Infectious hematopoietic necrosis virus detection system established based on the multienzyme isothermal rapid amplification (MIRA) method

Infectious hematopoietic necrosis virus (IHNV) is an important pathogen of salmonid fish. In this study, a multienzyme isothermal rapid amplification (MIRA) assay targeting a conserved region of the IHNV N gene was established for rapid detection. The performance of the assay was evaluated using IHNV RNA standards, and further compared with digital PCR (dPCR) using 8 IHNV isolates, 12 cell culture supernatants, and 67 tissue samples from rainbow trout suspected of IHNV infection. The MIRA assay produced detectable signals within approximately 20 min. A detection rate of 100% was achieved at 102 copies μl-1 using RNA standards. The assay successfully detected all 8 IHNV isolates and showed no cross-reaction with non-target fish viruses. For cell culture supernatants and tissue samples, the MIRA assay showed a high level of agreement with dPCR results. These findings indicate that the MIRA assay may serve as a rapid and practical method for IHNV detection, particularly for on-site screening and preliminary diagnosis in resource-limited settings.