Optimization and validation of LAMP technique for the detection of leptospiral DNA

Autores
Hamer, Micaela; Saraullo, Vanina Rosa; Esteban, Micaela; Sanchez, Maria Cristina; Brihuega, Bibiana Felicitas; Martinez, Mara Leila
Año de publicación
2024
Idioma
inglés
Tipo de recurso
documento de conferencia
Estado
versión publicada
Descripción
Poster
Leptospira spp. is the second most important abortifacient pathogen in livestock pro duction in Argentina, responsible for the loss of millions of dollars in the beef and dairy industry in the Pampas region (Cantón et al., 2022). It is crucial to have diagnostic tools that allow simple and effective detection of the agent, in order to implement neces sary measures to prevent spread of the pathogen and mitigate economic and public health consequences. The aim of this study was to develop a method for detecting leptospiral DNA based on loop-mediated isothermal amplification (LAMP) technique, designed for application in low-complexity laboratories: Lepto-LAMP. Molecular target of Lepto-LAMP was rrs gene (koizumi et al., 2012). After optimization, Lepto-LAMP in corporated calcein as an indicator and presented high analytical sensitivity (10 pg of DNA per reaction, or 0.1; 1 and 10 leptospires/mL in bovine serum, urine, and kidney macerate, respectively). Lepto-LAMP specifically detected DNA from pathogenic and intermediate strains of Leptospira spp. In a trial using hamsters (Mesocricetus auratus) as a model of animal leptospirosis, Lepto-LAMP allowed for the detection of a greater number of positive samples in infected animals compared with end-point PCR lipL32. Both molecular diagnostic techniques showed substantial agreement according to Cohen’s kappa coefficient. Employing clinical samples (serum, urine, and organs) from domestic and wild animals, Lepto-LAMP demonstrated high diagnostic sensitivity (100%, 95% CI:93.6–100%) and high diagnostic specificity (96.6%, 95% CI: 95.2–97.7%). Lepto-LAMP had a lower implementation cost compared to the end-point PCR, mak ing it more accessible for low-complexity laboratories. The use of Lepto-LAMP for the diagnosis of animal leptospirosis has great potential to complement the microscopic agglutination test (MAT) as it can be implemented in most non-specialized laborato ries and provides a short time to obtain results (between 75–90 minutes from DNA extraction to final result reading).
Instituto de Patobiología
Fil: Hamer, Micaela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología Veterinaria. Laboratorio de Leptospirosis; Argentina
Fil: Hamer, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Saraullo, Vanina Rosa. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología Veterinaria. Laboratorio de Leptospirosis; Argentina
Fil: Saraullo, Vanina Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Esteban, Micaela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología Veterinaria. Laboratorio de Leptospirosis; Argentina
Fil: Esteban, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Sanchez, Maria Cristina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología Veterinaria. Laboratorio de Leptospirosis; Argentina
Fil: Sanchez, Maria Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Brihuega, Bibiana Felicitas. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología Veterinaria. Laboratorio de Leptospirosis; Argentina
Fil: Brihuega, Bibiana Felicitas. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Brihuega, Bibiana Felicitas. Universidad del Salvador. Facultad de Ciencias Agrarias y Veterinarias. Instituto de Investigación en Veterinaria; Argentina
Fil: Martinez, Mara Leila. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología Veterinaria. Laboratorio de Leptospirosis; Argentina
Fil: Martinez, Mara Leila. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Martinez, Mara Leila. Universidad del Salvador. Facultad de Ciencias Agrarias y Veterinarias. Instituto de Investigación en Veterinaria; Argentina
Fuente
The 13th Conference of the International Leptospirosis Society and The 4th Meeting of the European Leptospirosis and other Roden-Born Haemorrhagic fevers Society, Brussels, 2-4 September 2024
Materia
Diagnostic Techniques
Leptospirosis
DNA
Técnica de Diagnóstico
ADN
Mesocricetus auratus
PCR
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:20.500.12123/20486
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spelling Optimization and validation of LAMP technique for the detection of leptospiral DNAHamer, MicaelaSaraullo, Vanina RosaEsteban, MicaelaSanchez, Maria CristinaBrihuega, Bibiana FelicitasMartinez, Mara LeilaDiagnostic TechniquesLeptospirosisDNATécnica de DiagnósticoADNMesocricetus auratusPCRPosterLeptospira spp. is the second most important abortifacient pathogen in livestock pro duction in Argentina, responsible for the loss of millions of dollars in the beef and dairy industry in the Pampas region (Cantón et al., 2022). It is crucial to have diagnostic tools that allow simple and effective detection of the agent, in order to implement neces sary measures to prevent spread of the pathogen and mitigate economic and public health consequences. The aim of this study was to develop a method for detecting leptospiral DNA based on loop-mediated isothermal amplification (LAMP) technique, designed for application in low-complexity laboratories: Lepto-LAMP. Molecular target of Lepto-LAMP was rrs gene (koizumi et al., 2012). After optimization, Lepto-LAMP in corporated calcein as an indicator and presented high analytical sensitivity (10 pg of DNA per reaction, or 0.1; 1 and 10 leptospires/mL in bovine serum, urine, and kidney macerate, respectively). Lepto-LAMP specifically detected DNA from pathogenic and intermediate strains of Leptospira spp. In a trial using hamsters (Mesocricetus auratus) as a model of animal leptospirosis, Lepto-LAMP allowed for the detection of a greater number of positive samples in infected animals compared with end-point PCR lipL32. Both molecular diagnostic techniques showed substantial agreement according to Cohen’s kappa coefficient. Employing clinical samples (serum, urine, and organs) from domestic and wild animals, Lepto-LAMP demonstrated high diagnostic sensitivity (100%, 95% CI:93.6–100%) and high diagnostic specificity (96.6%, 95% CI: 95.2–97.7%). Lepto-LAMP had a lower implementation cost compared to the end-point PCR, mak ing it more accessible for low-complexity laboratories. The use of Lepto-LAMP for the diagnosis of animal leptospirosis has great potential to complement the microscopic agglutination test (MAT) as it can be implemented in most non-specialized laborato ries and provides a short time to obtain results (between 75–90 minutes from DNA extraction to final result reading).Instituto de PatobiologíaFil: Hamer, Micaela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología Veterinaria. Laboratorio de Leptospirosis; ArgentinaFil: Hamer, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Saraullo, Vanina Rosa. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología Veterinaria. Laboratorio de Leptospirosis; ArgentinaFil: Saraullo, Vanina Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Esteban, Micaela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología Veterinaria. Laboratorio de Leptospirosis; ArgentinaFil: Esteban, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sanchez, Maria Cristina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología Veterinaria. Laboratorio de Leptospirosis; ArgentinaFil: Sanchez, Maria Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Brihuega, Bibiana Felicitas. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología Veterinaria. Laboratorio de Leptospirosis; ArgentinaFil: Brihuega, Bibiana Felicitas. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Brihuega, Bibiana Felicitas. Universidad del Salvador. Facultad de Ciencias Agrarias y Veterinarias. Instituto de Investigación en Veterinaria; ArgentinaFil: Martinez, Mara Leila. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología Veterinaria. Laboratorio de Leptospirosis; ArgentinaFil: Martinez, Mara Leila. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Martinez, Mara Leila. Universidad del Salvador. Facultad de Ciencias Agrarias y Veterinarias. Instituto de Investigación en Veterinaria; ArgentinaInternational Leptospirosis Society2024-12-05T11:23:13Z2024-12-05T11:23:13Z2024-09info:eu-repo/semantics/conferenceObjectinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_5794info:ar-repo/semantics/documentoDeConferenciaapplication/pdfhttp://hdl.handle.net/20.500.12123/20486The 13th Conference of the International Leptospirosis Society and The 4th Meeting of the European Leptospirosis and other Roden-Born Haemorrhagic fevers Society, Brussels, 2-4 September 2024reponame: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)2025-09-29T13:47:00Zoai:localhost:20.500.12123/20486instacron: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 13:47:00.341INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuariafalse
dc.title.none.fl_str_mv Optimization and validation of LAMP technique for the detection of leptospiral DNA
title Optimization and validation of LAMP technique for the detection of leptospiral DNA
spellingShingle Optimization and validation of LAMP technique for the detection of leptospiral DNA
Hamer, Micaela
Diagnostic Techniques
Leptospirosis
DNA
Técnica de Diagnóstico
ADN
Mesocricetus auratus
PCR
title_short Optimization and validation of LAMP technique for the detection of leptospiral DNA
title_full Optimization and validation of LAMP technique for the detection of leptospiral DNA
title_fullStr Optimization and validation of LAMP technique for the detection of leptospiral DNA
title_full_unstemmed Optimization and validation of LAMP technique for the detection of leptospiral DNA
title_sort Optimization and validation of LAMP technique for the detection of leptospiral DNA
dc.creator.none.fl_str_mv Hamer, Micaela
Saraullo, Vanina Rosa
Esteban, Micaela
Sanchez, Maria Cristina
Brihuega, Bibiana Felicitas
Martinez, Mara Leila
author Hamer, Micaela
author_facet Hamer, Micaela
Saraullo, Vanina Rosa
Esteban, Micaela
Sanchez, Maria Cristina
Brihuega, Bibiana Felicitas
Martinez, Mara Leila
author_role author
author2 Saraullo, Vanina Rosa
Esteban, Micaela
Sanchez, Maria Cristina
Brihuega, Bibiana Felicitas
Martinez, Mara Leila
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv Diagnostic Techniques
Leptospirosis
DNA
Técnica de Diagnóstico
ADN
Mesocricetus auratus
PCR
topic Diagnostic Techniques
Leptospirosis
DNA
Técnica de Diagnóstico
ADN
Mesocricetus auratus
PCR
dc.description.none.fl_txt_mv Poster
Leptospira spp. is the second most important abortifacient pathogen in livestock pro duction in Argentina, responsible for the loss of millions of dollars in the beef and dairy industry in the Pampas region (Cantón et al., 2022). It is crucial to have diagnostic tools that allow simple and effective detection of the agent, in order to implement neces sary measures to prevent spread of the pathogen and mitigate economic and public health consequences. The aim of this study was to develop a method for detecting leptospiral DNA based on loop-mediated isothermal amplification (LAMP) technique, designed for application in low-complexity laboratories: Lepto-LAMP. Molecular target of Lepto-LAMP was rrs gene (koizumi et al., 2012). After optimization, Lepto-LAMP in corporated calcein as an indicator and presented high analytical sensitivity (10 pg of DNA per reaction, or 0.1; 1 and 10 leptospires/mL in bovine serum, urine, and kidney macerate, respectively). Lepto-LAMP specifically detected DNA from pathogenic and intermediate strains of Leptospira spp. In a trial using hamsters (Mesocricetus auratus) as a model of animal leptospirosis, Lepto-LAMP allowed for the detection of a greater number of positive samples in infected animals compared with end-point PCR lipL32. Both molecular diagnostic techniques showed substantial agreement according to Cohen’s kappa coefficient. Employing clinical samples (serum, urine, and organs) from domestic and wild animals, Lepto-LAMP demonstrated high diagnostic sensitivity (100%, 95% CI:93.6–100%) and high diagnostic specificity (96.6%, 95% CI: 95.2–97.7%). Lepto-LAMP had a lower implementation cost compared to the end-point PCR, mak ing it more accessible for low-complexity laboratories. The use of Lepto-LAMP for the diagnosis of animal leptospirosis has great potential to complement the microscopic agglutination test (MAT) as it can be implemented in most non-specialized laborato ries and provides a short time to obtain results (between 75–90 minutes from DNA extraction to final result reading).
Instituto de Patobiología
Fil: Hamer, Micaela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología Veterinaria. Laboratorio de Leptospirosis; Argentina
Fil: Hamer, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Saraullo, Vanina Rosa. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología Veterinaria. Laboratorio de Leptospirosis; Argentina
Fil: Saraullo, Vanina Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Esteban, Micaela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología Veterinaria. Laboratorio de Leptospirosis; Argentina
Fil: Esteban, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Sanchez, Maria Cristina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología Veterinaria. Laboratorio de Leptospirosis; Argentina
Fil: Sanchez, Maria Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Brihuega, Bibiana Felicitas. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología Veterinaria. Laboratorio de Leptospirosis; Argentina
Fil: Brihuega, Bibiana Felicitas. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Brihuega, Bibiana Felicitas. Universidad del Salvador. Facultad de Ciencias Agrarias y Veterinarias. Instituto de Investigación en Veterinaria; Argentina
Fil: Martinez, Mara Leila. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología Veterinaria. Laboratorio de Leptospirosis; Argentina
Fil: Martinez, Mara Leila. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Martinez, Mara Leila. Universidad del Salvador. Facultad de Ciencias Agrarias y Veterinarias. Instituto de Investigación en Veterinaria; Argentina
description Poster
publishDate 2024
dc.date.none.fl_str_mv 2024-12-05T11:23:13Z
2024-12-05T11:23:13Z
2024-09
dc.type.none.fl_str_mv info:eu-repo/semantics/conferenceObject
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_5794
info:ar-repo/semantics/documentoDeConferencia
format conferenceObject
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/20.500.12123/20486
url http://hdl.handle.net/20.500.12123/20486
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 International Leptospirosis Society
publisher.none.fl_str_mv International Leptospirosis Society
dc.source.none.fl_str_mv The 13th Conference of the International Leptospirosis Society and The 4th Meeting of the European Leptospirosis and other Roden-Born Haemorrhagic fevers Society, Brussels, 2-4 September 2024
reponame:INTA Digital (INTA)
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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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