Rapid discrimination of fish pathogenic Vibrio and Photobacterium species by oligonucleotide DNA array

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 oligonucleotide 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 database and the results in this study, three oligonucleotide probes from a bacterial species were designed 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 carried out within a day. Our method enables a global screening of fish pathogenic Vibrio and Photobacterium species © 2006 The Japanese Society of Fish Pathology.