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Título
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
Autor(es)
Palabras clave
Loop-mediated isothermal amplification
Schistosoma mansoni
Strongyloides
Point-of-care
Duplex LAMP assay
Multiplex LAMP
Clasificación UNESCO
24 Ciencias de la vida
3207.12 Parasitología
Fecha de publicación
2023-01-03
Editor
MDPI
Citación
Crego-Vicente, B., Fernández-Soto, P., García-Bernalt Diego, J., Febrer-Sendra, B., & Muro, A. (2023). 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. International Journal of Molecular Sciences, 24(1), 893.
Resumen
[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.
URI
ISSN
1661-6596
DOI
10.3390/ijms24010893
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