Development and comparative evaluation of TaqMan real-time PCR assays for detecting Enterocytozoon hepatopenaei (EHP) in penaeid shrimp

Enterocytozoon hepatopenaei (EHP) is a microsporidian that retards shrimp growth and threatens global aquaculture, making reliable, sensitive detection essential for surveillance and control. Herein, we compared six TaqMan real-time PCR assays targeting distinct EHP loci, including three published assays (SSU rDNA, β-tubulin, and cysteine desulfurase/NFS1), two unpublished internal Aquaculture Pathology Laboratory assays (DNA polymerase and ATP/ADP translocase), and a newly designed spore wall protein (SWP) assay. Assay development and validation followed the World Organisation for Animal Health (WOAH) framework, assessing analytical specificity, analytical sensitivity/limit of detection (LOD), repeatability, and reproducibility using defined plasmid DNA standards. All assays were specific for EHP, with no cross-reactivity to other crustacean pathogens causing enteric disease (e.g., NHP, AHPND, TPD) or systemic infections (e.g., IHHNV, WSSV nor with the crustacean microsporidian Perezia sp. or a novel intranuclear Enterocytozoon sp. The only exception was the SSU rDNA assay, which also amplified the novel intranuclear Enterocytozoon sp. All assays achieved an LOD of 10 copies/µL. Repeatability was strong (%CV < 1.65%) and reproducibility good (%CV < 5.70%); however, qPCR efficiency fell outside the 90–110% range for the β-tubulin and ATP/ADP translocase assays in repeatability tests, and for the β-tubulin, NFS1, and ATP/ADP translocase assays in reproducibility tests. These three assays also showed relatively lower reproducibility, with occasional reductions in linearity (R² < 0.99). Screening of experimentally challenged shrimp with a wide dynamic infection range yielded 100% diagnostic specificity (95% CI 69.15–100) for all assays. Diagnostic sensitivity was lower for the β-tubulin (90.00%; 95% CI 73.47–97.89) and ATP/ADP translocase (80.00%; 95% CI 61.43–92.29) assays, whereas the SSU rDNA, NFS1, DNA polymerase, and SWP assays each reached 100% sensitivity (95% CI 88.43–100). The SSU rDNA and SWP assays were the most sensitive at low infection levels but SSU rDNA may cross-react with related microsporidia. By targeting an EHP-unique region, the SWP assay currently provides higher specificity and strengthen EHP surveillance to help diminish its global spread, as EHP is listed by WOAH as an emerging pathogen.