Real-time on-site detection of the three ‘Candidatus Liberibacter’ species associated with HLB disease: a rapid and validated method
Huanglongbing (HLB) is a devastating disease that affects all commercial citrus species worldwide. The disease is associated with bacteria of three species of the genus ‘Candidatus Liberibacter’ transmitted by psyllid vectors. To date, HLB has no cure, so preventing its introduction into HLB-free ar...
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| Format: | Artículo |
| Language: | Inglés |
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Frontiers
2023
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| Online Access: | https://hdl.handle.net/20.500.11939/8644 https://www.frontiersin.org/articles/10.3389/fpls.2023.1176513/full |
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| author | Morán, Félix Herrero-Cervera, Mario Carvajal-Rojas, Sofía Marco-Noales, Ester |
| author_browse | Carvajal-Rojas, Sofía Herrero-Cervera, Mario Marco-Noales, Ester Morán, Félix |
| author_facet | Morán, Félix Herrero-Cervera, Mario Carvajal-Rojas, Sofía Marco-Noales, Ester |
| author_sort | Morán, Félix |
| collection | ReDivia |
| description | Huanglongbing (HLB) is a devastating disease that affects all commercial citrus species worldwide. The disease is associated with bacteria of three species of the genus ‘Candidatus Liberibacter’ transmitted by psyllid vectors. To date, HLB has no cure, so preventing its introduction into HLB-free areas is the best strategy to control its spread. For that, the use of accurate, sensitive, specific, and reliable detection methods is critical for good integrated management of this serious disease. This study presents a new real-time recombinase polymerase amplification (RPA) protocol able to detect the three ‘Ca. Liberibacter’ species associated with HLB in both plant and insect samples, validated according to European and Mediterranean Plant Protection Organization (EPPO) guidelines and tested on 365 samples from nine different geographic origins. This new protocol does not require nucleic acid purification or specialized equipment, making it ideal to be used under field conditions. It is based on specific primers and probe targeting a region of fusA gene, which shows a specificity of 94%– 100%, both in silico and in vitro, for the ‘Ca. Liberibacter’ species associated with HLB. The analytical sensitivity of the new protocol is excellent, with a reliable detection limit in the order of 101 copies per microliter in HLB-infected plant and insect material. The repeatability and reproducibility of the new methods showed consistent results. Diagnostic parameters of the new RPA protocol were calculated and compared with the gold standard technique, a quantitative real-time PCR, in both crude extracts of citrus plants and insect vectors. The agreement between the two techniques was almost perfect according to the estimated Cohen’s kappa index, with a diagnostic sensitivity and specificity of 83.89% and 100%, respectively, and a relative accuracy of 91.59%. Moreover, the results are obtained in less than 35 min. All these results indicate the potential of this new RPA protocol to be implemented as a reliable on-site detection kit for HLB due to its simplicity, speed, and portability. |
| format | Artículo |
| id | ReDivia8644 |
| institution | Instituto Valenciano de Investigaciones Agrarias (IVIA) |
| language | Inglés |
| publishDate | 2023 |
| publishDateRange | 2023 |
| publishDateSort | 2023 |
| publisher | Frontiers |
| publisherStr | Frontiers |
| record_format | dspace |
| spelling | ReDivia86442025-04-25T14:49:12Z Real-time on-site detection of the three ‘Candidatus Liberibacter’ species associated with HLB disease: a rapid and validated method Morán, Félix Herrero-Cervera, Mario Carvajal-Rojas, Sofía Marco-Noales, Ester Huanglongbing rapid-screening test in situ KIT RPA H20 Plant diseases Pathogenic bacteria Liberibacter Phytopathology Huanglongbing (HLB) is a devastating disease that affects all commercial citrus species worldwide. The disease is associated with bacteria of three species of the genus ‘Candidatus Liberibacter’ transmitted by psyllid vectors. To date, HLB has no cure, so preventing its introduction into HLB-free areas is the best strategy to control its spread. For that, the use of accurate, sensitive, specific, and reliable detection methods is critical for good integrated management of this serious disease. This study presents a new real-time recombinase polymerase amplification (RPA) protocol able to detect the three ‘Ca. Liberibacter’ species associated with HLB in both plant and insect samples, validated according to European and Mediterranean Plant Protection Organization (EPPO) guidelines and tested on 365 samples from nine different geographic origins. This new protocol does not require nucleic acid purification or specialized equipment, making it ideal to be used under field conditions. It is based on specific primers and probe targeting a region of fusA gene, which shows a specificity of 94%– 100%, both in silico and in vitro, for the ‘Ca. Liberibacter’ species associated with HLB. The analytical sensitivity of the new protocol is excellent, with a reliable detection limit in the order of 101 copies per microliter in HLB-infected plant and insect material. The repeatability and reproducibility of the new methods showed consistent results. Diagnostic parameters of the new RPA protocol were calculated and compared with the gold standard technique, a quantitative real-time PCR, in both crude extracts of citrus plants and insect vectors. The agreement between the two techniques was almost perfect according to the estimated Cohen’s kappa index, with a diagnostic sensitivity and specificity of 83.89% and 100%, respectively, and a relative accuracy of 91.59%. Moreover, the results are obtained in less than 35 min. All these results indicate the potential of this new RPA protocol to be implemented as a reliable on-site detection kit for HLB due to its simplicity, speed, and portability. 2023-06-09T07:08:58Z 2023-06-09T07:08:58Z 2023 article PublishedVersion Morán, F., Herrero-Cervera, M., Carvajal-Rojas, S., & Marco-Noales, E. Real-Time On-site Detection of the threeCandidatus Liberibacter´ Species associated with HLB disease: A Rapid and Validated Method. Frontiers in Plant Science, 14, 1772. 1664-462X https://hdl.handle.net/20.500.11939/8644 10.3389/fpls.2023.1176513 https://www.frontiersin.org/articles/10.3389/fpls.2023.1176513/full en LIFE18 CCA/ES/001109 (Development of sustainable control strategies for citric under threat of climate change AND preventing entry of HLB in EU-LIFE Vida for Citrus-), PID2021-124145ORC21 Breeding, selection and evaluation of new citrus materials for more sustainable plantations in the face of emerging threats due to global change— FORTECITRUS), and IVIA-GVA 52202D (SOSTENIBLE) from Instituto Valenciano de Investigaciones Agrarias (project susceptible of being co-financed by the European Union through the ERDF Program 2021-2027 Comunitat Valenciana) info:eu-repo/grantAgreement/AEI/Programa Estatal de Generación de Conocimiento y Fortalecimiento Científico y Tecnológico del Sistema de I+D+i/PID2021-124145OR-C21/ES/Obtención, selección y evaluación de cítricos para conseguir plantaciones más sostenibles frente a las nuevas amenazas debidas al cambio global info:eu-repo/grantAgreement/ERDF/PCV 2021-2027/52202/ES/Control biológico de plagas y enfermedades como pieza clave en el impulso de la Sostenibilidad de los cultivos agrarios de la Comunidad Valenciana/SOSTENIBLE info:eu-repo/grantAgreement/EC/LIFE 18/001109/ES/Development of sustainable control strategies for citric under threat of climate change & preventing entry of HLB in EU/Life Vida for citrus Attribution-NonCommercial-NoDerivatives 4.0 Internacional http://creativecommons.org/licenses/by-nc-nd/4.0/ openAccess Frontiers electronico |
| spellingShingle | Huanglongbing rapid-screening test in situ KIT RPA H20 Plant diseases Pathogenic bacteria Liberibacter Phytopathology Morán, Félix Herrero-Cervera, Mario Carvajal-Rojas, Sofía Marco-Noales, Ester Real-time on-site detection of the three ‘Candidatus Liberibacter’ species associated with HLB disease: a rapid and validated method |
| title | Real-time on-site detection of the three ‘Candidatus Liberibacter’ species associated with HLB disease: a rapid and validated method |
| title_full | Real-time on-site detection of the three ‘Candidatus Liberibacter’ species associated with HLB disease: a rapid and validated method |
| title_fullStr | Real-time on-site detection of the three ‘Candidatus Liberibacter’ species associated with HLB disease: a rapid and validated method |
| title_full_unstemmed | Real-time on-site detection of the three ‘Candidatus Liberibacter’ species associated with HLB disease: a rapid and validated method |
| title_short | Real-time on-site detection of the three ‘Candidatus Liberibacter’ species associated with HLB disease: a rapid and validated method |
| title_sort | real time on site detection of the three candidatus liberibacter species associated with hlb disease a rapid and validated method |
| topic | Huanglongbing rapid-screening test in situ KIT RPA H20 Plant diseases Pathogenic bacteria Liberibacter Phytopathology |
| url | https://hdl.handle.net/20.500.11939/8644 https://www.frontiersin.org/articles/10.3389/fpls.2023.1176513/full |
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