Development and Evaluation of a Rapid Visualisation Detection Method for Ameson portunus Based on RPA-LFD.

Abstract

The Ameson portunus is a prevalent pathogen affecting Portunus trituberculatus, which can infect P. trituberculatus and cause albinism, seriously damaging its economic value. Therefore, there is a pressing need for an efficient detection platform to rapidly and sensitively identify A. portunus. In this study, we have developed a method known as recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) for the swift detection of A. portunus. Three sets of specific primer pairs and a probe were designed according to the spore wall protein (SWP) sequence of A. portunus, in which one of the primer pair (ApSWP-F1/R1) showed the best amplification effects. The optimal reaction temperature was ultimately determined to be 39°C and the optimal reaction time was set at 10 min after careful optimisation of both variables. The sensitivity of the RPA-LFD method was better than that of polymerase chain reaction (PCR), with a limit of 1.71 × 10^-4 ng/μL. This RPA-LFD detection method has good specificity for the detection of A. portunus, and tests for other parasites such as Zschokkella ophiocephali, Myxobolus drjagini, Myxidium lieberkuhni and Pelteobagrus fulvidraeo are negative. The above results show that the RPA-LFD detection method of A. portunus established in this study has strong specificity, high sensitivity, simple operation and visual results, which can be widely used for rapid detection on site.