Enterocytozoon hepatopenaei (EHP) is a microsporidian that causes growth retardation in shrimp and threatens global aquaculture. A sensitive and robust detection method is essential for surveillance and control of EHP. In this study, we compared six TaqMan real-time PCR (qPCR) assays targeting different EHP loci: 18S rRNA, β-tubulin, and cysteine desulfurase (NFS1) from published sources; DNA polymerase and ATP–ADP translocase from an Aquaculture Pathology Laboratory panel (unpublished); and a newly designed spore wall protein (SWP) assay. Diagnostic validation followed the World Organisation for Animal Health (WOAH) framework, evaluating analytical specificity, analytical sensitivity/limit of detection (LOD), repeatability (intra-assay), and reproducibility (inter-assay) using plasmid DNA standards.
All assays were specific for EHP, with no cross-reactivity to the non-target pathogens tested (i.e., WSSV, IHHNV, NHP, AHPND). The LOD was 10 plasmid copies/µL across assays. Repeatability was good (%CV < 10%) and reproducibility acceptable (%CV < 15%) for all assays. However, qPCR efficiency fell outside the acceptable range (90%–110%) for the β-tubulin and ATP–ADP translocase assays in repeatability testing, and for the β-tubulin, NFS1, and ATP–ADP translocase assays in reproducibility testing.
Using a panel of biological shrimp samples spanning different EHP infection statuses, diagnostic specificity was 100% (95% CI: 69.15%–100%) for all assays. Diagnostic sensitivity was lower for β-tubulin at 90.00% (95% CI: 73.47%–97.89%) and for ATP–ADP translocase at 80.00% (95% CI: 61.43%–92.29%), whereas the remaining four assays each achieved 100% sensitivity (95% CI: 88.43%–100%). Notably, the 18S rRNA and SWP assays showed superior sensitivity, particularly at low infection levels. Given the potential for microsporidian cross-reactivity at the 18S rRNA locus, the new SWP assay is expected to provide high specificity, underscoring its diagnostic value. These results support optimization and standardization of EHP molecular diagnostics to strengthen surveillance and management in shrimp aquaculture