Pyrococcus furiosus Argonaute Combined with Recombinase Polymerase Amplification for Rapid and Sensitive Detection of Enterocytozoon hepatopenaei

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 ( Pf Ago) 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 Pf Ago 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 Pf Ago.