Development of a Phage-Display Derived Peptide-Based Colloidal Gold Strip for Rapid Detection of Enterocytozoon hepatopenaei via Spore Wall Protein Interaction.

Abstract

Infection by Enterocytozoon hepatopenaei (EHP) leads to stunted or arrested growth in shrimp, causing significant economic losses in shrimp aquaculture. In this study, the spore wall protein EhSW12 was expressed using a prokaryotic expression system. Subsequently, phage display technology was employed to screen for peptides that interact with EhSW12. A rapid detection method was developed that bypasses the laborious procedure of extracting tissue DNA samples. Instead, enzymatic digestion was used to lyse the spore wall protein present in tissue homogenates, thereby enabling direct detection of EhSW12 via antigen colloidal gold rapid test strips and dot blot assays. The colloidal gold test strips, noted for their convenience and rapidity, satisfy the requirements for on-site testing, while the dot blot method-with its simple operation-is well suited for high-throughput sample analysis. Both methods were compared with the national standard qPCR assay. Specificity was validated using various shrimp antigens prepared in the laboratory; the results demonstrated that only the EHP antigen yielded a positive reaction, with no cross-reaction observed with other common shrimp pathogens (e.g., white spot syndrome virus, decapod iridescent virus and highly pathogenic Vibrio). Sensitivity tests indicated that the minimum detection limits for the test strip and dot blot methods were, respectively, 7 × 10⁶ copies and 5.2 × 10⁴ copies. In summary, both methods exhibit strong specificity, high sensitivity, rapid detection speed and operational simplicity-characteristics that make them highly suitable for large-scale on-site rapid testing.