Pyrococcus furiosus Argonaute with isothermal amplification for fast and ultra-sensitive diagnosis of acute hepatopancreatic necrosis disease in shrimp

The acute hepatopancreas necrosis disease (AHPND) caused by Vibrio species containing the virulence Photorhabdus insecticidal-related genes, PirAvp and PirBvp, is a high-mortality-rate disease in shrimp farming with strong transmission, quick onset, and severe symptoms. It is vital to control the disease transmission with frequent screening and early diagnosis; hence, developing fast diagnostic methods for AHPND with point-of-care testing (POCT) capacity is particularly important. In this study, we have established a method for fast and ultra-sensitive diagnosis of AHPND by combining a nuclease, Pyrococcus furiosus Argonaute (PfAgo), with recombinase polymerase amplification (RPA). Through systematic screening and optimizations, a strategy of using two guide DNA (gDNA) molecules to program PfAgo is realized for precisely tailored nuclease cleavage. The fabricated procedure can be completed in 75 min with a sensitivity of one copy per reaction, which is better than all the currently available molecular diagnosis methods for AHPND. The procedure requires only simple device support, which is suitable for POCT. This study exhibits the advantages of the PfAgo cleavage system concerning flexibility and sensitivity. The established RPA-PfAgo method is valuable for the prevention of AHPND in shrimp farms, presenting a good example of utilizing PfAgo for highly sensitive molecular diagnosis.