Development of a rapid and cost-effective multiplex PCR-Nanopore sequencing assay for accurate diagnosis of four tilapia pathogens

Abstract

AbstractTilapia aquaculture faces significant threats posed by four prominent pathogens: tilapia lake virus (TiLV), infectious spleen and kidney necrosis virus (ISKNV),Francisella orientalis, andStreptococcus agalactiae. Currently, employed molecular diagnostic methods for these pathogens rely on multiple singleplex PCR reactions, which are both time-consuming and expensive. In this study, we present a pioneering approach utilizing a novel multiplex PCR (mPCR) assay, coupled with rapid Nanopore sequencing, enabling for the one-tube simultaneous detection and one-reaction Nanopore sequencing-based identification of all four pathogens. Our one-tube multiplex assay exhibits a detection limit of 1,000 copies per reaction for TiLV, ISKNV, andS. agalactiae, while forF. orientalis, the detection limit is 10,000 copies per reaction. This capability allows for the detection of single infections as well as co-infections in clinical samples within a single day. Moreover, the consensus sequences generated from the amplicons of each sample demonstrate 100% sequence identity with publicly available data, providing strong support for the improving accuracy of Nanopore sequencing. The integration of multiplex PCR and Nanopore sequencing provides a promising and cost-effective platform for rapid and precise diagnostics of major tilapia pathogens, making it a valuable tool for enhancing health management practices within the aquaculture industry, ultimately contributing to improved disease control and prevention.