16S rDNA sequences of 16 Vibrio species Ž Õulnificus ATCC 27562, Õulnificus C7184, alginolyticus, anguillarum, campbelli, carchariae, cholerae, diazotropicus, fluÕialis, parahaemolyticus, proteolyticus, mimi- cus, ordalii, hollisae, penaeicida, furnissii and damsela re-classified as Photobac- . Ž terium damselae recently retrieved from the Entrez database accession numbers; X56582, X76334, X56576, X71819, X56575, AF134581, X74696, X56577, X76335, X56580, X56579, X74713, X74718, X56583, AJ249719, X74704 and X74700, respec- . tively . Three primers were designed: Vib 1, Vib 2, and Vib 3R, which were comple- mentary to regions of 16S rRNA of V. Õulnificus ATCC 27562; base positions 454 to Ž 473, 1006 to 1023, and 1278 to 1258 in Escherichia coli 16S rRNA Kita-Tsukamoto et . Ž . al., 1993 . Cyt1 and Cyt2 primers Table 2 were derived from the nucleotide sequence Ž of the cytotoxin–hemolysin structural gene base positions 1277 to 1297 and 1684 to . Ž . 1665, respectively cloned by Morris et al. 1987 and used for the detection of V. Õ ulnificus by PCR gene amplification. All primers were synthesized with an automated Ž . DNA synthesizer Bioneer, Taejon, Korea by the phosphoramidite method. PCR amplification was carried out in a 50 ml reaction mixture containing the extracted Ž . bacterial nucleic acids 100 ng of the isolated total nucleic acids , 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl , 0.001 wrv gelatin, 0.5 Tween-20, 200 mM each 2 Ž dNTP, 1 mM each primer, 1.25 U AmpliTaq DNA polymerase Perkin-Elmer, Norwalk, . Ž . CT, USA with an Perkin-Elmer 2400 thermal cycler Perkin-Elmer . Amplification consisted of 35 cycles at 948C for 30 s, 558C for 30 s, and 728C for 30 s in 0.2 ml thin-walled tubes. The results of amplification were analyzed by 2 agarose gel electrophoresis. The PCR products were purified by agarose gel electrophoresis using a Ž . Prep-A-Gene DNA Purification systems Bio-Rad and sequenced using Big dye termi- Ž nator cycle DNA sequencing kit ABI PRISM, PE Applied Biosystems, Foster City, CA, . USA and an automatic sequencer. RT-PCR amplification was performed for the isolated total nucleic acids from V. Õulnificus using the EZ-rTth DNA polymerase Ž . Perkin-Elmer . The cDNAs to the 16S rRNA were synthesized in a reaction mixture Ž . containing 10 mM Tris–HCl, 50 mM Bicine, 115 mM potassium acetate, 8 wrv Ž . glycerol, pH 8.2, 2.5 mM Mn OAc , 1 mM of each Vib 2 and Vib 3R primer, 300 mM 2 each dNTP, 1 Urml RNase inhibitor and 2.5 U EZ-rTth DNA polymerase at 608C for 45 min followed PCR amplification on the same tube containing cDNA produced using 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