title: Characterization of ISR region and development of a PCR assay for rapid detection of the fish pathogen Tenacibaculum soleae.
abstract: The aims of this work were to characterize the 16S-23S internal spacer region of the fish pathogen Tenacibaculum soleae and to develop a PCR assay for its identification and detection. All T. soleae strains tested displayed a single internal spacer region class, containing tRNA(I)             (le)  and tRNA(A)             (la)  genes; nevertheless, a considerable intraspecific heterogeneity was observed. However, this region proved to be useful for differentiation of T. soleae from related and non-related species. Species-specific primers were designed targeting the 16S rRNA gene and the internal spacer region region, yielding a 1555-bp fragment. Detection limit was of 1 pg DNA per reaction (< 30 bacterial cells) when using pure cultures. The detection level in the presence of DNA from fish or other bacteria was lower; however, 10 pg were detected at a target/background ratio of 1 : 10(5) . The PCR assay proved to be more sensitive than agar cultivation for the detection of T. soleae from naturally diseased fish, offering a useful tool for diagnosis and for understanding the epidemiology of this pathogen.
authors.affiliation: IFAPA Centro Agua del Pino, Huelva, Spain. lasacias@yahoo.es
authors.forename: Jose R
authors.initials: JR
authors.lastname: López
authors.forename: Abdel M
authors.initials: AM
authors.lastname: Hamman-Khalifa
authors.forename: José I
authors.initials: JI
authors.lastname: Navas
authors.forename: Roberto
authors.initials: R
authors.lastname: de la Herran
mesh_terms: D000818:Animals; D015373:Bacterial Typing Techniques; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics*; D005393:Fish Diseases / Q000382:microbiology*; D005399:Fishes / Q000382:microbiology; D045826:Flavobacteriaceae Infections / Q000382:microbiology / Q000662:veterinary*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D060493:Tenacibaculum / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: D004269:DNA, Bacterial; D021903:DNA, Ribosomal Spacer; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S