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

Abstract

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 ~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. KEY WORDS: Francisella · Tilapia · qPCRResale or republication not permitted without written consent of the publisher

Outbreaks and genetic diversity of Francisella noatunensis subsp orientalis isolated from farm-raised Nile tilapia (Oreochromis niloticus) in Brazil

“…The identity of all of the isolates was confirmed using FNO-specific qPCR, as previously described (Soto et al, 2010). The primers and probe were obtained from Sigma-Aldrich (USA).…”

First identification of Francisella noatunensis subsp. orientalis causing mortality in Mexican tilapia Oreochromis spp.

“…It is important to note that since 2006, the identification of novel Francisella species recovered from distinct fish species has not been easy; therefore, these isolates are classified as Francisella-like (Hsieh et al 2006, Soto et al 2010. Some of these isolates have been classified as belonging to F. noatunensis within the new subspecies orientalis (Mikalsen et al 2007, Ottem et al 2007, 2009, Soto et al 2011.…”

“…6A) was confirmed by nucleotide sequencing from both bacterial cultures and infected tissues, showing 100% identity with iglC from the F. noatunensis subsp. orientalis reference strain LADL 07-285A (Soto et al 2010) (Fig. 6B).…”

“…Real time-PCR assays were performed in a final volume of 20 µl using the SYBR ® Green PCR Master Mix Kit (Applied Biosystems), which included all of the reagents needed for the PCR reactions, except for 2 µl of DNA and 1 µM of each primer. Samples were amplified using a One Step Applied Biosystems Thermocycler, and the PCR conditions were the same as previously described by Soto et al (2010): 10 min at 95°C; followed by 40 cycles of 15 s at 95°C, and 60 s at 60°C. The real-time PCR amplicon was confirmed by the melting temperature of the amplicon and by electrophoresis on 2% SeaKem ® LE agarose gel and ethidium bromide staining.…”

“…orientalis, realtime PCRs were performed using the IglCR and IglCF primers described by Soto et al (2010), together with the DNA obtained from the bacterial colonies and from splenic tissue of symptomatic fish. Real time-PCR assays were performed in a final volume of 20 µl using the SYBR ® Green PCR Master Mix Kit (Applied Biosystems), which included all of the reagents needed for the PCR reactions, except for 2 µl of DNA and 1 µM of each primer.…”