Rapid point-of-need blood fluke detection in Southern Bluefin Tuna samples using recombinase polymerase amplification coupled with lateral flow test (RPA-LF)

Aporocotylid blood flukes Cardicola forsteri and C. orientalis are considered one of the most significant health concerns for Southern Bluefin Tuna (SBT) Thunnus maccoyii ranched in Australia. There is a need for rapid point-of-need diagnostics to detect Cardicola spp. in SBT to allow the industry to make timely management decisions. Recombinase polymerase amplification (RPA) is an isothermal technique which operates at constant low temperature (25-42˚C), and when coupled with a lateral flow (LF) strip, makes an ideal diagnostic tool for rapid, specific, and sensitive identification of pathogens in field applications. RPA-LF assays were designed and validated for detection of C. forsteri and C. orientalis. For each assay, no cross-species amplification was seen and detection as low as 30-50 gene copy equivalents was achieved. Reactions can be completed in 10 minutes. Similar specificity and sensitivity were demonstrated for SBT samples when compared to qPCR analysis, and use of equipment-free incubation using body heat outside of laboratory settings was demonstrated. By developing rapid, ready-to-use diagnostics, the SBT industry can identify risks relating to blood flukes far quicker than is currently possible.