Multiplex PCR with Nanopore Sequencing for Sequence-Based Detection of Four Tilapia Pathogens

Abstract

Tilapia lake virus (TiLV), Infectious spleen and kidney necrosis virus (ISKNV), Francisella orientalis, and Streptococcus agalactiae (Group B Streptococcus, GBS) are significant pathogens that affect farmed tilapia (Oreochromis spp.) in Southeast Asia and Africa. Currently, detection of these pathogens for disease surveillance or during outbreak investigations relies on multiple singleplex PCR runs. In this study, we present an innovative multiplex PCR (mPCR) method capable of simultaneous detection of all four pathogens in a single PCR reaction. The mPCR method produces expected amplicons of 137 bp for TiLV, 190 bp for ISKNV, 203 bp for F. orientalis, and 351 bp for S. agalactiae. The assay has detection limits of 103 copies/reaction for ISKNV and S. agalactiae, and 104 copies/reaction for TiLV and F. orientalis, making it capable of detecting both single and dual infections in clinical samples within a day. The accuracy of the consensus sequences generated was confirmed to be 100%, making this method ideal for genotyping. Amplicon-based Nanopore sequencing approach is a promising, cost-effective platform for rapid and accurate diagnostics of major pathogens of tilapia and can be used to facilitate timely responses to prevent their widespread dissemination.