Development of a Duplex LAMP Assay with Probe-Based Readout for Simultaneous Real-Time Detection of Schistosoma mansoni and Strongyloides spp. -A Laboratory Approach to Point-Of-Care
[ENG] isothermal amplification (LAMP) is the most popular technology for point-of-care testing applications due its rapid, sensitive and specific detection with simple instrumentation compared to PCR-based methods. Many systems for reading the results of LAMP amplifications exist, including real-tim...
| Autores: | , , , , |
|---|---|
| Tipo de recurso: | artículo |
| Estado: | Versión publicada |
| Fecha de publicación: | 2023 |
| País: | España |
| Institución: | Universidad de Salamanca (USAL) |
| Repositorio: | GREDOS. Repositorio Institucional de la Universidad de Salamanca |
| OAI Identifier: | oai:gredos.usal.es:10366/154618 |
| Acceso en línea: | http://hdl.handle.net/10366/154618 |
| Access Level: | acceso abierto |
| Palabra clave: | Loop-mediated isothermal amplification Schistosoma mansoni Strongyloides Point-of-care Duplex LAMP assay Multiplex LAMP Schistosomiasis mansoni Point-of-Care Systems Molecular Diagnostic Techniques 24 Ciencias de la vida 3207.12 Parasitología esquistosomiasis intestinal técnicas de diagnóstico molecular sistemas de atención en la cabecera del paciente |
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Development of a Duplex LAMP Assay with Probe-Based Readout for Simultaneous Real-Time Detection of Schistosoma mansoni and Strongyloides spp. -A Laboratory Approach to Point-Of-CareCrego Vicente, BeatrizFernández Soto, PedroGarcía-Bernalt Diego, JuanFebrer Sendra, BegoñaMuro Álvarez, AntonioLoop-mediated isothermal amplificationSchistosoma mansoniStrongyloidesPoint-of-careDuplex LAMP assayMultiplex LAMPSchistosomiasis mansoniPoint-of-Care SystemsSchistosoma mansoniMolecular Diagnostic TechniquesStrongyloides24 Ciencias de la vida3207.12 ParasitologíaSchistosoma mansoniStrongyloidesesquistosomiasis intestinaltécnicas de diagnóstico molecularsistemas de atención en la cabecera del paciente[ENG] isothermal amplification (LAMP) is the most popular technology for point-of-care testing applications due its rapid, sensitive and specific detection with simple instrumentation compared to PCR-based methods. Many systems for reading the results of LAMP amplifications exist, including real-time fluorescence detection using fluorophore-labelled probes attached to oligonucleotide sequences complementary to the target nucleic acid. This methodology allows the simultaneous detection of multiple targets (multiplexing) in one LAMP assay. A method for multiplexing LAMP is the amplification by release of quenching (DARQ) technique by using a 5′-quencher modified LAMP primer annealed to 3′-fluorophore-labelled acting as detection oligonucleotide. The main application of multiplex LAMP is the rapid and accurate diagnosis of infectious diseases, allowing differentiation of co-infecting pathogens in a single reaction. Schistosomiasis, caused among other species by Schistosoma mansoni and strongyloidiasis, caused by Strongyloides stercoralis, are the most common helminth-parasite infections worldwide with overlapping distribution areas and high possibility of coinfections in the human population. It would be of great interest to develop a duplex LAMP to detect both pathogens in the same reaction. In this study, we investigate the use of our two previously developed and well-stablished LAMP assays for S. mansoni and Strongyloides spp. DNA detection in a new duplex real-time eight-primer system based on a modified DARQ probe method that can be performed in a portable isothermal fluorimeter with minimal laboratory resources. We also applied a strategy to stabilize the duplexed DARQ-LAMP mixtures at room temperature for use as ready-to-use formats facilitating analysis in field settings as point-of-care diagnostics for schistosomiasis and strongyloidiasis.MDPI202420242023info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionapplication/pdfhttp://hdl.handle.net/10366/154618reponame:GREDOS. Repositorio Institucional de la Universidad de Salamancainstname:Universidad de Salamanca (USAL)InglésThis work was funded by the Institute of Health Carlos III, ISCIII, through the project PI19/01727 and cofunded by the European UnionCC0 1.0 Universalhttp://creativecommons.org/publicdomain/zero/1.0/info:eu-repo/semantics/openAccessoai:gredos.usal.es:10366/1546182026-06-07T06:28:51Z |
| dc.title.none.fl_str_mv |
Development of a Duplex LAMP Assay with Probe-Based Readout for Simultaneous Real-Time Detection of Schistosoma mansoni and Strongyloides spp. -A Laboratory Approach to Point-Of-Care |
| title |
Development of a Duplex LAMP Assay with Probe-Based Readout for Simultaneous Real-Time Detection of Schistosoma mansoni and Strongyloides spp. -A Laboratory Approach to Point-Of-Care |
| spellingShingle |
Development of a Duplex LAMP Assay with Probe-Based Readout for Simultaneous Real-Time Detection of Schistosoma mansoni and Strongyloides spp. -A Laboratory Approach to Point-Of-Care Crego Vicente, Beatriz Loop-mediated isothermal amplification Schistosoma mansoni Strongyloides Point-of-care Duplex LAMP assay Multiplex LAMP Schistosomiasis mansoni Point-of-Care Systems Schistosoma mansoni Molecular Diagnostic Techniques Strongyloides 24 Ciencias de la vida 3207.12 Parasitología Schistosoma mansoni Strongyloides esquistosomiasis intestinal técnicas de diagnóstico molecular sistemas de atención en la cabecera del paciente |
| title_short |
Development of a Duplex LAMP Assay with Probe-Based Readout for Simultaneous Real-Time Detection of Schistosoma mansoni and Strongyloides spp. -A Laboratory Approach to Point-Of-Care |
| title_full |
Development of a Duplex LAMP Assay with Probe-Based Readout for Simultaneous Real-Time Detection of Schistosoma mansoni and Strongyloides spp. -A Laboratory Approach to Point-Of-Care |
| title_fullStr |
Development of a Duplex LAMP Assay with Probe-Based Readout for Simultaneous Real-Time Detection of Schistosoma mansoni and Strongyloides spp. -A Laboratory Approach to Point-Of-Care |
| title_full_unstemmed |
Development of a Duplex LAMP Assay with Probe-Based Readout for Simultaneous Real-Time Detection of Schistosoma mansoni and Strongyloides spp. -A Laboratory Approach to Point-Of-Care |
| title_sort |
Development of a Duplex LAMP Assay with Probe-Based Readout for Simultaneous Real-Time Detection of Schistosoma mansoni and Strongyloides spp. -A Laboratory Approach to Point-Of-Care |
| dc.creator.none.fl_str_mv |
Crego Vicente, Beatriz Fernández Soto, Pedro García-Bernalt Diego, Juan Febrer Sendra, Begoña Muro Álvarez, Antonio |
| author |
Crego Vicente, Beatriz |
| author_facet |
Crego Vicente, Beatriz Fernández Soto, Pedro García-Bernalt Diego, Juan Febrer Sendra, Begoña Muro Álvarez, Antonio |
| author_role |
author |
| author2 |
Fernández Soto, Pedro García-Bernalt Diego, Juan Febrer Sendra, Begoña Muro Álvarez, Antonio |
| author2_role |
author author author author |
| dc.subject.none.fl_str_mv |
Loop-mediated isothermal amplification Schistosoma mansoni Strongyloides Point-of-care Duplex LAMP assay Multiplex LAMP Schistosomiasis mansoni Point-of-Care Systems Schistosoma mansoni Molecular Diagnostic Techniques Strongyloides 24 Ciencias de la vida 3207.12 Parasitología Schistosoma mansoni Strongyloides esquistosomiasis intestinal técnicas de diagnóstico molecular sistemas de atención en la cabecera del paciente |
| topic |
Loop-mediated isothermal amplification Schistosoma mansoni Strongyloides Point-of-care Duplex LAMP assay Multiplex LAMP Schistosomiasis mansoni Point-of-Care Systems Schistosoma mansoni Molecular Diagnostic Techniques Strongyloides 24 Ciencias de la vida 3207.12 Parasitología Schistosoma mansoni Strongyloides esquistosomiasis intestinal técnicas de diagnóstico molecular sistemas de atención en la cabecera del paciente |
| description |
[ENG] isothermal amplification (LAMP) is the most popular technology for point-of-care testing applications due its rapid, sensitive and specific detection with simple instrumentation compared to PCR-based methods. Many systems for reading the results of LAMP amplifications exist, including real-time fluorescence detection using fluorophore-labelled probes attached to oligonucleotide sequences complementary to the target nucleic acid. This methodology allows the simultaneous detection of multiple targets (multiplexing) in one LAMP assay. A method for multiplexing LAMP is the amplification by release of quenching (DARQ) technique by using a 5′-quencher modified LAMP primer annealed to 3′-fluorophore-labelled acting as detection oligonucleotide. The main application of multiplex LAMP is the rapid and accurate diagnosis of infectious diseases, allowing differentiation of co-infecting pathogens in a single reaction. Schistosomiasis, caused among other species by Schistosoma mansoni and strongyloidiasis, caused by Strongyloides stercoralis, are the most common helminth-parasite infections worldwide with overlapping distribution areas and high possibility of coinfections in the human population. It would be of great interest to develop a duplex LAMP to detect both pathogens in the same reaction. In this study, we investigate the use of our two previously developed and well-stablished LAMP assays for S. mansoni and Strongyloides spp. DNA detection in a new duplex real-time eight-primer system based on a modified DARQ probe method that can be performed in a portable isothermal fluorimeter with minimal laboratory resources. We also applied a strategy to stabilize the duplexed DARQ-LAMP mixtures at room temperature for use as ready-to-use formats facilitating analysis in field settings as point-of-care diagnostics for schistosomiasis and strongyloidiasis. |
| publishDate |
2023 |
| dc.date.none.fl_str_mv |
2023 2024 2024 |
| dc.type.none.fl_str_mv |
info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion |
| format |
article |
| status_str |
publishedVersion |
| dc.identifier.none.fl_str_mv |
http://hdl.handle.net/10366/154618 |
| url |
http://hdl.handle.net/10366/154618 |
| dc.language.none.fl_str_mv |
Inglés |
| language_invalid_str_mv |
Inglés |
| dc.relation.none.fl_str_mv |
This work was funded by the Institute of Health Carlos III, ISCIII, through the project PI19/01727 and cofunded by the European Union |
| dc.rights.none.fl_str_mv |
CC0 1.0 Universal http://creativecommons.org/publicdomain/zero/1.0/ info:eu-repo/semantics/openAccess |
| rights_invalid_str_mv |
CC0 1.0 Universal http://creativecommons.org/publicdomain/zero/1.0/ |
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openAccess |
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application/pdf |
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MDPI |
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MDPI |
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reponame:GREDOS. Repositorio Institucional de la Universidad de Salamanca instname:Universidad de Salamanca (USAL) |
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Universidad de Salamanca (USAL) |
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GREDOS. Repositorio Institucional de la Universidad de Salamanca |
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GREDOS. Repositorio Institucional de la Universidad de Salamanca |
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