Species-specific loop-mediated isothermal amplification (LAMP) assays for the detection of three blood flukes affecting bluefin tuna aquaculture

Blood flukes of the genus Cardicola (Digenea: Aporocotylidae) are well-documented health threats to farmed and ranched bluefin tuna (BFT), where infections can result in substantial economic losses. While recombinase polymerase assays (RPA) have been developed for two species of Cardicola , the high degree of sequence conservation across all three species reported from BFT requires an alternative method to enable confident species-level discrimination. The aims of this study were to design, optimise and validate loop-mediated isothermal amplification (LAMP) for the detection of the three Cardicola species which infect BFT, and apply the assays to Pacific bluefin tuna (PBT) cultured in Japan. Species-specific LAMP assays were successfully designed, optimised and validated for C. forsteri , C. opisthorchis and C. orientalis targeting ITS-2 ribosomal DNA. In addition, these assays were applied to DNA from three sample types targeting sites of significant pathology, the heart, gill necropsy and gill biopsy of 15 PBT. Results were compared to outputs of quantitative polymerase chain reactions (qPCR), the gold standard for blood fluke detection in ranched Australian SBT, where LAMP assays were shown to be more sensitive. This research constitutes the first development and application of species-specific LAMP diagnostics for the three described Cardicola species infecting BFT, providing a rapid, cost-effective and accessible alternative to standard molecular diagnostics, informing management and treatment decisions.