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

Autores
Stolowicz, Fabiana Gisela; Larocca, Luciana; Werbajh, Santiago Enrique; Parma, Yanil Renee; Carrillo, Carolina; Ogas Castells, Maria Lorena; Agostini, Juan Pedro; Redes, Jonathan; Welin, Björn; Castagnaro, Atilio Pedro; Vojnov, Adrián Alberto
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.
Fil: Stolowicz, Fabiana Gisela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; Argentina
Fil: Larocca, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; Argentina
Fil: Werbajh, Santiago Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; Argentina
Fil: Parma, Yanil Renee. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; Argentina
Fil: Carrillo, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; Argentina
Fil: Ogas Castells, Maria Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; Argentina
Fil: Agostini, Juan Pedro. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Misiones. Estación Experimental Agropecuaria Montecarlo; Argentina
Fil: Redes, Jonathan. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Misiones. Estación Experimental Agropecuaria Montecarlo; Argentina
Fil: Welin, Björn. Gobierno de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial Obispo Colombres; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Castagnaro, Atilio Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial Obispo Colombres; Argentina
Fil: Vojnov, Adrián Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; Argentina
Materia
CANDIDATUS LIBERIBACTER ASIATICUS
CITRUS
DIAGNOSIS
HIDROXYNAPHTOL BLUE
HUANGLONGBING
LAMP
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/208735
Acceder
id CONICETDig_f181215805acba47067b3a6b71e06222
oai_identifier_str oai:ri.conicet.gov.ar:11336/208735
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling A colorimetric, sensitive, rapid, and simple diagnostic kit for the HLB putative causal agent detectionStolowicz, Fabiana GiselaLarocca, LucianaWerbajh, Santiago EnriqueParma, Yanil ReneeCarrillo, CarolinaOgas Castells, Maria LorenaAgostini, Juan PedroRedes, JonathanWelin, BjörnCastagnaro, Atilio PedroVojnov, Adrián AlbertoCANDIDATUS LIBERIBACTER ASIATICUSCITRUSDIAGNOSISHIDROXYNAPHTOL BLUEHUANGLONGBINGLAMPhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Huanglongbing (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.Fil: Stolowicz, Fabiana Gisela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFil: Larocca, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFil: Werbajh, Santiago Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFil: Parma, Yanil Renee. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFil: Carrillo, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFil: Ogas Castells, Maria Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFil: Agostini, Juan Pedro. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Misiones. Estación Experimental Agropecuaria Montecarlo; ArgentinaFil: Redes, Jonathan. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Misiones. Estación Experimental Agropecuaria Montecarlo; ArgentinaFil: Welin, Björn. Gobierno de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial Obispo Colombres; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Castagnaro, Atilio Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial Obispo Colombres; ArgentinaFil: Vojnov, Adrián Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFrontiers Media2022-10info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/208735Stolowicz, Fabiana Gisela; Larocca, Luciana; Werbajh, Santiago Enrique; Parma, Yanil Renee; Carrillo, Carolina; et al.; A colorimetric, sensitive, rapid, and simple diagnostic kit for the HLB putative causal agent detection; Frontiers Media; Frontiers in Agronomy; 4; 2; 10-2022; 1-112673-3218CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.frontiersin.org/articles/10.3389/fagro.2022.984360/fullinfo:eu-repo/semantics/altIdentifier/doi/10.3389/fagro.2022.984360info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-17T11:10:14Zoai:ri.conicet.gov.ar:11336/208735instacron: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:34982025-09-17 11:10:14.35CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
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 Gisela
CANDIDATUS LIBERIBACTER ASIATICUS
CITRUS
DIAGNOSIS
HIDROXYNAPHTOL BLUE
HUANGLONGBING
LAMP
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 Gisela
Larocca, Luciana
Werbajh, Santiago Enrique
Parma, Yanil Renee
Carrillo, Carolina
Ogas Castells, Maria Lorena
Agostini, Juan Pedro
Redes, Jonathan
Welin, Björn
Castagnaro, Atilio Pedro
Vojnov, Adrián Alberto
author Stolowicz, Fabiana Gisela
author_facet Stolowicz, Fabiana Gisela
Larocca, Luciana
Werbajh, Santiago Enrique
Parma, Yanil Renee
Carrillo, Carolina
Ogas Castells, Maria Lorena
Agostini, Juan Pedro
Redes, Jonathan
Welin, Björn
Castagnaro, Atilio Pedro
Vojnov, Adrián Alberto
author_role author
author2 Larocca, Luciana
Werbajh, Santiago Enrique
Parma, Yanil Renee
Carrillo, Carolina
Ogas Castells, Maria Lorena
Agostini, Juan Pedro
Redes, Jonathan
Welin, Björn
Castagnaro, Atilio Pedro
Vojnov, Adrián Alberto
author2_role author
author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv CANDIDATUS LIBERIBACTER ASIATICUS
CITRUS
DIAGNOSIS
HIDROXYNAPHTOL BLUE
HUANGLONGBING
LAMP
topic CANDIDATUS LIBERIBACTER ASIATICUS
CITRUS
DIAGNOSIS
HIDROXYNAPHTOL BLUE
HUANGLONGBING
LAMP
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
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.
Fil: Stolowicz, Fabiana Gisela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; Argentina
Fil: Larocca, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; Argentina
Fil: Werbajh, Santiago Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; Argentina
Fil: Parma, Yanil Renee. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; Argentina
Fil: Carrillo, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; Argentina
Fil: Ogas Castells, Maria Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; Argentina
Fil: Agostini, Juan Pedro. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Misiones. Estación Experimental Agropecuaria Montecarlo; Argentina
Fil: Redes, Jonathan. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Misiones. Estación Experimental Agropecuaria Montecarlo; Argentina
Fil: Welin, Björn. Gobierno de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial Obispo Colombres; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Castagnaro, Atilio Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial Obispo Colombres; Argentina
Fil: Vojnov, Adrián Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; 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
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/11336/208735
Stolowicz, Fabiana Gisela; Larocca, Luciana; Werbajh, Santiago Enrique; Parma, Yanil Renee; Carrillo, Carolina; et al.; A colorimetric, sensitive, rapid, and simple diagnostic kit for the HLB putative causal agent detection; Frontiers Media; Frontiers in Agronomy; 4; 2; 10-2022; 1-11
2673-3218
CONICET Digital
CONICET
url http://hdl.handle.net/11336/208735
identifier_str_mv Stolowicz, Fabiana Gisela; Larocca, Luciana; Werbajh, Santiago Enrique; Parma, Yanil Renee; Carrillo, Carolina; et al.; A colorimetric, sensitive, rapid, and simple diagnostic kit for the HLB putative causal agent detection; Frontiers Media; Frontiers in Agronomy; 4; 2; 10-2022; 1-11
2673-3218
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://www.frontiersin.org/articles/10.3389/fagro.2022.984360/full
info:eu-repo/semantics/altIdentifier/doi/10.3389/fagro.2022.984360
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
application/pdf
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dc.publisher.none.fl_str_mv Frontiers Media
publisher.none.fl_str_mv Frontiers Media
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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