Development of a real-time enzymatic recombinase amplification assay (RT-ERA) and an ERA combined with lateral flow dipsticks (LFD) assay (ERA-LFD) for rapid detection of acute hepatopancreatic necrosis disease (AHPND) in shrimp Penaeus vannamei

Acute hepatopancreatic necrosis disease (AHPND) is a major bacterial acute disease of shrimp caused by Vibrio infection, which can lead to high mortality and cause huge economic losses to the shrimp farming industry. Therefore, the development of early, rapid, and accurate AHPND diagnostic methods plays an important role in disease prevention in shrimp aquaculture. In this study, a real-time enzymatic recombinase amplification assay (RT-ERA) and an ERA combined with lateral flow dipsticks (LFD) assay (ERA-LFD) based on the conserved sequence of the pirA and pirB gene was developed and evaluated. Both the two methods can complete the detection within 30 min at a constant temperature of 37–42 °C. Using plasmid standards as template, the detection limits of the both methods are 101 copies/μL, which is 10 times more sensitive than those of nested PCR and RT-PCR. Taking DNA extracted from the hepatopancreas of diseased shrimp as template, the detection limit of the both methods is 1 pg/μL, which is as the same as nested PCR and 1000 times more sensitive than that of RT-PCR. The specificities of both assays were tested and no cross-reaction with white spot syndrome virus (WSSV), Enterocytozoon hepatopenaei (EHP), infectious hypodermitis and hematopoietic necrosis virus (IHHNV) and healthy shrimp was exhibited. The practical applicability of the both assays were evaluated using 24 field samples. The coincidence rates of RT-ERA, ERA-LFD and nested PCR on samples collected from shrimp farms were all 100%, which were higher than RT-PCR. Taken together, the developed RT-ERA and ERA-LFD assays are simple, rapid, sensitive, and affordable on-site diagnostic methods for AHPND infection, with great potential to help control AHPND infection and reduce economic losses in the shrimp industry.