Rapid and sensitive detection of Candidatus Liberibacter asiaticus by loop mediated isothermal amplification combined with a lateral flow dipstick

Autores
Rigano, Luciano Ariel; Malamud, Florencia; Orce, Ingrid Georgina; Filippone, María Paula; Marano, Maria Rosa; Do Amaral, Alexandre Morais; Castagnaro, Atilio Pedro; Vojnov, Adrian Alberto
Año de publicación
2014
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Background: Citrus Huanglongbing (HLB) is the most devastating bacterial citrus disease worldwide. Three Candidatus Liberibacter species are associated with different forms of the disease: Candidatus Liberibacter asiaticus, Candidatus Liberibacter americanus and Candidatus Liberibacter africanus. Amongst them, Candidatus Liberibacter asiaticus is the most widespread and economically important. These Gram-negative bacterial plant pathogens are phloem-limited and vectored by citrus psyllids. The current management strategy of HLB is based on early and accurate detection of Candidatus Liberibacter asiaticus in both citrus plants and vector insects. Nowadays, real time PCR is the method of choice for this task, mainly because of its sensitivity and reliability. However, this methodology has several drawbacks, namely high equipment costs, the need for highly trained personnel, the time required to conduct the whole process, and the difficulty in carrying out the detection reactions in field conditions. Results: A recent DNA amplification technique known as Loop Mediated Isothermal Amplification (LAMP) was adapted for the detection of Candidatus Liberibacter asiaticus. This methodology was combined with a Lateral Flow Dipstick (LFD) device for visual detection of the resulting amplicons, eliminating the need for gel electrophoresis. The assay was highly specific for the targeted bacterium. No cross-reaction was observed with DNA from any of the other phytopathogenic bacteria or fungi assayed. By serially diluting purified DNA from an infected plant, the sensitivity of the assay was found to be 10 picograms. This sensitivity level was proven to be similar to the values obtained running a real time PCR in parallel. This methodology was able to detect Candidatus Liberibacter asiaticus from different kinds of samples including infected citrus plants and psyllids. Conclusions: Our results indicate that the methodology here reported constitutes a step forward in the development of new tools for the management, control and eradication of this destructive citrus disease. This system constitutes a potentially field-capable approach for the detection of the most relevant HLB-associated bacteria in plant material and psyllid vectors.
Fil: Rigano, Luciano Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; Argentina. University of Otago. Department of Microbiology and Immunology; Nueva Zelanda
Fil: Malamud, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura; Argentina
Fil: Orce, Ingrid Georgina. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Estación Experimental Agroindustrial Obispo Colombres; Argentina
Fil: Filippone, María Paula. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Estación Experimental Agroindustrial Obispo Colombres; Argentina
Fil: Marano, Maria Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina
Fil: Do Amaral, Alexandre Morais. Ministerio Da Agricultura Pecuaria E Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; Brasil
Fil: Castagnaro, Atilio Pedro. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Estación Experimental Agroindustrial Obispo Colombres; Argentina
Fil: Vojnov, Adrian Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; Argentina
Materia
Huanglongbing
Candidatus Liberibacter asiaticus
Diaphorina citri
Detection by LAMP
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/5282
Acceder
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repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Rapid and sensitive detection of Candidatus Liberibacter asiaticus by loop mediated isothermal amplification combined with a lateral flow dipstickRigano, Luciano ArielMalamud, FlorenciaOrce, Ingrid GeorginaFilippone, María PaulaMarano, Maria RosaDo Amaral, Alexandre MoraisCastagnaro, Atilio PedroVojnov, Adrian AlbertoHuanglongbingCandidatus Liberibacter asiaticusDiaphorina citriDetection by LAMPhttps://purl.org/becyt/ford/4.4https://purl.org/becyt/ford/4Background: Citrus Huanglongbing (HLB) is the most devastating bacterial citrus disease worldwide. Three Candidatus Liberibacter species are associated with different forms of the disease: Candidatus Liberibacter asiaticus, Candidatus Liberibacter americanus and Candidatus Liberibacter africanus. Amongst them, Candidatus Liberibacter asiaticus is the most widespread and economically important. These Gram-negative bacterial plant pathogens are phloem-limited and vectored by citrus psyllids. The current management strategy of HLB is based on early and accurate detection of Candidatus Liberibacter asiaticus in both citrus plants and vector insects. Nowadays, real time PCR is the method of choice for this task, mainly because of its sensitivity and reliability. However, this methodology has several drawbacks, namely high equipment costs, the need for highly trained personnel, the time required to conduct the whole process, and the difficulty in carrying out the detection reactions in field conditions. Results: A recent DNA amplification technique known as Loop Mediated Isothermal Amplification (LAMP) was adapted for the detection of Candidatus Liberibacter asiaticus. This methodology was combined with a Lateral Flow Dipstick (LFD) device for visual detection of the resulting amplicons, eliminating the need for gel electrophoresis. The assay was highly specific for the targeted bacterium. No cross-reaction was observed with DNA from any of the other phytopathogenic bacteria or fungi assayed. By serially diluting purified DNA from an infected plant, the sensitivity of the assay was found to be 10 picograms. This sensitivity level was proven to be similar to the values obtained running a real time PCR in parallel. This methodology was able to detect Candidatus Liberibacter asiaticus from different kinds of samples including infected citrus plants and psyllids. Conclusions: Our results indicate that the methodology here reported constitutes a step forward in the development of new tools for the management, control and eradication of this destructive citrus disease. This system constitutes a potentially field-capable approach for the detection of the most relevant HLB-associated bacteria in plant material and psyllid vectors.Fil: Rigano, Luciano Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; Argentina. University of Otago. Department of Microbiology and Immunology; Nueva ZelandaFil: Malamud, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura; ArgentinaFil: Orce, Ingrid Georgina. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Estación Experimental Agroindustrial Obispo Colombres; ArgentinaFil: Filippone, María Paula. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Estación Experimental Agroindustrial Obispo Colombres; ArgentinaFil: Marano, Maria Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Do Amaral, Alexandre Morais. Ministerio Da Agricultura Pecuaria E Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; BrasilFil: Castagnaro, Atilio Pedro. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Estación Experimental Agroindustrial Obispo Colombres; ArgentinaFil: Vojnov, Adrian Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; ArgentinaBiomed Central2014-03info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/5282Rigano, Luciano Ariel; Malamud, Florencia; Orce, Ingrid Georgina; Filippone, María Paula; Marano, Maria Rosa; et al.; Rapid and sensitive detection of Candidatus Liberibacter asiaticus by loop mediated isothermal amplification combined with a lateral flow dipstick; Biomed Central; Bmc Microbiology; 14; 86; 3-2014; 1-91471-2180enginfo:eu-repo/semantics/altIdentifier/url/http://bmcmicrobiol.biomedcentral.com/articles/10.1186/1471-2180-14-86info:eu-repo/semantics/altIdentifier/url/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4021466/info:eu-repo/semantics/altIdentifier/doi/10.1186%2F1471-2180-14-86info:eu-repo/semantics/altIdentifier/doi/info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:12:48Zoai:ri.conicet.gov.ar:11336/5282instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 10:12:48.928CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Rapid and sensitive detection of Candidatus Liberibacter asiaticus by loop mediated isothermal amplification combined with a lateral flow dipstick
title Rapid and sensitive detection of Candidatus Liberibacter asiaticus by loop mediated isothermal amplification combined with a lateral flow dipstick
spellingShingle Rapid and sensitive detection of Candidatus Liberibacter asiaticus by loop mediated isothermal amplification combined with a lateral flow dipstick
Rigano, Luciano Ariel
Huanglongbing
Candidatus Liberibacter asiaticus
Diaphorina citri
Detection by LAMP
title_short Rapid and sensitive detection of Candidatus Liberibacter asiaticus by loop mediated isothermal amplification combined with a lateral flow dipstick
title_full Rapid and sensitive detection of Candidatus Liberibacter asiaticus by loop mediated isothermal amplification combined with a lateral flow dipstick
title_fullStr Rapid and sensitive detection of Candidatus Liberibacter asiaticus by loop mediated isothermal amplification combined with a lateral flow dipstick
title_full_unstemmed Rapid and sensitive detection of Candidatus Liberibacter asiaticus by loop mediated isothermal amplification combined with a lateral flow dipstick
title_sort Rapid and sensitive detection of Candidatus Liberibacter asiaticus by loop mediated isothermal amplification combined with a lateral flow dipstick
dc.creator.none.fl_str_mv Rigano, Luciano Ariel
Malamud, Florencia
Orce, Ingrid Georgina
Filippone, María Paula
Marano, Maria Rosa
Do Amaral, Alexandre Morais
Castagnaro, Atilio Pedro
Vojnov, Adrian Alberto
author Rigano, Luciano Ariel
author_facet Rigano, Luciano Ariel
Malamud, Florencia
Orce, Ingrid Georgina
Filippone, María Paula
Marano, Maria Rosa
Do Amaral, Alexandre Morais
Castagnaro, Atilio Pedro
Vojnov, Adrian Alberto
author_role author
author2 Malamud, Florencia
Orce, Ingrid Georgina
Filippone, María Paula
Marano, Maria Rosa
Do Amaral, Alexandre Morais
Castagnaro, Atilio Pedro
Vojnov, Adrian Alberto
author2_role author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Huanglongbing
Candidatus Liberibacter asiaticus
Diaphorina citri
Detection by LAMP
topic Huanglongbing
Candidatus Liberibacter asiaticus
Diaphorina citri
Detection by LAMP
purl_subject.fl_str_mv https://purl.org/becyt/ford/4.4
https://purl.org/becyt/ford/4
dc.description.none.fl_txt_mv Background: Citrus Huanglongbing (HLB) is the most devastating bacterial citrus disease worldwide. Three Candidatus Liberibacter species are associated with different forms of the disease: Candidatus Liberibacter asiaticus, Candidatus Liberibacter americanus and Candidatus Liberibacter africanus. Amongst them, Candidatus Liberibacter asiaticus is the most widespread and economically important. These Gram-negative bacterial plant pathogens are phloem-limited and vectored by citrus psyllids. The current management strategy of HLB is based on early and accurate detection of Candidatus Liberibacter asiaticus in both citrus plants and vector insects. Nowadays, real time PCR is the method of choice for this task, mainly because of its sensitivity and reliability. However, this methodology has several drawbacks, namely high equipment costs, the need for highly trained personnel, the time required to conduct the whole process, and the difficulty in carrying out the detection reactions in field conditions. Results: A recent DNA amplification technique known as Loop Mediated Isothermal Amplification (LAMP) was adapted for the detection of Candidatus Liberibacter asiaticus. This methodology was combined with a Lateral Flow Dipstick (LFD) device for visual detection of the resulting amplicons, eliminating the need for gel electrophoresis. The assay was highly specific for the targeted bacterium. No cross-reaction was observed with DNA from any of the other phytopathogenic bacteria or fungi assayed. By serially diluting purified DNA from an infected plant, the sensitivity of the assay was found to be 10 picograms. This sensitivity level was proven to be similar to the values obtained running a real time PCR in parallel. This methodology was able to detect Candidatus Liberibacter asiaticus from different kinds of samples including infected citrus plants and psyllids. Conclusions: Our results indicate that the methodology here reported constitutes a step forward in the development of new tools for the management, control and eradication of this destructive citrus disease. This system constitutes a potentially field-capable approach for the detection of the most relevant HLB-associated bacteria in plant material and psyllid vectors.
Fil: Rigano, Luciano Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; Argentina. University of Otago. Department of Microbiology and Immunology; Nueva Zelanda
Fil: Malamud, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura; Argentina
Fil: Orce, Ingrid Georgina. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Estación Experimental Agroindustrial Obispo Colombres; Argentina
Fil: Filippone, María Paula. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Estación Experimental Agroindustrial Obispo Colombres; Argentina
Fil: Marano, Maria Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina
Fil: Do Amaral, Alexandre Morais. Ministerio Da Agricultura Pecuaria E Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; Brasil
Fil: Castagnaro, Atilio Pedro. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Estación Experimental Agroindustrial Obispo Colombres; Argentina
Fil: Vojnov, Adrian Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; Argentina
description Background: Citrus Huanglongbing (HLB) is the most devastating bacterial citrus disease worldwide. Three Candidatus Liberibacter species are associated with different forms of the disease: Candidatus Liberibacter asiaticus, Candidatus Liberibacter americanus and Candidatus Liberibacter africanus. Amongst them, Candidatus Liberibacter asiaticus is the most widespread and economically important. These Gram-negative bacterial plant pathogens are phloem-limited and vectored by citrus psyllids. The current management strategy of HLB is based on early and accurate detection of Candidatus Liberibacter asiaticus in both citrus plants and vector insects. Nowadays, real time PCR is the method of choice for this task, mainly because of its sensitivity and reliability. However, this methodology has several drawbacks, namely high equipment costs, the need for highly trained personnel, the time required to conduct the whole process, and the difficulty in carrying out the detection reactions in field conditions. Results: A recent DNA amplification technique known as Loop Mediated Isothermal Amplification (LAMP) was adapted for the detection of Candidatus Liberibacter asiaticus. This methodology was combined with a Lateral Flow Dipstick (LFD) device for visual detection of the resulting amplicons, eliminating the need for gel electrophoresis. The assay was highly specific for the targeted bacterium. No cross-reaction was observed with DNA from any of the other phytopathogenic bacteria or fungi assayed. By serially diluting purified DNA from an infected plant, the sensitivity of the assay was found to be 10 picograms. This sensitivity level was proven to be similar to the values obtained running a real time PCR in parallel. This methodology was able to detect Candidatus Liberibacter asiaticus from different kinds of samples including infected citrus plants and psyllids. Conclusions: Our results indicate that the methodology here reported constitutes a step forward in the development of new tools for the management, control and eradication of this destructive citrus disease. This system constitutes a potentially field-capable approach for the detection of the most relevant HLB-associated bacteria in plant material and psyllid vectors.
publishDate 2014
dc.date.none.fl_str_mv 2014-03
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
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info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/5282
Rigano, Luciano Ariel; Malamud, Florencia; Orce, Ingrid Georgina; Filippone, María Paula; Marano, Maria Rosa; et al.; Rapid and sensitive detection of Candidatus Liberibacter asiaticus by loop mediated isothermal amplification combined with a lateral flow dipstick; Biomed Central; Bmc Microbiology; 14; 86; 3-2014; 1-9
1471-2180
url http://hdl.handle.net/11336/5282
identifier_str_mv Rigano, Luciano Ariel; Malamud, Florencia; Orce, Ingrid Georgina; Filippone, María Paula; Marano, Maria Rosa; et al.; Rapid and sensitive detection of Candidatus Liberibacter asiaticus by loop mediated isothermal amplification combined with a lateral flow dipstick; Biomed Central; Bmc Microbiology; 14; 86; 3-2014; 1-9
1471-2180
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/http://bmcmicrobiol.biomedcentral.com/articles/10.1186/1471-2180-14-86
info:eu-repo/semantics/altIdentifier/url/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4021466/
info:eu-repo/semantics/altIdentifier/doi/10.1186%2F1471-2180-14-86
info:eu-repo/semantics/altIdentifier/doi/
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dc.publisher.none.fl_str_mv Biomed Central
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