An isothermal enzymatic recombinase amplification (ERA) assay for rapid and accurate detection of Enterocytozoon hepatopenaei infection in shrimp

Abstract

Enterocytozoon hepatopenaei (EHP) is a kind of microsporidian parasite belonging to fungi, and poses a serious threat to prawn farmers. Due to the lack of effective treatments for EHP, the establishment of a rapid and sensitive detection method would be beneficial to the control and prevention of this prawn parasitic disease. In this study, an isothermal enzymatic recombinase amplification (EHP-ERA) assay that could diagnose EHP within 20 min at 42 °C was developed and evaluated. The determined final concentrations of primers and probe in the reaction system were 400 nM and 120 nM, respectively. EHP-ERA was carried out within 13 min (24.31 ± 0.37 Ct) with a detection limit of 10 copies/μL. The results of specificity test showed that EHP-ERA had no cross-reactivity with white spot syndrome virus (WSSV), Vibrio parahaemolyticus strain causing acute hepatopancreatic necrosis disease (VpAHPND), and infectious hypodermal and hematopoietic necrosis virus (IHHNV) and specific pathogen free (SPF) shrimp. Using 32 clinical samples, the practical diagnostic results of EHP-ERA was consistent with nested PCR and real-time PCR (qPCR) under the premise of less time-consuming and simpler operation. In summary, we established a simple, rapid, and effective ERA assay for the detection of EHP, which had great potential to be widely used in both lab and practical usage.