Development of a recombinase polymerase amplification assay for rapid detection of <i>Francisella noatunensis</i> subsp. <i>orientalis</i>

<div><p><i>Francisella noatunensis</i> subsp. <i>orientalis</i> (<i>Fno</i>) is the causative agent of piscine francisellosis in warm water fish including tilapia. The disease induces chronic granulomatous inflammation with high morbidity and can result in high mortality. Early and accurate detection of <i>Fno</i> is crucial to set appropriate outbreak control measures in tilapia farms. Laboratory detection of <i>Fno</i> mainly depends on bacterial culture and molecular techniques. Recombinase polymerase amplification (RPA) is a novel isothermal technology that has been widely used for the molecular diagnosis of various infectious diseases. In this study, a recombinase polymerase amplification (RPA) assay for rapid detection of <i>Fno</i> was developed and validated. The RPA reaction was performed at a constant temperature of 42°C for 20 min. The RPA assay was performed using a quantitative plasmid standard containing a unique <i>Fno</i> gene sequence. Validation of the assay was performed not only by using DNA from <i>Fno</i>, closely related <i>Francisella</i> species and other common bacterial pathogens in tilapia farms, but also by screening 78 Nile tilapia and 5 water samples. All results were compared with those obtained by previously established real-time qPCR. The developed RPA showed high specificity in detection of <i>Fno</i> with no cross-detection of either the closely related <i>Francisella</i> spp. or the other tested bacteria. The <i>Fno-</i>RPA performance was highly comparable to the published qPCR with detection limits at 15 and 11 DNA molecules detected, respectively. The RPA gave quicker results in approximately 6 min in contrast to the qPCR that needed about 90 min to reach the same detection limit, taking only 2.7–3 min to determine <i>Fno</i> in clinical samples. Moreover, RPA was more tolerant to reaction inhibitors than qPCR when tested with field samples. The fast reaction, simplicity, cost-effectiveness, sensitivity and specificity make the RPA an attractive diagnostic tool that will contribute to controlling the infection through prompt on-site detection of <i>Fno</i>.</p>