Quantitative PCR coupled with melting curve analysis for rapid detection and quantification of Tenacibaculum maritimum in fish and environmental samples

The development of a rapid and accurate method for the diagnosis of tenacibaculosis caused by Tenacibaculum maritimum, is of great interest to improve the management and control of an outbreak of this fish disease. In this study, a real-time polymerase chain reaction assay coupled with melting curve analysis was designed for the detection and quantification of T. maritimum targeting a fragment of 164 bp of the sequence of the 16S rRNA gene. Validation of the PCR system demonstrated that the method was able to specifically detect clinical isolates of T. maritimum, as well as to discriminate this species from other related Tenacibaculum fish pathogens and other non-related pathogens. All T. maritimum strains showed a species-specific melting temperature (Tm) of 80.25 +/- 0.35 degrees C. The sensitivity of the assay was determined by constructing a standard curve plotting the Cq values versus the quantity (ng) of the amplicon. The detection sensitivity of the protocol was determined to be 4x10(-10) ng/mu l of the 16S rRNA equivalent to 2.22 copies number of the gene (Cq 33.05) (R-2 of 0.99, m = -3.3527) with an efficiency of 98.73%. The protocol was tested using laboratory-generated samples, including bacterial cultures, samples of kidney, blood and mucus of fish experimentally infected with different concentrations of T. maritimum. The assay was highly reproducible as indicated by the low variation coefficient values for intra-run and inter-run assays. The assay also allowed the detection of T. maritimum in infected tissues as well as in samples of ulcers of cultured turbot Scophthalmus maximus, sole Solea senegalensis and sea bass Dicentrarchus labrax affected by tenacibaculosis. Overall, the real-time PCR assay developed in this study was found to be a rapid, sensitive and reliable diagnosis tool that could allow the identification of T. maritimum bacterial cultures, the detection and quantification of T. maritimum in lethal and non-lethal fish samples as well as the monitorization of the quality of the water and the presence of pathogenic microorganisms.