Enterocytozoon hepatopenaei (EHP) is one of the most serious pathogens in shrimp farming. This study combines recombinase polymerase amplification (RPA) with the Argonaute from Pyrococcus furiosus (PfAgo) and establishes a sensitive and reliable method for on-site detection of EHP. With careful screening of gDNA and optimization of the reaction, the method shows a good specificity and reaches a sensitivity of single copy per reaction, which is higher than the sensitivity of the currently available molecular assays. The whole procedure can be finished within 1.5 h including the sample processing time and only requires minimum laboratory support, which is user-friendly for on-site environments. This is the first application of PfAgo for the diagnosis of infectious diseases in seafood supply chains. It provides a reliable method for on-site detection of EHP in shrimp farms and establishes a groundwork for multiplex detection of important pathogens in seafood farming using PfAgo.
Keywords: Argonaute; Enterocytozoon hepatopenaei; Pyrococcus furiosus; on-site detection; recombinase polymerase amplification.