Development and validation of a PCR-based protocol for the detection of Pseudomonas anguilliseptica

Abstract

A PCR-based protocol for the detection of the emerging fish pathogen Pseudomonas anguilliseptica was developed, using the primer set Psan-F/Psan-R designed on the basis of the 16S rRNA sequence. This PCR protocol was optimized and validated by testing 50 target and 38 non-target pure cultures. The method showed a 100% specificity and the detection limit obtained was of 0.7 pgDNA/PCR tube, which equates to 20 or lower bacterial cells. A similar level of sensitivity was observed when the PCR protocol was applied to fish tissues seeded with bacteria. In addition, the developed PCR assay was also capable of direct detection of P. anguilliseptica from tissues obtained from experimentally infected fish and fish obtained from natural outbreaks in gilthead seabream (Sparus aurata) and turbot (Scophthalmus maximus) farms. This PCR protocol can be a useful tool for a rapid, specific and sensitive diagnosis of fish pseudomonadiasis caused by P. anguilliseptica.