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A multiplexed RT-PCR assay for nanopore whole genome sequencing of Tilapia lake virus (TiLV)
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Document Title
A multiplexed RT-PCR assay for nanopore whole genome sequencing of Tilapia lake virus (TiLV)
Author
Delamare-Deboutteville J. Meemetta W. Pimsannil K. Sangpo P. Gan H.M. Mohan C.V. Dong H.T. Senapin S.
Affiliations
WorldFish Penang Bayan Lepas Malaysia; Fish Health Platform Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp) Faculty of Science Mahidol University Rama VI Rd. Bangkok 10400 Thailand; Patriot Biotech Sdn Bhd Bandar Sunway Selangor Subang Jaya 47500 Malaysia; School of Environment Resources and Development Asian Institute of Technology Pathum Thani 12120 Thailand; National Center for Genetic Engineering and Biotechnology (BIOTEC) National Science and Technology Development Agency (NSTDA) Pathum Thani 12120 Thailand
Type
Article
Source Title
Scientific Reports
ISSN
20452322
Year
2023
Volume
13
Issue
1
Open Access
All Open Access Gold Green
Publisher
Nature Research
DOI
10.1038/s41598-023-47425-w
Abstract
Tilapia lake virus (TiLV) is a highly contagious viral pathogen that affects tilapia a globally significant and affordable source of fish protein. To prevent 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 rapid 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 were immediately subjected to real-time sequencing on the Nanopore system. By using this approach we have recovered and assembled 10 TiLV genomes from total RNA extracted from naturally TiLV-infected tilapia fish concentrated tilapia rearing water and cell culture. 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 genetic diversity of TiLV. This work is an essential steppingstone towards integrating rapid and real-time Nanopore-based amplicon sequencing into routine genomic surveillance of TiLV as well as future vaccine development. ? 2023 The Author(s).
Industrial Classification
Knowledge Taxonomy Level 1
Knowledge Taxonomy Level 2
Knowledge Taxonomy Level 3
License
CC BY
Rights
Authors
Publication Source
WOS