A colorimetric, sensitive, rapid, and simple diagnostic kit for the HLB putative causal agent detection

Autores
Stolowicz, Fabiana; Larocca, Luciana; Werbajh, Santiago; Parma, Yanil; Carrillo, Carolina; Ogas, Lorena; Agostini, Juan Pedro; Redes, Jonathan; Welin, Bjorn; Castagnaro, Atilio; Vojnov, Adrian
Año de publicación
2022
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Huanglongbing (HLB) is one of the most devastating diseases in citrus worldwide. The Gram-negative bacterial plant pathogen “Candidatus Liberibacter spp.” is phloem-limited and vectored by citrus psyllids. The species “Candidatus Liberibacter asiaticus” (C.Las) has been detected in Argentina, and its vector has been found in at least nine provinces. Early detection of C.Las is critical for a successful management of HLB disease. Currently, HLB molecular diagnosis is carried out by PCR, nested PCR, real-time PCR, or another combination of these techniques, which require purification of genomic DNA, sophisticated equipment, and highly trained personnel. We have developed a prototype of a sensitive colorimetric kit to detect C.Las based on the specific DNA isothermal amplification of this microorganism. The reaction buffer contains hydroxynaphthol blue (HNB), an indicator dye that turns from violet to blue/light blue when the DNA amplification reaction is positive. Similar sensitivity to visualize a positive reaction was observed between HNB loop-mediated isothermal amplification and agarose gel electrophoresis analysis. The detection of C.Las-infected plants was up to 8 ng of total infected plant genomic DNA, similar to quantitative PCR. A blind validation test of the prototype kit was performed with purified DNA extracted from healthy or C.Las-infected midrib plants. Our kit showed 100% concordance with the results of a gold-standard quantitative PCR technique applied by the Laboratorio de Biología Molecular de EEA Montecarlo. The analysis of samples, without DNA purification to detect C.Las, showed a similar sensitivity to the analysis of the same samples in which C.Las DNA was previously purified.
EEA Montecarlo
Fil: Larocca, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencia y Tecnología Dr. Cesar Milstein. Fundación Pablo Cassará; Argentina
Fil: Werbajh, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencia y Tecnología Dr. Cesar Milstein. Fundación Pablo Cassará; Argentina
Fil: Parma, Yanil. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencia y Tecnología Dr. Cesar Milstein. Fundación Pablo Cassará; Argentina
Fil: Carrillo, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencia y Tecnología Dr. Cesar Milstein. Fundación Pablo Cassará; Argentina
Fil: Ogas, Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencia y Tecnología Dr. Cesar Milstein. Fundación Pablo Cassará; Argentina
Fil: Agostini, Juan P. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Montecarlo; Argentina
Fil: Redes, Jonathan. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Montecarlo; Argentina
Fil: Welin, Bjorn. Estación Experimental Agroindustrial Obispo Colombres; Argentina
Fil: Castagnaro, Atilio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Centro Científico Tecnológico Tucumán; Argentina
Fil: Vojnov, Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencia y Tecnología Dr. Cesar Milstein. Fundación Pablo Cassará; Argentina
Fuente
Frontiers in Agronomy 4 : 984360. (October 2022)
Materia
Huanglongbing de los Cítricos
Enfermedades de las Plantas
Sanidad Vegetal
Huanglongbing
Plant Diseases
Plant Health
Citrus
Candidatus liberibacter
Prototipo Colorimétrico
Colorimetric Prototype
Nivel de accesibilidad
acceso abierto
Condiciones de uso
http://creativecommons.org/licenses/by-nc-sa/4.0/
Repositorio
INTA Digital (INTA)
Institución
Instituto Nacional de Tecnología Agropecuaria
OAI Identificador
oai:localhost:20.500.12123/21386
Acceder
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oai_identifier_str oai:localhost:20.500.12123/21386
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network_name_str INTA Digital (INTA)
spelling A colorimetric, sensitive, rapid, and simple diagnostic kit for the HLB putative causal agent detectionStolowicz, FabianaLarocca, LucianaWerbajh, SantiagoParma, YanilCarrillo, CarolinaOgas, LorenaAgostini, Juan PedroRedes, JonathanWelin, BjornCastagnaro, AtilioVojnov, AdrianHuanglongbing de los CítricosEnfermedades de las PlantasSanidad VegetalHuanglongbingPlant DiseasesPlant HealthCitrusCandidatus liberibacterPrototipo ColorimétricoColorimetric PrototypeHuanglongbing (HLB) is one of the most devastating diseases in citrus worldwide. The Gram-negative bacterial plant pathogen “Candidatus Liberibacter spp.” is phloem-limited and vectored by citrus psyllids. The species “Candidatus Liberibacter asiaticus” (C.Las) has been detected in Argentina, and its vector has been found in at least nine provinces. Early detection of C.Las is critical for a successful management of HLB disease. Currently, HLB molecular diagnosis is carried out by PCR, nested PCR, real-time PCR, or another combination of these techniques, which require purification of genomic DNA, sophisticated equipment, and highly trained personnel. We have developed a prototype of a sensitive colorimetric kit to detect C.Las based on the specific DNA isothermal amplification of this microorganism. The reaction buffer contains hydroxynaphthol blue (HNB), an indicator dye that turns from violet to blue/light blue when the DNA amplification reaction is positive. Similar sensitivity to visualize a positive reaction was observed between HNB loop-mediated isothermal amplification and agarose gel electrophoresis analysis. The detection of C.Las-infected plants was up to 8 ng of total infected plant genomic DNA, similar to quantitative PCR. A blind validation test of the prototype kit was performed with purified DNA extracted from healthy or C.Las-infected midrib plants. Our kit showed 100% concordance with the results of a gold-standard quantitative PCR technique applied by the Laboratorio de Biología Molecular de EEA Montecarlo. The analysis of samples, without DNA purification to detect C.Las, showed a similar sensitivity to the analysis of the same samples in which C.Las DNA was previously purified.EEA MontecarloFil: Larocca, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencia y Tecnología Dr. Cesar Milstein. Fundación Pablo Cassará; ArgentinaFil: Werbajh, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencia y Tecnología Dr. Cesar Milstein. Fundación Pablo Cassará; ArgentinaFil: Parma, Yanil. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencia y Tecnología Dr. Cesar Milstein. Fundación Pablo Cassará; ArgentinaFil: Carrillo, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencia y Tecnología Dr. Cesar Milstein. Fundación Pablo Cassará; ArgentinaFil: Ogas, Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencia y Tecnología Dr. Cesar Milstein. Fundación Pablo Cassará; ArgentinaFil: Agostini, Juan P. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Montecarlo; ArgentinaFil: Redes, Jonathan. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Montecarlo; ArgentinaFil: Welin, Bjorn. Estación Experimental Agroindustrial Obispo Colombres; ArgentinaFil: Castagnaro, Atilio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Centro Científico Tecnológico Tucumán; ArgentinaFil: Vojnov, Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencia y Tecnología Dr. Cesar Milstein. Fundación Pablo Cassará; ArgentinaFrontiers Media2025-02-21T12:12:50Z2025-02-21T12:12:50Z2022-10-03info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfhttp://hdl.handle.net/20.500.12123/21386https://www.frontiersin.org/journals/agronomy/articles/10.3389/fagro.2022.984360/fullStolowicz F, Larocca L, Werbajh S, Parma Y, Carrillo C, Ogas L, Agostini JP, Redes J, Welin B, Castagnaro A and Vojnov A (2022) A colorimetric, sensitive, rapid, and simple diagnostic kit for the HLB putative causal agent detection. Front. Agron. 4:984360. doi: 10.3389/fagro.2022.9843602673-3218https://doi.org/10.3389/fagro.2022.984360Frontiers in Agronomy 4 : 984360. (October 2022)reponame:INTA Digital (INTA)instname:Instituto Nacional de Tecnología Agropecuariaenginfo:eu-repo/semantics/openAccesshttp://creativecommons.org/licenses/by-nc-sa/4.0/Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)2025-09-29T13:47:09Zoai:localhost:20.500.12123/21386instacron:INTAInstitucionalhttp://repositorio.inta.gob.ar/Organismo científico-tecnológicoNo correspondehttp://repositorio.inta.gob.ar/oai/requesttripaldi.nicolas@inta.gob.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:l2025-09-29 13:47:09.543INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuariafalse
dc.title.none.fl_str_mv A colorimetric, sensitive, rapid, and simple diagnostic kit for the HLB putative causal agent detection
title A colorimetric, sensitive, rapid, and simple diagnostic kit for the HLB putative causal agent detection
spellingShingle A colorimetric, sensitive, rapid, and simple diagnostic kit for the HLB putative causal agent detection
Stolowicz, Fabiana
Huanglongbing de los Cítricos
Enfermedades de las Plantas
Sanidad Vegetal
Huanglongbing
Plant Diseases
Plant Health
Citrus
Candidatus liberibacter
Prototipo Colorimétrico
Colorimetric Prototype
title_short A colorimetric, sensitive, rapid, and simple diagnostic kit for the HLB putative causal agent detection
title_full A colorimetric, sensitive, rapid, and simple diagnostic kit for the HLB putative causal agent detection
title_fullStr A colorimetric, sensitive, rapid, and simple diagnostic kit for the HLB putative causal agent detection
title_full_unstemmed A colorimetric, sensitive, rapid, and simple diagnostic kit for the HLB putative causal agent detection
title_sort A colorimetric, sensitive, rapid, and simple diagnostic kit for the HLB putative causal agent detection
dc.creator.none.fl_str_mv Stolowicz, Fabiana
Larocca, Luciana
Werbajh, Santiago
Parma, Yanil
Carrillo, Carolina
Ogas, Lorena
Agostini, Juan Pedro
Redes, Jonathan
Welin, Bjorn
Castagnaro, Atilio
Vojnov, Adrian
author Stolowicz, Fabiana
author_facet Stolowicz, Fabiana
Larocca, Luciana
Werbajh, Santiago
Parma, Yanil
Carrillo, Carolina
Ogas, Lorena
Agostini, Juan Pedro
Redes, Jonathan
Welin, Bjorn
Castagnaro, Atilio
Vojnov, Adrian
author_role author
author2 Larocca, Luciana
Werbajh, Santiago
Parma, Yanil
Carrillo, Carolina
Ogas, Lorena
Agostini, Juan Pedro
Redes, Jonathan
Welin, Bjorn
Castagnaro, Atilio
Vojnov, Adrian
author2_role author
author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Huanglongbing de los Cítricos
Enfermedades de las Plantas
Sanidad Vegetal
Huanglongbing
Plant Diseases
Plant Health
Citrus
Candidatus liberibacter
Prototipo Colorimétrico
Colorimetric Prototype
topic Huanglongbing de los Cítricos
Enfermedades de las Plantas
Sanidad Vegetal
Huanglongbing
Plant Diseases
Plant Health
Citrus
Candidatus liberibacter
Prototipo Colorimétrico
Colorimetric Prototype
dc.description.none.fl_txt_mv Huanglongbing (HLB) is one of the most devastating diseases in citrus worldwide. The Gram-negative bacterial plant pathogen “Candidatus Liberibacter spp.” is phloem-limited and vectored by citrus psyllids. The species “Candidatus Liberibacter asiaticus” (C.Las) has been detected in Argentina, and its vector has been found in at least nine provinces. Early detection of C.Las is critical for a successful management of HLB disease. Currently, HLB molecular diagnosis is carried out by PCR, nested PCR, real-time PCR, or another combination of these techniques, which require purification of genomic DNA, sophisticated equipment, and highly trained personnel. We have developed a prototype of a sensitive colorimetric kit to detect C.Las based on the specific DNA isothermal amplification of this microorganism. The reaction buffer contains hydroxynaphthol blue (HNB), an indicator dye that turns from violet to blue/light blue when the DNA amplification reaction is positive. Similar sensitivity to visualize a positive reaction was observed between HNB loop-mediated isothermal amplification and agarose gel electrophoresis analysis. The detection of C.Las-infected plants was up to 8 ng of total infected plant genomic DNA, similar to quantitative PCR. A blind validation test of the prototype kit was performed with purified DNA extracted from healthy or C.Las-infected midrib plants. Our kit showed 100% concordance with the results of a gold-standard quantitative PCR technique applied by the Laboratorio de Biología Molecular de EEA Montecarlo. The analysis of samples, without DNA purification to detect C.Las, showed a similar sensitivity to the analysis of the same samples in which C.Las DNA was previously purified.
EEA Montecarlo
Fil: Larocca, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencia y Tecnología Dr. Cesar Milstein. Fundación Pablo Cassará; Argentina
Fil: Werbajh, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencia y Tecnología Dr. Cesar Milstein. Fundación Pablo Cassará; Argentina
Fil: Parma, Yanil. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencia y Tecnología Dr. Cesar Milstein. Fundación Pablo Cassará; Argentina
Fil: Carrillo, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencia y Tecnología Dr. Cesar Milstein. Fundación Pablo Cassará; Argentina
Fil: Ogas, Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencia y Tecnología Dr. Cesar Milstein. Fundación Pablo Cassará; Argentina
Fil: Agostini, Juan P. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Montecarlo; Argentina
Fil: Redes, Jonathan. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Montecarlo; Argentina
Fil: Welin, Bjorn. Estación Experimental Agroindustrial Obispo Colombres; Argentina
Fil: Castagnaro, Atilio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Centro Científico Tecnológico Tucumán; Argentina
Fil: Vojnov, Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencia y Tecnología Dr. Cesar Milstein. Fundación Pablo Cassará; Argentina
description Huanglongbing (HLB) is one of the most devastating diseases in citrus worldwide. The Gram-negative bacterial plant pathogen “Candidatus Liberibacter spp.” is phloem-limited and vectored by citrus psyllids. The species “Candidatus Liberibacter asiaticus” (C.Las) has been detected in Argentina, and its vector has been found in at least nine provinces. Early detection of C.Las is critical for a successful management of HLB disease. Currently, HLB molecular diagnosis is carried out by PCR, nested PCR, real-time PCR, or another combination of these techniques, which require purification of genomic DNA, sophisticated equipment, and highly trained personnel. We have developed a prototype of a sensitive colorimetric kit to detect C.Las based on the specific DNA isothermal amplification of this microorganism. The reaction buffer contains hydroxynaphthol blue (HNB), an indicator dye that turns from violet to blue/light blue when the DNA amplification reaction is positive. Similar sensitivity to visualize a positive reaction was observed between HNB loop-mediated isothermal amplification and agarose gel electrophoresis analysis. The detection of C.Las-infected plants was up to 8 ng of total infected plant genomic DNA, similar to quantitative PCR. A blind validation test of the prototype kit was performed with purified DNA extracted from healthy or C.Las-infected midrib plants. Our kit showed 100% concordance with the results of a gold-standard quantitative PCR technique applied by the Laboratorio de Biología Molecular de EEA Montecarlo. The analysis of samples, without DNA purification to detect C.Las, showed a similar sensitivity to the analysis of the same samples in which C.Las DNA was previously purified.
publishDate 2022
dc.date.none.fl_str_mv 2022-10-03
2025-02-21T12:12:50Z
2025-02-21T12:12:50Z
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/20.500.12123/21386
https://www.frontiersin.org/journals/agronomy/articles/10.3389/fagro.2022.984360/full
Stolowicz F, Larocca L, Werbajh S, Parma Y, Carrillo C, Ogas L, Agostini JP, Redes J, Welin B, Castagnaro A and Vojnov A (2022) A colorimetric, sensitive, rapid, and simple diagnostic kit for the HLB putative causal agent detection. Front. Agron. 4:984360. doi: 10.3389/fagro.2022.984360
2673-3218
https://doi.org/10.3389/fagro.2022.984360
url http://hdl.handle.net/20.500.12123/21386
https://www.frontiersin.org/journals/agronomy/articles/10.3389/fagro.2022.984360/full
https://doi.org/10.3389/fagro.2022.984360
identifier_str_mv Stolowicz F, Larocca L, Werbajh S, Parma Y, Carrillo C, Ogas L, Agostini JP, Redes J, Welin B, Castagnaro A and Vojnov A (2022) A colorimetric, sensitive, rapid, and simple diagnostic kit for the HLB putative causal agent detection. Front. Agron. 4:984360. doi: 10.3389/fagro.2022.984360
2673-3218
dc.language.none.fl_str_mv eng
language eng
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
http://creativecommons.org/licenses/by-nc-sa/4.0/
Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)
eu_rights_str_mv openAccess
rights_invalid_str_mv http://creativecommons.org/licenses/by-nc-sa/4.0/
Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Frontiers Media
publisher.none.fl_str_mv Frontiers Media
dc.source.none.fl_str_mv Frontiers in Agronomy 4 : 984360. (October 2022)
reponame:INTA Digital (INTA)
instname:Instituto Nacional de Tecnología Agropecuaria
reponame_str INTA Digital (INTA)
collection INTA Digital (INTA)
instname_str Instituto Nacional de Tecnología Agropecuaria
repository.name.fl_str_mv INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuaria
repository.mail.fl_str_mv tripaldi.nicolas@inta.gob.ar
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