Rapid genotyping of tilapia lake virus (TiLV) using Nanopore sequencing

Delamare-Deboutteville, J.; Taengphu, Suwimon; Gan, Han Ming; Kayansamruaj, Pattanapon; Debnath, Partho Pratim; Barnes, Andrew C.; Wilkinson, Shaun P.; Kawasaki, Minami; Mohan, C. V.; Senapin, Saengchan; Dong, Ha Thanh

doi:10.1111/jfd.13467

Cited by 11 publications

(8 citation statements)

References 39 publications

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“…These diseases continue to evolve and spread under intense selective pressures, particularly in regions with high-density tilapia farming. To address this issue, we built upon our previous study 18 and improved the detection and genotyping of multiple pathogens by switching to multiplex PCR coupled with Nanopore sequencing. Our updated approach enabled the simultaneous and sequence-based detection and verification of four major tilapia pathogens: ISKNV, TiLV, S. agalactiae, and F. orientalis.…”

Section: Discussionmentioning

confidence: 99%

“…Sequence-based verification is particularly valuable in multiplex PCR since nonspecific amplification is more likely to occur in a multiplex PCR reaction due to the presence of multiple primer combinations. Fortunately, this is now possible with Oxford nanopore technology (ONT) that allows on-site amplicon sequencing 18 . Proper integration of multiplex PCR and ONT-based genotyping may therefore present a promising tool for the cost-effective, efficient, and precise detection and characterization of pathogens in tilapia aquaculture.…”

Section: Introductionmentioning

confidence: 99%

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

Delamare-Deboutteville,

Meemetta,

Pimsannil

et al. 2023

Preprint

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Delamare-Deboutteville,

Meemetta,

Pimsannil

et al. 2023

Preprint

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“…Nanopore sequencing offers several advantages, including high throughput, portability, cost-effectiveness, and real-time sequencing, which can greatly facilitate the detection and sequencing of viral genomes in remote locations (Alathari et al, 2023). Rapid amplicon-based reverse transcription polymerase chain reaction (RT-PCR) assays coupled with Nanopore technologies can provide a sensitive and specific means of detecting and genotyping TiLV in field samples, allowing for fine epidemiological surveillance and timely management and control of outbreaks (Delamare-Deboutteville et al, 2021). To date, Nanopore has been used to sequence the genomes of non-segmented fish viruses such as infectious spleen and kidney necrosis virus (ISKNV) (Alathari et al, 2023), salmonid alphavirus (SAV) and infectious salmon anaemia virus (ISAV) (Gallagher et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

A multiplexed RT-PCR Assay for Nanopore Whole Genome Sequencing of Tilapia lake virus (TiLV)

Delamare-Deboutteville

Meemetta

Gan³

et al. 2023

Preprint

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Tilapia lake virus (TiLV) is a highly contagious viral disease that affects tilapia, a globally significant and affordable source of fish protein. To combat the introduction and spread of TiLV and its impact, there is an urgent need for increased surveillance, improved biosecurity measures, and continuous development of effective diagnostic and sequencing methods. In this study, we have developed a multiplexed RT-PCR assay that can amplify all ten complete genomic segments of TiLV from various sources of isolation. The amplicons generated using this approach are immediately compatible with real-time sequencing on the Nanopore system. By using this approach, we have recovered and assembled 13 TiLV genomes from total RNA extracted from naturally TiLV-infected tilapia fish, concentrated tilapia rearing water, and other isolation sources. Our phylogenetic analysis, consisting of more than 36 TiLV genomes from both newly sequenced and publicly available TiLV genomes, provides new insights into the high phylogenetic diversity of TiLV from Thailand, suggesting multiple genetic origins. This work is an essential stepping stone towards integrating rapid and real-time Nanopore-based amplicon sequencing into routine genomic surveillance of TiLV, as well as future vaccine development.

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“…Several molecular methods have been developed for the detection of TiLV, including RT-PCR ( Eyngor et al, 2014 ), nested and semi-nested PCR ( Dong et al, 2017a ; Kembou Tsofack et al, 2017 ; Taengphu et al, 2020 ), RT-qPCR ( Tattiyapong, Sirikanchana & Surachetpong, 2018 ; Waiyamitra et al, 2018 ), loop-mediated isothermal amplification (LAMP) ( Kampeera et al, 2021 ; Phusantisampan et al, 2019 ; Yin et al, 2019 ) and the nanopore-based PCR amplicon approach ( Delamare-Deboutteville et al, 2021 ). However, all of these methods require fish tissues for diagnosis and none reported any use for TiLV detection from environmental water samples.…”

Section: Introductionmentioning

confidence: 99%

Concentration and quantification of Tilapia tilapinevirus from water using a simple iron flocculation coupled with probe-based RT-qPCR

Taengphu

Kayansamruaj

Kawato³

et al. 2022

PeerJ

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Background Tilapia tilapinevirus, also known as tilapia lake virus (TiLV), is a significant virus that is responsible for the die-off of farmed tilapia across the globe. The detection and quantification of the virus using environmental RNA (eRNA) from pond water samples represents a potentially non-invasive and routine strategy for monitoring pathogens and early disease forecasting in aquaculture systems. Methods Here, we report a simple iron flocculation method for concentrating viruses in water, together with a newly-developed hydrolysis probe quantitative RT-qPCR method for the detection and quantification of TiLV. Results The RT-qPCR method designed to target a conserved region of the TiLV genome segment 9 has a detection limit of 10 viral copies per µL of template. The method had a 100% analytical specificity and sensitivity for TiLV. The optimized iron flocculation method was able to recover 16.11 ± 3.3% of the virus from water samples spiked with viral cultures. Tilapia and water samples were collected for use in the detection and quantification of TiLV disease during outbreaks in an open-caged river farming system and two earthen fish farms. TiLV was detected from both clinically sick and asymptomatic fish. Most importantly, the virus was successfully detected from water samples collected from different locations in the affected farms (i.e., river water samples from affected cages (8.50 × 103 to 2.79 × 105 copies/L) and fish-rearing water samples, sewage, and reservoir (4.29 × 103 to 3.53 × 104 copies/L)). By contrast, TiLV was not detected in fish or water samples collected from two farms that had previously experienced TiLV outbreaks and from one farm that had never experienced a TiLV outbreak. In summary, this study suggests that the eRNA detection system using iron flocculation, coupled with probe based-RT-qPCR, is feasible for use in the concentration and quantification of TiLV from water. This approach may be useful for the non-invasive monitoring of TiLV in tilapia aquaculture systems and may support evidence-based decisions on biosecurity interventions needed.

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Rapid genotyping of tilapia lake virus (TiLV) using Nanopore sequencing

Abstract: This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Cited by 11 publications

References 39 publications

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

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

A multiplexed RT-PCR Assay for Nanopore Whole Genome Sequencing of Tilapia lake virus (TiLV)

Concentration and quantification of Tilapia tilapinevirus from water using a simple iron flocculation coupled with probe-based RT-qPCR

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