On-Site Detection of Enterocytozoon hepatopenaei in Shrimp: A Rapid and Reliable Colorimetric PSR Method

Enterocytozoon hepatopenaei (EHP) is a microsporidian parasite that significantly threatens global shrimp farming, particularly affecting species like Penaeus monodon and Penaeus vannamei. Early detection of EHP is crucial for effective disease control in aquaculture environments. This study aimed to develop a rapid, sensitive and specific diagnostic method for identifying EHP in shrimp populations through an isothermal and direct colorimetric polymerase spiral reaction (PSR) assay. The PSR assay was optimized for performance, yielding the best results at 65°C for 40 minutes. We validated the specificity of our PSR primer set by testing it against a range of aquatic pathogens, confirming no crossreactivity. Furthermore, the limit of detection with a 95% probability was remarkably low, at just 7.142 copies of the target gene per reaction. Clinical trials on shrimp samples yielded encouraging results, with the direct colorimetric PSR method achieving 100% sensitivity and specificity compared to traditional qPCR assays. Notably, the PSR assay eliminates the need for DNA extraction, facilitating on-site detection at shrimp farms. In summary, the newly developed colorimetric PSR assay offers a quick and reliable option for detecting EHP, enhancing disease management in shrimp aquaculture through its simplicity, accuracy and cost-effectiveness.