Development of a real-time PCR assay for identification and quantification of the fish pathogen Francisella...

Members of the genus Francisella are small Gram-negative facultative intracellular bacteria that cause francisellosis in a wide variety of fish species worldwide. F. noatunensis subsp. orientalis has been recently described as a warm-water pathogen of tilapia Oreochromis spp. In this study, a quantitative real-time polymerase chain reaction (qPCR) TaqMan probe assay was developed to rapidly and accurately detect and quantify F. noatunensis subsp. orientalis from fish tissue. The target region of the assay was the F. tularensis iglC gene homologue previously found in F. noatunensis subsp. orientalis. Probe specificity was confirmed by the lack of signal and cross-reactivity with 12 common fish pathogens, 2 subspecies of F. tularensis, F. noatunensis subsp. noatunensis, and tilapia tissue. The range of linearity was determined to be 50 fg to 1.4 mg, and the lower limit of detection was 50 fg of DNA (equivalent to approximately 25 genome equivalents) per reaction. A similar sensitivity was observed with DNA extracted from a mixture of F. noatunensis subsp. orientalis and fish tissue. The assay was also able to detect and quantify F. noatunensis subsp. orientalis from the spleens of experimentally infected tilapia. No signal was observed in the control groups. In conclusion, we have developed a highly sensitive and specific assay that can be used for the specific identification and quantification of F. noatunensis subsp. orientalis.