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Rapid Discrimination of Fish Pathogenic Vibrio and Photobacterium Species by Oligonucleotide DNA Array

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Rapid Discrimination of Fish Pathogenic Vibrio and Photobacterium Species by Oligonucleotide DNA Array 魚病研究 Fish Pathology, 41 (3), 105–112, 2006. 9 © 2006 The Japanese Society of Fish Pathology Rapid Discrimination of Fish Pathogenic Vibrio and Photobacterium Species by Oligonucleotide DNA Array Tomomasa Matsuyama*, Takashi Kamaishi and Norihisa Oseko Aquatic Animal Health Division, National Research Institute of Aquaculture, Fisheries Research Agency, Minami-Ise, Mie 516-0193 Japan (Received April 17, 2006) ABSTRACT—We developed a low-density oligonucleotide DNA array for the discrimination of 17 Vibrio and 1 Photobacterium species that are pathogenic to aquatic animals. We designed oligo- nucleotide probes targeting non-coding regions of the intergenic transcribed spacers (ITS) between 16S and 23S ribosomal DNA. Based on the alignment of ITS sequences obtained from the data- base and the results in this study, three oligonucleotide probes from a bacterial species were de- signed and immobilized on a nylon membrane. The ITS regions were PCR-amplified using a pair of universal primers, followed by hybridization of the digoxigenin (DIG)-labeled PCR products to the membrane. Specific signals were produced with alkaline phosphatase-conjugated anti-DIG anti- body and chemiluminescent substrate. Although cross hybridization was observed, the patterns from each species were different among the species. This DNA array is useful for a rapid and easy discrimination of Vibrio species. The total process, including PCR amplification, can be car- ried out within a day. Our method enables a global screening of fish pathogenic Vibrio and Photobacterium species Key words: DNA array, discrimination, Vibrio, Photobacterium Vibrio species are widely distributed in aquatic envi- reported (Buller, 2004), they are not suitable for exhaus- ronments and many of the species are causative agents tive screening. of infection in humans and aquatic animals (Buller, DNA array allows the simultaneous screening of a 2004). In fish pathology, the identification of Vibrio-like large amount of genes in a short assay time. Because organisms depends first on their growth on a selective of their usefulness, DNA arrays have been utilized for medium: thiosulphate citrate bile salt sucrose (TCBS). bacterial identification (Call, 2005). Warsen et al. Generally, further identification depends on their physi- (2004) developed a DNA array based on 16S ribosomal ological and biochemical characteristics. However, RNA gene for the discrimination of fish pathogens. commercial bacterial identification systems based on the Gonzalez et al. (2004) reported the detection of marine characteristics need extreme care to take correct an- fish pathogenic bacteria using multiplex PCR and DNA swers, because misidentification by these systems has microarray. They targeted the genes encoding cytol- been reported (Pillot et al., 2002; O’Hara et al., ysin, DNA gyrase B subunit, urease, phospholipase and 2003). Alternative serological identification of Vibrio plasmid borne-loci. Yet, their method is not enough for species with polyclonal antisera (Tajima et al., 1987; an exhaustive screening of Vibrio species, because Martin and Siebeling, 1991; Mutharia et al., 1993; Listonella anguillarum, V. parahaemolyticus and V. Martinez-Govea et al., 2001) and monoclonal antibodies vulnificus has only been targeted. (Ito and Yokota, 1987; Chen et al., 1992; Sung et al., Intergenic transcribed spacers (ITS) between 16S 1993) has been reported, but the number of detectable and 23S rRNA gene were thought to be under less species by the serological method is limited. Although selective constrains and, therefore, to provide higher some species-specific PCR primers has also been genetic variation than rRNA gene sequences (Gürtler and Stanisich, 1996). ITS have been utilized to differ- * Corresponding author entiate various bacteria (Gürtler and Stanisich, 1996; E-mail: [email protected] Kong et al., 1999; Chen et al., 2005) including Vibrio 106 T. Matsuyama, T. Kamaishi and N. Oseko species (Chun et al., 1999; Maeda et al., 2000; Simon et Table 1. Strains used in this study and results of PCR amplifi- al., 2002). In this study, we utilized species-specific cation sequences in the ITS non-coding region to design oligo- Strain numbers Results usedfor sequence of PCR nucleotide probes for DNA array for the discrimination of Species genus Vibrio at the species level. analysis and/or amplifi- PCR cation P. damselae subsp. damselae ATCC33539 + Materials and Methods P. damselae subsp. piscicida ATCC17911 + V. alginolyticus IID950 + PCR amplification of ITS non-coding region and se- V. anguillarum NCMB6 + quence analysis (Listonella anguillarum) The bacterial strains used in this study are listed in V. harveyi ATCC35084 + Table 1. Bacteria were cultured on Marine agar 2216 HUFP9111 + V. cholerae WA1 + (Difco, USA). Chromosomal DNA was extracted by a V. fluvialis IAM14403 + method using proteinase K and phenol-chloroform V. ichthyoenteri IFO15847 + (Sambrook et al., 1989). The PCR primer pair used for V. nigripulchritudo ATCC27043 + amplification of the ITS non-coding region consisted of V. ordalii ATCC33509 + VITSF, 5’ - wrctctttaacaatttgga-3’ (designed based on V. parahaemolyticus IFO12711 + V. pelagius (L. pelagia) ATCC25916 + the consensus sequence found in the box A (Simon et V. penaeicida KH1 + al., 2002) of ITS non-coding region) and VITSR, 5’ - V. salmonicida NCIMB2262 + cgstkagycacttaaccata-3’ (designed based on the con- V. splendidus IAM14411 + sensus sequence in 23S rRNA gene). The expression V. tapetis NCIMB13622 + of the primer sequence is based on the IUPAC V. tubiashii ATCC19109 + V. vulnificus ATCC27562 + code. Temperature cycling on an iCycler (Bio-Rad, Vibrio sp. * —+ ° USA) included an initial denaturation step (94 C for 4 Aeromonas caviae JCM1060 + min), 30 cycles of core PCR reaction (30 s at 94°C, 30 s A. hydrophila IAM12337 + at 55°C, 60 s at 72°C), and a final elongation step (7 min A. jandaei JCM8316 + at 72°C). PCR products were cloned into the plasmid A. salmonicida JCM7874 – A. sobria JCM2139 + vector pDrive (Qiagen, Germany), and amplified for Bacillus subtilis IAM1069 – nucleotide sequencing. Sequence was performed Citrobacter freundii FPC349 – using the BigDye Terminator Cycle Sequencing kit Edwardsiella ictaluri JCM1679 – (Applied Biosystems, USA) and analyzed in the ABI 377 E. tarda ATCC15947 – automated DNA sequencer (Applied Biosystems, USA). Escherichia coli IAM1239 – E. vulneris IAM14239 – The collected sequencing data were aligned using the Flavobacterium branchiophilum FPC520 – Genetyx software package (version 6, Genetyx, F. columnare ATCC43622 – Japan). F. psychrophilum NCMB1947 – Hafnia alvei JCM1666 – DNA array composed with oligonucleotides Klebsiella pneumoniae IAM12015 – Lactococcus garvieae FPC136 – The sequences of oligonucleotide probes are listed Micrococcus luteus IAM1056 – in Table 2. The unique regions to one particular Vibrio Nocardia seriolae TC 258 – and Photobacterium species were identified using the Pseudomonas anguilliseptica NCMB 1949 – sequence alignment from the sequences obtained in this P. chlororaphis IAM12354 – study and the sequences from a GenBank database. P. fluorescens IAM12022 – P. plecoglossicida ATCC700383 – Oligonucleotides were commercially synthesized. P. putida ATCC12633 – Because most Vibrio and Photobacterium species were Serratia marcescens IAM12142 – hardly identified with a single probe, we used a combina- S. plymuthica IAM13543 – tion of three probes to identify individual species. The Staphylococcus aureus IAM1011 – positive control probe was designed based on the 5’-end S. epidermidis IAM12012 – Streptococcus iniae ATCC29178 – of the 23S rRNA gene sequence. The oligonucleotide Tenacibaculum maritimum ATCC43397 – µ probes (0.1 M in sterilized DW) were transferred onto a Yersinia ruckeri ATCC29473 – positively charged nylon membrane using multi-pin-blot- *: Isolated from diseased striped jack Pseudocaranx dentex ter (Atto, Japan) according to the layout illustrated in Fig. (unpublished). 1. The spotted nylon membrane was baked at 120°C Only extracted genomic DNA exist. for 20 min and stored at room temperature. Sample labeling and hybridization with DNA array The ITS non-coding region of sample DNA was Discrimination of Vibrio species by DNA array 107 108 T. Matsuyama, T. Kamaishi and N. Oseko with 0.1 × SSC-0.1% SDS at room temperature. The membrane was then processed with alkaline phos- phatase-conjugated anti-DIG antibody (Roche Diagnos- tics, Switzerland). Signals were produced with the chemiluminescent substrate CDP-Star (Amersham Bio- sciences, USA). To detect the signal, instant films were exposed for 1 to 5 minutes to the arrays using an ECL Mini-Camera (Amersham Biosciences, USA). Diagnosis using oligonucleotide DNA array Ayu (Plecoglossus altivelis), which were suspected of being infected with Vibrio organisms, were diagnosed using oligonucleotide DNA array. Bacteria were iso- lated from the diseased fish on brain heart infusion agar (Difco) with 1.5% NaCl. DNA was extracted from the isolated bacterium and the spleen of the fish, and then subjected to oligonucleotide DNA array analysis. API 20E strip (bioMerieux, France) was used to identify the biochemical characteristics of the isolated bacterium. Results PCR amplification of the ITS non-coding region and sequence analysis Our newly designed PCR primers amplified the ITS
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