Simultaneous visual diagnosis of acute hepatopancreatic necrosis disease and Enterocytozoon hepatopenaei infection in shrimp with duplex recombinase polymerase amplification

Shrimp is a globally popular seafood. Shrimp farming has been challenged by various infectious diseases that lead to significant economic losses. The prevention of two important shrimp infectious diseases, the acute hepatopancreatic necrosis disease (AHPND) and the Enterocytozoon hepatopenaei (EHP) infection, is highly dependent on early and accurate diagnostic. On-site monitoring of the two diseases in shrimp farming facilities demands point-of-care-testing (POCT) type of diagnostic assays. This study established a duplex recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD) combined assay that could simultaneously diagnose the two diseases. The optimized RPA-LFD assay could finish the diagnostic in 35 min with good specificity, and the sensitivity reached 10(1) and 10(2) gene copies per reaction for EHP and AHPND, respectively, which were at the same level as the currently available molecular diagnostic assays. Test results of clinical samples showed 100% agreement of this assay with the industrial standard nested polymerase chain reaction (PCR) assays, and samples with both diseases were simultaneously identified. Because of the isothermal 37celcius amplification and the visual reading of the signal on dipsticks, the dependence on equipment is minimal. This duplex RPA-LFD assay is well suited for simultaneous POCT diagnostic of the two important shrimp infectious diseases. Moreover, the principle can be applied to multiplex POCT diagnostic of other infectious diseases in aquaculture.

Automated summary

This study developed a duplex RPA-LFD combined assay for the simultaneous diagnosis of AHPND and EHP in shrimp farming. The assay showed good specificity, completed the diagnostic in 35 min, and had sensitivities comparable to current molecular diagnostic assays. Clinical sample testing demonstrated 100% agreement with standard PCR assays and simultaneous detection of both diseases.