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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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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