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 1pg 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, 10pg 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.