Lateral flow nucleic acid assay for Ecytonucleospora hepatopenaei based on recombinase polymerase amplification and strand displacement reaction.

Abstract

The incidence of Ecytonucleospora hepatopenaei (EHP) infections in farmed shrimp has increased markedly in recent years, resulting in significant economic losses for the global shrimp farming industry. The lack of an efficacious drug for EHP infection has led to the development of a strategy based on the timely screening and elimination of EHP-carrying shrimp seeds as a means of preventing financial loss. This strategy requires portable, accurate and rapid detection methods for EHP, especially when applied to sites such as farms. However, the current lack of user-friendly devices capable of real-time detection under field conditions represents a significant challenge in the implementation of this strategy. In this study, an isothermal amplification nucleic acid biosensor for EHP detection was developed. The biosensor targeted the spore wall protein gene of EHP and amplified the target gene by recombinase polymerase amplification (RPA) combined with strand displacement reaction (SDR). The amplified products were applied on gold nanoparticle-based lateral flow nucleic acid strips (LFNAS) for visual signal conversion. The limit of detection of the SDR-RPA-LFNAS assay was 7 copies reaction-1, and the entire process could be completed in 30 min without cross-reaction. In contrast to existing conventional RPA-related detection methods, the introduction of SDR, which is used to eliminate the background signal produced by long primers, avoids the use of endonucleases and reduces costs. Moreover, the biosensor is straightforward to operate and does not require the use of expensive machinery, rendering it more suitable for the in situ detection of EHP in shrimp farms or aquaculture facilities.