Characterization of ISR region and development of a PCR assay for rapid detection of the fish pathogen Tenacibaculum soleae

Authors


Correspondence: Jose R. López, IFAPA Centro Agua del Pino, Junta de Andalucia, Carretera El Portil-El Rompido s/n, 21450 Cartaya, Huelva, Spain. Tel.: +34 959 024900; fax: +34 959 024929; e-mail: lasacias@yahoo.es

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 tRNAIle and tRNAAla 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 : 105. 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.

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