Real-time PCR assays targeting unique DNA sequences of fish-pathogenic Francisella noatunensis subspecies noatunensis and orientalis

Abstract

Specific identification and differentiation of the 2 subspecies of the fish pathogen Francisella noatunensis, namely, F. noatunensis subsp. noatunensis and F. noatunensis subsp. orientalis, remains a major diagnostic challenge. Following whole-genome sequencing and analysis of representatives of all major subclades of the genus Francisella, specific genomic regions were identified for each of the subspecies of this fish pathogen. Two specific real-time quantitative PCR assays, directed at hypothetical genes within these regions were developed. Specificity was confirmed by lack of signal and cross-reactivity with the closest relative, F. philomiragia, and other common bacterial fish pathogens. Both assays, used either as monoplex or multiplex, have a limit of detection of 10 genome equivalents. The quantitative sensitivity of the assays was not affected by the presence of kidney tissues or DNA from Atlantic cod Gadus morhua or tilapia Oreochromis sp. KEY WORDS: Francisella noatunensis · Fish · DNA · qPCR Resale or republication not permitted without written consent of the publisherDis Aquat Org 101: [225][226][227][228][229][230][231][232][233][234] 2012 real-time PCR has become the preferred methodology for Francisella detection due to its sensitivity and rapid turnaround time. However, the high degree of genetic similarity between the fish-pathogenic strains and their almost ubiquitous close relative F. philomiragia (Barns et al. 2005, Berrada & Telford 2010, Duodu et al. 2012) has made development of specific assays challenging. Nevertheless, several PCR assays exhibiting varying degrees of specificity now exist for the detection of members of the genus Francisella in fish. Ottem et al. (2008) described a combination of 2 real-time PCR assays targeting the 16S rRNA gene and the outer membrane protein fopA gene, which enabled detection and differentiation of the 2 fish pathogens when used together in separate analyses. More recently, a realtime PCR for specific detection of F. noatunensis subsp. orientalis targeting the intracellular growth locus gene, iglC, has been developed (Soto et al. 2010). A common feature of these assays is the dependency on low levels of nucleotide variation between the recognized fish-pathogenic species, which conceivably may lead to cross-reaction and false identification. The high prevalence of closely related Francisella bacteria in the environment may also complicate PCR detection of these pathogens (Duodu et al. 2012).Our objectives were, therefore, to develop realtime quantitative PCR (qPCR) assays based on unique sequences found only in respective target genes of Francisella noatunensis subspecies. Selection of target genes was achieved by comparison of whole genome sequences from all known subspecies of this fish pathogen and representatives of all major subclades of the Francisella genus. We present results showing that the 2 real-time qPCR assays developed in this study are highly sensitive and specifically detect F. noatunensis subsp. noatunensis and F. noatune...

Whole cell inactivated autogenous vaccine effectively protects red Nile tilapia (Oreochromis niloticus) against francisellosis via intraperitoneal injection

“…The bacterial load in the spleen of surviving fish was determined at the end of the challenge experiment using a qPCR protocol previously described by Duodu et al (). For this, 10 of the spleens previously preserved in 95% ethanol were randomly selected from each treatment for genomic DNA extraction.…”

Dissection of Francisella-Host Cell Interactions in Dictyostelium discoideum

“…noatunensis-specific primers (see Table S1 in the supplemental material, Q9 and Q10) for quantification of F. noatunensis subsp. noatunensis genomes were obtained from Duodu et al (72). F. noatunensis subsp.…”

“…noatunensis gDNA was used as an equilibrator in the qPCR run, and absolute quantification was performed so that 20 fg corresponded to 10 F. noatunensis subsp. noatunensis genome equivalents, as described previously (72).…”

Development of a real‐time PCR assay for rapid detection and quantification of Photobacterium damselae subsp. piscicida in fish tissues

“…(); however, its sensitivity has only been tested on pure bacterial DNA and an additional RFLP protocol on amplified DNA is needed to discriminate between Phdp and Phdd subspecies. The use of closed‐tube, SYBR Green chemistry represents a practical and robust tool for rapid screening and diagnosis of bacterial fish diseases (e.g., Duodu et al., ), which is cost‐effective and avoids contamination‐prone post‐PCR manipulations.…”

“…The only previous assay with a LOD lower than 10 copies was the one proposed byZappulli et al (2005); however, its sensitivity has only been tested on pure bacterial DNA and an additional RFLP protocol on amplified DNA is needed to discriminate between Phdp and Phdd subspecies. The use of closed-tube, SYBR Green chemistry represents a practical and robust tool for rapid screening and diagnosis of bacterial fish diseases (e.g.,Duodu et al, 2012), which is cost-effective and avoids contamination-prone post-PCR manipulations.In addition to providing a highly sensitive, cost-effective and robust diagnostic test, the proposed real-time PCR protocol guarantees accurate quantification of the target bacteria. The ability to quantify Phdp is important to improve our knowledge on infection dynamics.…”