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

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 degrees 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/mu L. The results of specificity test showed that EHP-ERA had no cross -reactivity with white spot syndrome virus (WSSV), Vibrio parahaemolyticus strain causing acute hepatopancre-atic 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.

Automated summary

In this study, a rapid and sensitive detection method for the prawn parasitic disease EHP was developed and evaluated. The EHP-ERA assay could diagnose EHP within 20 minutes at 42 degrees C, with a detection limit of 10 copies/mu L.