Multiplex recombinase polymerase amplification assay developed using unique genomic regions for rapid on-site detection of genus Clavibacter and C. nebraskensis

Abstract Clavibacter is an agriculturally important bacterial genus comprising nine host-specific species/subspecies including C. nebraskensis (Cn), which causes Goss's wilt and blight of maize. A robust, simple, and field-deployable method is required to specifically detect Cn in infected plan...

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Main Authors: Adriana Larrea-Sarmiento, James P. Stack, Anne M. Alvarez, Mohammad Arif
Format: Article
Language:English
Published: Nature Publishing Group 2021-06-01
Series:Scientific Reports
Online Access:https://doi.org/10.1038/s41598-021-91336-7
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spelling doaj-434548ae9bfd4fccabf848d9d568001e2021-06-13T11:42:03ZengNature Publishing GroupScientific Reports2045-23222021-06-0111111010.1038/s41598-021-91336-7Multiplex recombinase polymerase amplification assay developed using unique genomic regions for rapid on-site detection of genus Clavibacter and C. nebraskensisAdriana Larrea-Sarmiento0James P. Stack1Anne M. Alvarez2Mohammad Arif3Department of Plant and Environmental Protection Sciences, University of Hawaii at ManoaDepartment of Plant Pathology, Kansas State UniversityDepartment of Plant and Environmental Protection Sciences, University of Hawaii at ManoaDepartment of Plant and Environmental Protection Sciences, University of Hawaii at ManoaAbstract Clavibacter is an agriculturally important bacterial genus comprising nine host-specific species/subspecies including C. nebraskensis (Cn), which causes Goss's wilt and blight of maize. A robust, simple, and field-deployable method is required to specifically detect Cn in infected plants and distinguish it from other Clavibacter species for quarantine purposes and timely disease management. A multiplex Recombinase Polymerase Amplification (RPA) coupled with a Lateral Flow Device (LFD) was developed for sensitive and rapid detection of Clavibacter and Cn directly from infected host. Unique and conserved genomic regions, the ABC transporter ATP-binding protein CDS/ABC-transporter permease and the MFS transporter gene, were used to design primers/probes for specific detection of genus Clavibacter and Cn, respectively. The assay was evaluated using 52 strains, representing all nine species/subspecies of Clavibacter, other closely related bacterial species, and naturally- and artificially-infected plant samples; no false positives or negatives were detected. The RPA reactions were also incubated in a closed hand at body temperature; results were again specific. The assay does not require DNA isolation and can be directly performed using host sap. The detection limit of 10 pg (~ 3000 copies) and 100 fg (~ 30 copies) was determined for Clavibacter- and Cn-specific primers/probes, respectively. The detection limit for Cn-specific primer/probe set was decreased to 1 pg (~ 300 copies) when 1 µL of host sap was added into the RPA reaction containing tenfold serially diluted genomic DNA; though no effect was observed on Clavibacter-specific primer/probe set. The assay is accurate and has applications at point-of-need diagnostics. This is the first multiplex RPA assay for any plant pathogen.https://doi.org/10.1038/s41598-021-91336-7
collection DOAJ
language English
format Article
sources DOAJ
author Adriana Larrea-Sarmiento
James P. Stack
Anne M. Alvarez
Mohammad Arif
spellingShingle Adriana Larrea-Sarmiento
James P. Stack
Anne M. Alvarez
Mohammad Arif
Multiplex recombinase polymerase amplification assay developed using unique genomic regions for rapid on-site detection of genus Clavibacter and C. nebraskensis
Scientific Reports
author_facet Adriana Larrea-Sarmiento
James P. Stack
Anne M. Alvarez
Mohammad Arif
author_sort Adriana Larrea-Sarmiento
title Multiplex recombinase polymerase amplification assay developed using unique genomic regions for rapid on-site detection of genus Clavibacter and C. nebraskensis
title_short Multiplex recombinase polymerase amplification assay developed using unique genomic regions for rapid on-site detection of genus Clavibacter and C. nebraskensis
title_full Multiplex recombinase polymerase amplification assay developed using unique genomic regions for rapid on-site detection of genus Clavibacter and C. nebraskensis
title_fullStr Multiplex recombinase polymerase amplification assay developed using unique genomic regions for rapid on-site detection of genus Clavibacter and C. nebraskensis
title_full_unstemmed Multiplex recombinase polymerase amplification assay developed using unique genomic regions for rapid on-site detection of genus Clavibacter and C. nebraskensis
title_sort multiplex recombinase polymerase amplification assay developed using unique genomic regions for rapid on-site detection of genus clavibacter and c. nebraskensis
publisher Nature Publishing Group
series Scientific Reports
issn 2045-2322
publishDate 2021-06-01
description Abstract Clavibacter is an agriculturally important bacterial genus comprising nine host-specific species/subspecies including C. nebraskensis (Cn), which causes Goss's wilt and blight of maize. A robust, simple, and field-deployable method is required to specifically detect Cn in infected plants and distinguish it from other Clavibacter species for quarantine purposes and timely disease management. A multiplex Recombinase Polymerase Amplification (RPA) coupled with a Lateral Flow Device (LFD) was developed for sensitive and rapid detection of Clavibacter and Cn directly from infected host. Unique and conserved genomic regions, the ABC transporter ATP-binding protein CDS/ABC-transporter permease and the MFS transporter gene, were used to design primers/probes for specific detection of genus Clavibacter and Cn, respectively. The assay was evaluated using 52 strains, representing all nine species/subspecies of Clavibacter, other closely related bacterial species, and naturally- and artificially-infected plant samples; no false positives or negatives were detected. The RPA reactions were also incubated in a closed hand at body temperature; results were again specific. The assay does not require DNA isolation and can be directly performed using host sap. The detection limit of 10 pg (~ 3000 copies) and 100 fg (~ 30 copies) was determined for Clavibacter- and Cn-specific primers/probes, respectively. The detection limit for Cn-specific primer/probe set was decreased to 1 pg (~ 300 copies) when 1 µL of host sap was added into the RPA reaction containing tenfold serially diluted genomic DNA; though no effect was observed on Clavibacter-specific primer/probe set. The assay is accurate and has applications at point-of-need diagnostics. This is the first multiplex RPA assay for any plant pathogen.
url https://doi.org/10.1038/s41598-021-91336-7
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