Development of a Duplex LAMP Assay with Probe-Based Readout for Simultaneous Real-Time Detection of <i>Schistosoma mansoni</i> and <i>Strongyloides</i> spp. -A Laboratory Approach to Point-Of-Care
Loop-mediated 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...
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MDPI AG
2023-01-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/1/893 |
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| author | Beatriz Crego-Vicente Pedro Fernández-Soto Juan García-Bernalt Diego Begoña Febrer-Sendra Antonio Muro |
| author_facet | Beatriz Crego-Vicente Pedro Fernández-Soto Juan García-Bernalt Diego Begoña Febrer-Sendra Antonio Muro |
| author_sort | Beatriz Crego-Vicente |
| collection | DOAJ |
| description | Loop-mediated 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 <i>Schistosoma mansoni</i> and strongyloidiasis, caused by <i>Strongyloides stercoralis</i>, 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 <i>S. mansoni</i> and <i>Strongyloides</i> 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. |
| first_indexed | 2024-03-11T09:58:10Z |
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| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-11T09:58:10Z |
| publishDate | 2023-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-b5d236d3e892476dbb6e6fed876b1bfb2023-11-16T15:39:58ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-01-0124189310.3390/ijms24010893Development of a Duplex LAMP Assay with Probe-Based Readout for Simultaneous Real-Time Detection of <i>Schistosoma mansoni</i> and <i>Strongyloides</i> spp. -A Laboratory Approach to Point-Of-CareBeatriz Crego-Vicente0Pedro Fernández-Soto1Juan García-Bernalt Diego2Begoña Febrer-Sendra3Antonio Muro4Infectious and Tropical Diseases Research Group (e-INTRO), Biomedical Research Institute of Salamanca Research Centre for Tropical Diseases at the University of Salamanca (IBSAL-CIETUS), Faculty of Pharmacy, University of Salamanca, 37007 Salamanca, SpainInfectious and Tropical Diseases Research Group (e-INTRO), Biomedical Research Institute of Salamanca Research Centre for Tropical Diseases at the University of Salamanca (IBSAL-CIETUS), Faculty of Pharmacy, University of Salamanca, 37007 Salamanca, SpainInfectious and Tropical Diseases Research Group (e-INTRO), Biomedical Research Institute of Salamanca Research Centre for Tropical Diseases at the University of Salamanca (IBSAL-CIETUS), Faculty of Pharmacy, University of Salamanca, 37007 Salamanca, SpainInfectious and Tropical Diseases Research Group (e-INTRO), Biomedical Research Institute of Salamanca Research Centre for Tropical Diseases at the University of Salamanca (IBSAL-CIETUS), Faculty of Pharmacy, University of Salamanca, 37007 Salamanca, SpainInfectious and Tropical Diseases Research Group (e-INTRO), Biomedical Research Institute of Salamanca Research Centre for Tropical Diseases at the University of Salamanca (IBSAL-CIETUS), Faculty of Pharmacy, University of Salamanca, 37007 Salamanca, SpainLoop-mediated 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 <i>Schistosoma mansoni</i> and strongyloidiasis, caused by <i>Strongyloides stercoralis</i>, 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 <i>S. mansoni</i> and <i>Strongyloides</i> 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.https://www.mdpi.com/1422-0067/24/1/893LAMPmultiplex LAMPduplex LAMPDARQ-LAMP<i>Schistosoma mansoni</i><i>Strongyloides</i> spp. |
| spellingShingle | Beatriz Crego-Vicente Pedro Fernández-Soto Juan García-Bernalt Diego Begoña Febrer-Sendra Antonio Muro Development of a Duplex LAMP Assay with Probe-Based Readout for Simultaneous Real-Time Detection of <i>Schistosoma mansoni</i> and <i>Strongyloides</i> spp. -A Laboratory Approach to Point-Of-Care International Journal of Molecular Sciences LAMP multiplex LAMP duplex LAMP DARQ-LAMP <i>Schistosoma mansoni</i> <i>Strongyloides</i> spp. |
| title | Development of a Duplex LAMP Assay with Probe-Based Readout for Simultaneous Real-Time Detection of <i>Schistosoma mansoni</i> and <i>Strongyloides</i> 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 <i>Schistosoma mansoni</i> and <i>Strongyloides</i> 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 <i>Schistosoma mansoni</i> and <i>Strongyloides</i> 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 <i>Schistosoma mansoni</i> and <i>Strongyloides</i> spp. -A Laboratory Approach to Point-Of-Care |
| title_short | Development of a Duplex LAMP Assay with Probe-Based Readout for Simultaneous Real-Time Detection of <i>Schistosoma mansoni</i> and <i>Strongyloides</i> 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 i schistosoma mansoni i and i strongyloides i spp a laboratory approach to point of care |
| topic | LAMP multiplex LAMP duplex LAMP DARQ-LAMP <i>Schistosoma mansoni</i> <i>Strongyloides</i> spp. |
| url | https://www.mdpi.com/1422-0067/24/1/893 |
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