A colorimetric, sensitive, rapid, and simple diagnostic kit for the HLB putative causal agent detection
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 it...
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| Format: | info:ar-repo/semantics/artículo |
| Language: | Inglés |
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Frontiers Media
2025
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| Online Access: | 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 |
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| author | Stolowicz, Fabiana Larocca, Luciana Werbajh, Santiago Parma, Yanil Carrillo, Carolina Ogas, Lorena Agostini, Juan Pedro Redes, Jonathan Welin, Bjorn Castagnaro, Atilio Vojnov, Adrian |
| author_browse | Agostini, Juan Pedro Carrillo, Carolina Castagnaro, Atilio Larocca, Luciana Ogas, Lorena Parma, Yanil Redes, Jonathan Stolowicz, Fabiana Vojnov, Adrian Welin, Bjorn Werbajh, Santiago |
| 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_sort | Stolowicz, Fabiana |
| collection | INTA Digital |
| 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. |
| format | info:ar-repo/semantics/artículo |
| id | INTA21386 |
| institution | Instituto Nacional de Tecnología Agropecuaria (INTA -Argentina) |
| language | Inglés |
| publishDate | 2025 |
| publishDateRange | 2025 |
| publishDateSort | 2025 |
| publisher | Frontiers Media |
| publisherStr | Frontiers Media |
| record_format | dspace |
| spelling | INTA213862025-02-21T12:27:12Z A colorimetric, sensitive, rapid, and simple diagnostic kit for the HLB putative causal agent detection Stolowicz, Fabiana Larocca, Luciana Werbajh, Santiago Parma, Yanil Carrillo, Carolina Ogas, Lorena Agostini, Juan Pedro Redes, Jonathan Welin, Bjorn Castagnaro, Atilio Vojnov, Adrian Huanglongbing de los Cítricos Enfermedades de las Plantas Sanidad Vegetal Huanglongbing Plant Diseases Plant Health Citrus Candidatus liberibacter Prototipo Colorimétrico Colorimetric Prototype 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 2025-02-21T12:12:50Z 2025-02-21T12:12:50Z 2022-10-03 info:ar-repo/semantics/artículo info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion 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 eng 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) application/pdf Frontiers Media Frontiers in Agronomy 4 : 984360. (October 2022) |
| spellingShingle | Huanglongbing de los Cítricos Enfermedades de las Plantas Sanidad Vegetal Huanglongbing Plant Diseases Plant Health Citrus Candidatus liberibacter Prototipo Colorimétrico Colorimetric Prototype Stolowicz, Fabiana Larocca, Luciana Werbajh, Santiago Parma, Yanil Carrillo, Carolina Ogas, Lorena Agostini, Juan Pedro Redes, Jonathan Welin, Bjorn Castagnaro, Atilio Vojnov, Adrian 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 |
| 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_short | A colorimetric, sensitive, rapid, and simple diagnostic kit for the HLB putative causal agent detection |
| title_sort | colorimetric sensitive rapid and simple diagnostic kit for the hlb putative causal agent detection |
| topic | Huanglongbing de los Cítricos Enfermedades de las Plantas Sanidad Vegetal Huanglongbing Plant Diseases Plant Health Citrus Candidatus liberibacter Prototipo Colorimétrico Colorimetric Prototype |
| 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 |
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