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A Rapid and Cost-Effective Identification of Invertebrate Pests at the Borders Using MinION Sequencing of DNA Barcodes

The rapid and accurate identification of invertebrate pests detected at the border is a challenging task. Current diagnostic methods used at the borders are mainly based on time consuming visual and microscopic examinations. Here, we demonstrate a rapid in-house workflow for DNA extraction, PCR ampl...

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Autores principales: Abeynayake, Shamila Weerakoon, Fiorito, Sonia, Dinsdale, Adrian, Whattam, Mark, Crowe, Bill, Sparks, Kate, Campbell, Paul Richard, Gambley, Cherie
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8392835/
https://www.ncbi.nlm.nih.gov/pubmed/34440312
http://dx.doi.org/10.3390/genes12081138
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author Abeynayake, Shamila Weerakoon
Fiorito, Sonia
Dinsdale, Adrian
Whattam, Mark
Crowe, Bill
Sparks, Kate
Campbell, Paul Richard
Gambley, Cherie
author_facet Abeynayake, Shamila Weerakoon
Fiorito, Sonia
Dinsdale, Adrian
Whattam, Mark
Crowe, Bill
Sparks, Kate
Campbell, Paul Richard
Gambley, Cherie
author_sort Abeynayake, Shamila Weerakoon
collection PubMed
description The rapid and accurate identification of invertebrate pests detected at the border is a challenging task. Current diagnostic methods used at the borders are mainly based on time consuming visual and microscopic examinations. Here, we demonstrate a rapid in-house workflow for DNA extraction, PCR amplification of the barcode region of the mitochondrial cytochrome oxidase subunit I (COI) gene and Oxford Nanopore Technologies (ONT) MinION sequencing of amplified products multiplexed after barcoding on ONT Flongle flow cells. A side-by-side comparison was conducted of DNA barcode sequencing-based identification and morphological identification of both large (>0.5 mm in length) and small (<0.5 mm in length) invertebrate specimens intercepted at the Australian border. DNA barcode sequencing results supported the morphological identification in most cases and enabled immature stages of invertebrates and their eggs to be identified more confidently. Results also showed that sequencing the COI barcode region using the ONT rapid sequencing principle is a cost-effective and field-adaptable approach for the rapid and accurate identification of invertebrate pests. Overall, the results suggest that MinION sequencing of DNA barcodes offers a complementary tool to the existing morphological diagnostic approaches and provides rapid, accurate, reliable and defendable evidence for identifying invertebrate pests at the border.
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spelling pubmed-83928352021-08-28 A Rapid and Cost-Effective Identification of Invertebrate Pests at the Borders Using MinION Sequencing of DNA Barcodes Abeynayake, Shamila Weerakoon Fiorito, Sonia Dinsdale, Adrian Whattam, Mark Crowe, Bill Sparks, Kate Campbell, Paul Richard Gambley, Cherie Genes (Basel) Article The rapid and accurate identification of invertebrate pests detected at the border is a challenging task. Current diagnostic methods used at the borders are mainly based on time consuming visual and microscopic examinations. Here, we demonstrate a rapid in-house workflow for DNA extraction, PCR amplification of the barcode region of the mitochondrial cytochrome oxidase subunit I (COI) gene and Oxford Nanopore Technologies (ONT) MinION sequencing of amplified products multiplexed after barcoding on ONT Flongle flow cells. A side-by-side comparison was conducted of DNA barcode sequencing-based identification and morphological identification of both large (>0.5 mm in length) and small (<0.5 mm in length) invertebrate specimens intercepted at the Australian border. DNA barcode sequencing results supported the morphological identification in most cases and enabled immature stages of invertebrates and their eggs to be identified more confidently. Results also showed that sequencing the COI barcode region using the ONT rapid sequencing principle is a cost-effective and field-adaptable approach for the rapid and accurate identification of invertebrate pests. Overall, the results suggest that MinION sequencing of DNA barcodes offers a complementary tool to the existing morphological diagnostic approaches and provides rapid, accurate, reliable and defendable evidence for identifying invertebrate pests at the border. MDPI 2021-07-27 /pmc/articles/PMC8392835/ /pubmed/34440312 http://dx.doi.org/10.3390/genes12081138 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Abeynayake, Shamila Weerakoon
Fiorito, Sonia
Dinsdale, Adrian
Whattam, Mark
Crowe, Bill
Sparks, Kate
Campbell, Paul Richard
Gambley, Cherie
A Rapid and Cost-Effective Identification of Invertebrate Pests at the Borders Using MinION Sequencing of DNA Barcodes
title A Rapid and Cost-Effective Identification of Invertebrate Pests at the Borders Using MinION Sequencing of DNA Barcodes
title_full A Rapid and Cost-Effective Identification of Invertebrate Pests at the Borders Using MinION Sequencing of DNA Barcodes
title_fullStr A Rapid and Cost-Effective Identification of Invertebrate Pests at the Borders Using MinION Sequencing of DNA Barcodes
title_full_unstemmed A Rapid and Cost-Effective Identification of Invertebrate Pests at the Borders Using MinION Sequencing of DNA Barcodes
title_short A Rapid and Cost-Effective Identification of Invertebrate Pests at the Borders Using MinION Sequencing of DNA Barcodes
title_sort rapid and cost-effective identification of invertebrate pests at the borders using minion sequencing of dna barcodes
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8392835/
https://www.ncbi.nlm.nih.gov/pubmed/34440312
http://dx.doi.org/10.3390/genes12081138
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