Multiplex polymerase chain reaction (PCR) with Nanopore sequencing for sequence-based detection of four tilapia pathogens
Issued Date
2025-01-01
Resource Type
eISSN
21678359
Scopus ID
2-s2.0-105004807311
Journal Title
PeerJ
Volume
13
Issue
5
Rights Holder(s)
SCOPUS
Bibliographic Citation
PeerJ Vol.13 No.5 (2025)
Suggested Citation
Delamare-Deboutteville J., Meemetta W., Pimsannil K., Gan H.M., Khor L., Chadag M., Dong H.T., Senapin S. Multiplex polymerase chain reaction (PCR) with Nanopore sequencing for sequence-based detection of four tilapia pathogens. PeerJ Vol.13 No.5 (2025). doi:10.7717/peerj.19425 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/110243
Title
Multiplex polymerase chain reaction (PCR) with Nanopore sequencing for sequence-based detection of four tilapia pathogens
Corresponding Author(s)
Other Contributor(s)
Abstract
Background. Tilapia aquaculture faces significant threats posed by four prominent pathogens: tilapia lake virus (TiLV), infectious spleen and kidney necrosis virus (ISKNV), Francisella orientalis, and Streptococcus agalactiae. Currently, employed molecular diagnostic methods for these pathogens rely on multiple singleplex polymerase chain reactions (PCR), which are time-consuming and expensive. Methods. In this study, we present an approach utilizing a multiplex PCR (mPCR) assay, coupled with rapid Nanopore sequencing, enabling the one-tube simultaneous detection and one-reaction Nanopore sequencing-based validation of four pathogens. Results. Our one-tube multiplex assay exhibits a detection limit of 1,000 copies per reaction for TiLV, ISKNV, and S. agalactiae, while for F. orientalis, the detection limit is 10,000 copies per reaction. This sensitivity is sufficient for diagnosing infections and co-infections in clinical samples from sick fish, enabling rapid confirmation of the presence of pathogens. Integrating multiplex PCR and Nanopore sequencing provides an alternative approach platform for fast and precise diagnostics of major tilapia pathogens in clinically sick animals, adding to the available toolbox for disease diagnostics.