Duplex in-one-tube detection of two important shrimp farming pathogens with color indication using a method combining RPA and PfAgo

Abstract

White spot syndrome virus (WSSV) and acute hepatopancreatic necrosis disease caused by virulence gene-containing Vibrio bacteria (AHPND) are two important infectious diseases that affect shrimp, including the economically important species Penaeus vannamei. Because these pathogens can spread rapidly in shrimp aquaculture facilities and because effective therapies are presently lacking, WSSV and AHPND have resulted in significant economic losses in the shrimp farming industry. Thus, the early detection of WSSV and AHPND is essential for shrimp farming. This study combines recombinase polymerase amplification (RPA) technology with the nuclease Argonaute from Pyrococcus furiosus (PfAgo) to develop a duplex in-one-tube detection method with color indication (DIDCi) for WSSV and AHPND utilizing a combination of two fluorescence probes. The method successfully achieves a satisfactory sensitivity of a single gene copy per reaction for WSSV and 10² gene copies per reaction for AHPND. The detection exhibits good specificity and convenience, with the detection results indicated by different colors that can be read with the naked eye, thereby providing an effective tool for the early detection of WSSV and AHPND. This study marks the first application of PfAgo for multiplex nucleic acid detection of aquacultural pathogens and explores the strategy of using combinations of fluorescence probes for the identification of multiple targets in one tube.