Development of TaqMan qPCR and MIRA detection methods of Hepatospora eriocheir in Chinese mitten crab

Hepatopancreatic necrosis disease (HPND), which was linked to infections by the microsporidian Hepatospora eriocheir, was a disease of high mortality and prevalence that had caused significant losses in Chinese mitten crab (Eriocheir sinensis) aquaculture. In this study, two molecular detection methods, TaqMan quantitative PCR and multienzyme isothermal rapid amplification (MIRA) , were successfully established after designing specific primers and probes targeting the H. eriocheir CHI4 gene. The developed standard curve for TaqMan qPCR was y = -3.1654x + 43.925 (R² = 0.9977), with an amplification efficiency of 107%. After optimization of the reaction conditions, MIRA was able to detect H. eriocheir in 25 min. Specificity tests confirmed that TaqMan qPCR and MIRA targeting H. eriocheir had no cross-reactivity with Enterocytozoon hepatopenaei, Vibrio parahaemolyticus, Spiroplasma eriocheiris, white spot syndrome virus, and decapod iridescent virus I. The sensitivity detection result revealed that the minimum detection limit of TaqMan qPCR and MIRA was 4.6 × 10¹ copies/μL and 4.6 × 10⁰ copies/μL, respectively, while the minimum detection limit of PCR was 4.6 × 10³ copies/μL. TaqMan qPCR and MIRA detection methods of H. eriocheir were established in this study and could be used for early diagnosis of HPND.