multiple pathogens:Tilapia lake virus (TILV)Infectious spleen and kidney necrosis virus (ISKNV)Streptococcus agalactiaeFrancisella orientalis Raw sequence reads
Tilapia lake virus (TiLV), Infectious spleen and kidney necrosis virus (ISKNV), Francisella orientalis and Streptococcus agalactiae (Group B Streptococcus, GBS) are the top four most important pathogens affecting farmed tilapia (Oreochromis spp.) in Southeast Asia and Africa. Diagnosis of these pathogens during disease outbreaks currently relied on multiple PCR runs combined with Sanger sequencing of PCR amplicons, which is usually time consuming (approx. 5-7 days). Here, we developed an innovative multiplex PCR (mPCR) method capable of detecting the four pathogens simultaneously in a single PCR reaction coupled with rapid amplicons-targeted Nanopore sequencing for rapid and accurate diagnosis confirmation within a single day. Various sources of tilapia clinical samples were assessed by the mPCR that was able to detect all four pathogens with expected amplicons of 137 bp for TiLV, 190 bp for ISKNV, 203 bp for F. orientalis and 351 bp of S. agalactiae. Our method was capable to pick up both single and dual infections from clinical samples. Single and multiple PCR amplicons targeted Nanopore sequencing proved 100% accuracy of all consensus sequences. Taken together, this study suggests that mPCR-Nanopore is a promising cost-effective platform for fast and accurate confirmed diagnostics of four major pathogens of tilapia during disease outbreaks, which may facilitate appropriate action plan and timely response to disease outbreaks to prevent its widespread, especially in low- and middle-income countries (LMICs) in Southeast Asia and Africa.