Abstract：Infectious hematopoietic necrosis virus (IHNV) is an economically important pathogen causing significant mortalities in a wide variety of salmonid species, including the main aquaculture species, Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). Aimed to disease control and aquatic biosecurity, In this study, a new electrochemical biosensing method for IHNV on-site detection was developed to identify the IHNV-carrying salmonid fries or eggs. Based on evolutionary conservation analysis of 357 non-redundant IHNV sequences from different origins, the matrix gene (M) was found most conserved and selected as target sequence. Six IHNV specific primers were designed for reverse transcription isothermal amplification, according to M gene sequence of the IHNV Chinese strain Ch20101008. Biotinylated dUTP was mixed into dNTPs in the amplification system, thus amplification products were able to be captured and anchored on avidin modified electrode surfaces. And the 5' ends of 2 primers were labeled with ferrocene, which made the amplification products electrochemically active and capable to be detected by cyclic voltammetry (CV). The amplification for nucleic acid was able to be finished in 15 min, and the current value of oxidation peak had obvious positive correlation with the copy numbers of IHNV in the template. With amplification temperature at 63 ℃ and signal to noise ratio at 3, the limit of detection (LoD) was calculated to be 6.2 copies/μL, which was capable to distinguish asymptomatic IHNV carriers. Detectable products could be produced across a wide amplification temperature range from 59 to 65 ℃, thus the IHNV detection could be performed without precise thermal control. Therefore, the electrochemical biosensing method has promising application prospects in IHNV detection in aquaculture industry, especially salmonid farms and hatcheries.
