Development of real-time and lateral flow recombinase polymerase amplification assays for rapid detection of Schistosoma mansoni

dc.creatorMesquita, Silvia Gonçalves
dc.creatorLugli, Elena Birgitta
dc.creatorMatera, Giovanni
dc.creatorFonseca, Cristina Toscano
dc.creatorCaldeira, Roberta Lima
dc.creatorWebster, Bonnie
dc.date2023-01-20T16:48:55Z
dc.date2023-01-20T16:48:55Z
dc.date2022
dc.date.accessioned2023-09-26T21:02:30Z
dc.date.available2023-09-26T21:02:30Z
dc.identifierMESQUITA, Silvia Gonçalves et al. Development of real-time and lateral flow recombinase polymerase amplification assays for rapid detection of Schistosoma mansoni. Front Microbiol., v. 13, 1043596, 2022. doi: 10.3389/fmicb.2022.1043596.
dc.identifier1664-302X
dc.identifierhttps://www.arca.fiocruz.br/handle/icict/56536
dc.identifier.urihttps://repositorioslatinoamericanos.uchile.cl/handle/2250/8868184
dc.descriptionBackground: Accurate diagnosis followed by timely treatment is an effective strategy for the prevention of complications together with reducing schistosomiasis transmission. Recombinase Polymerase Amplification (RPA) is a simple, rapid, sensitive, and specific isothermal method with low resource needs. This research aimed at the development and optimisation of a real-time (RT) and a lateral flow (LF) RPA assay for the detection of Schistosoma mansoni. Methodology: Recombinase Polymerase Amplification reactions were performed at full- (as recommended) and half-volumes (to reduce costs), with RT or LF detection systems targeting the S. mansoni mitochondrial minisatellite region. The specificity was assessed using gDNA from other Schistosoma species, helminths co-endemic with S. mansoni, human stool, and urine, and Biomphalaria snail hosts. The analytical sensitivity was evaluated using serial dilutions of gDNA, synthetic copies of the target, and single eggs. The ability of both assays to detect the S. mansoni DNA in human urine and stool samples was also tested. The long-term stability of the RT-RPA reagents was evaluated by storing the reaction components in different temperature conditions for up to 3 weeks. Results: The RT- and the LF-RPA (SmMIT- and SmMIT-LF-RPA, respectively) presented similar results when used full- and half-volumes, thus the latter was followed in all experiments. The SmMIT-RPA was 100% specific to S. mansoni, able to detect a single egg, with a limit of detection (LOD) of down to 1 fg of gDNA and one synthetic copy of the target. The assay was able to detect S. mansoni DNA from stool containing 1 egg/g and in spiked urine at a concentration of 10 fg/μl. SmMIT-RPA reagents were stable for up to 3 weeks when kept at 19°C, and 2 weeks when stored at 27°C. The SmMIT-LF-RPA cross-reacted with Clinostomidae, presented the LOD of 10 fg and one synthetic copy of the target, being able to detect a single egg and 1 egg/g in a stool sample. The LOD in spiked urine samples was 10 pg/μl. Conclusion: The half-volume SmMIT-RPA is a promising method to be used in the field. It is specific, sensitive, robust, and tolerant to inhibitors, with a long-term stability of the reaction components and the real-time visualisation of results.
dc.formatapplication/pdf
dc.languageeng
dc.publisherFrontiers Research Foundation
dc.rightsopen access
dc.subjectRecombinase Polymerase Amplification
dc.subjectSchistosoma mansoni
dc.subjectSchistosomiasis
dc.subjectisothermal molecular diagnostics
dc.subjectlateral flow RPA
dc.subjectmitochondrial minisatellite region
dc.subjectreal-time RPA
dc.titleDevelopment of real-time and lateral flow recombinase polymerase amplification assays for rapid detection of Schistosoma mansoni
dc.typeArticle


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