Multiplex polymerase chain reaction (PCR) with Nanopore sequencing for sequence-based detection of four tilapia pathogens
Background: Tilapia aquaculture faces significant threats posed by four prominentpathogens: 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...
| Main Authors: | , , , , , , , |
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| Format: | Journal Article |
| Language: | Inglés |
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PeerJ
2025
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| Subjects: | |
| Online Access: | https://hdl.handle.net/10568/177740 |
| _version_ | 1855530298368851968 |
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| author | Delamare-Deboutteville, Jerome Meemetta, Watcharachai Pimsannil, Khaettareeya Han Ming, Gan Khor, Laura Chadag, Vishnumurthy Mohan Thanh, Dong Ha Saengchan, Senapin |
| author_browse | Chadag, Vishnumurthy Mohan Delamare-Deboutteville, Jerome Han Ming, Gan Khor, Laura Meemetta, Watcharachai Pimsannil, Khaettareeya Saengchan, Senapin Thanh, Dong Ha |
| author_facet | Delamare-Deboutteville, Jerome Meemetta, Watcharachai Pimsannil, Khaettareeya Han Ming, Gan Khor, Laura Chadag, Vishnumurthy Mohan Thanh, Dong Ha Saengchan, Senapin |
| author_sort | Delamare-Deboutteville, Jerome |
| collection | Repository of Agricultural Research Outputs (CGSpace) |
| description | Background: Tilapia aquaculture faces significant threats posed by four prominentpathogens: 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. |
| format | Journal Article |
| id | CGSpace177740 |
| institution | CGIAR Consortium |
| language | Inglés |
| publishDate | 2025 |
| publishDateRange | 2025 |
| publishDateSort | 2025 |
| publisher | PeerJ |
| publisherStr | PeerJ |
| record_format | dspace |
| spelling | CGSpace1777402025-12-13T02:10:07Z Multiplex polymerase chain reaction (PCR) with Nanopore sequencing for sequence-based detection of four tilapia pathogens Delamare-Deboutteville, Jerome Meemetta, Watcharachai Pimsannil, Khaettareeya Han Ming, Gan Khor, Laura Chadag, Vishnumurthy Mohan Thanh, Dong Ha Saengchan, Senapin qpcr bioinformatics tilapia streptococcus agalactiae fish multiplex pcr nanopore tilv francisella orientalis isknv amplicons sequencing Background: Tilapia aquaculture faces significant threats posed by four prominentpathogens: 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. 2025-11-11T03:16:56Z 2025-11-11T03:16:56Z Journal Article https://hdl.handle.net/10568/177740 en Open Access application/pdf PeerJ Jerome Delamare-Deboutteville, Watcharachai Meemetta, Khaettareeya Pimsannil, Gan Han Ming, Laura Khor, Vishnumurthy Mohan Chadag, Dong Ha Thanh, Senapin Saengchan. (13/5/2025). Multiplex polymerase chain reaction (PCR) with Nanopore sequencing for sequence-based detection of four tilapia pathogens. PeerJ. |
| spellingShingle | qpcr bioinformatics tilapia streptococcus agalactiae fish multiplex pcr nanopore tilv francisella orientalis isknv amplicons sequencing Delamare-Deboutteville, Jerome Meemetta, Watcharachai Pimsannil, Khaettareeya Han Ming, Gan Khor, Laura Chadag, Vishnumurthy Mohan Thanh, Dong Ha Saengchan, Senapin Multiplex polymerase chain reaction (PCR) with Nanopore sequencing for sequence-based detection of four tilapia pathogens |
| title | Multiplex polymerase chain reaction (PCR) with Nanopore sequencing for sequence-based detection of four tilapia pathogens |
| title_full | Multiplex polymerase chain reaction (PCR) with Nanopore sequencing for sequence-based detection of four tilapia pathogens |
| title_fullStr | Multiplex polymerase chain reaction (PCR) with Nanopore sequencing for sequence-based detection of four tilapia pathogens |
| title_full_unstemmed | Multiplex polymerase chain reaction (PCR) with Nanopore sequencing for sequence-based detection of four tilapia pathogens |
| title_short | Multiplex polymerase chain reaction (PCR) with Nanopore sequencing for sequence-based detection of four tilapia pathogens |
| title_sort | multiplex polymerase chain reaction pcr with nanopore sequencing for sequence based detection of four tilapia pathogens |
| topic | qpcr bioinformatics tilapia streptococcus agalactiae fish multiplex pcr nanopore tilv francisella orientalis isknv amplicons sequencing |
| url | https://hdl.handle.net/10568/177740 |
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