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...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Adriana Larrea-Sarmiento, James P. Stack, Anne M. Alvarez, Mohammad Arif
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/434548ae9bfd4fccabf848d9d568001e
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:434548ae9bfd4fccabf848d9d568001e
record_format	dspace
spelling	oai:doaj.org-article:434548ae9bfd4fccabf848d9d568001e2021-12-02T15:03:07ZMultiplex recombinase polymerase amplification assay developed using unique genomic regions for rapid on-site detection of genus Clavibacter and C. nebraskensis10.1038/s41598-021-91336-72045-2322https://doaj.org/article/434548ae9bfd4fccabf848d9d568001e2021-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-91336-7https://doaj.org/toc/2045-2322Abstract 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.Adriana Larrea-SarmientoJames P. StackAnne M. AlvarezMohammad ArifNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q 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
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.
format	article
author	Adriana Larrea-Sarmiento James P. Stack Anne M. Alvarez Mohammad Arif
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 Portfolio
publishDate	2021
url	https://doaj.org/article/434548ae9bfd4fccabf848d9d568001e
work_keys_str_mv	AT adrianalarreasarmiento multiplexrecombinasepolymeraseamplificationassaydevelopedusinguniquegenomicregionsforrapidonsitedetectionofgenusclavibacterandcnebraskensis AT jamespstack multiplexrecombinasepolymeraseamplificationassaydevelopedusinguniquegenomicregionsforrapidonsitedetectionofgenusclavibacterandcnebraskensis AT annemalvarez multiplexrecombinasepolymeraseamplificationassaydevelopedusinguniquegenomicregionsforrapidonsitedetectionofgenusclavibacterandcnebraskensis AT mohammadarif multiplexrecombinasepolymeraseamplificationassaydevelopedusinguniquegenomicregionsforrapidonsitedetectionofgenusclavibacterandcnebraskensis
_version_	1718389073692852224

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

Ejemplares similares