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Field-deployable recombinase polymerase amplification assay for specific, sensitive and rapid detection of the us select agent and toxigenic bacterium, rathayibacter toxicus

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posted on 08.09.2021, 01:48 by M Arif, GY Busot, R Mann, Brendan RodoniBrendan Rodoni, JP Stack
Rathayibacter toxicus is a toxigenic bacterial pathogen of several grass species and is responsible for massive livestock deaths in Australia and South Africa. Due to concern for animal health and livestock industries, it was designated a U.S. Select Agent. A rapid, accurate, and sensitive in-field detection method was designed to assist biosecurity surveillance surveys and to support export certification of annual ryegrass hay and seed. Complete genomes from all known R. toxicus populations were explored, unique diagnostic sequences identified, and target-specific primers and a probe for recombinase polymerase amplification (RPA) and endpoint PCR were designed. The RPA reaction ran at 37 °C and a lateral flow device (LFD) was used to visualize the amplified products. To enhance reliability and accuracy, primers and probes were also designed to detect portions of host ITS regions. RPA assay specificity and sensitivity were compared to endpoint PCR using appropriate inclusivity and exclusivity panels. The RPA assay sensitivity (10 fg) was 10 times more sensitive than endpoint PCR with and without a host DNA background. In comparative tests, the RPA assay was unaffected by plant-derived amplification inhibitors, unlike the LAMP and end-point PCR assays. In-field validation of the RPA assay at multiple sites in South Australia confirmed the efficiency, specificity, and applicability of the RPA assay. The RPA assay will support disease management and evidence-based in-field biosecurity decisions.

Funding

Australian Government's Cooperative Research Centres Program, Canberra, Australia. Contribution no. 20-127-J from the Kansas Agricultural Experiment Station.

History

Publication Date

03/07/2021

Journal

Biology

Volume

10

Issue

7

Article Number

620

Pagination

15p.

Publisher

MDPI

ISSN

2079-7737

Rights Statement

© 2021 by the authors. 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/).

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