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Development of a recombinase polymerase amplification (RPA) fluorescence assay for the detection of Schistosoma haematobium
Rostron, Penelope Bakar, Faki Knopp, Stefanie Allan, Fiona Kabole, Fatma Ali, Said M Ame, Shaali M
Rostron, Penelope
Bakar, Faki
Knopp, Stefanie
Allan, Fiona
Kabole, Fatma
Ali, Said M
Ame, Shaali M
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2019-11-04
Submitted Date
2019-06-02
Subject Terms
Schistosoma haematobium
Urogenital schistomiasis
diagnostics
RPA
isothermal
molecular
Point-of-need (PON)
surveillance
control
elimination
Urogenital schistomiasis
diagnostics
RPA
isothermal
molecular
Point-of-need (PON)
surveillance
control
elimination
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Abstract
Background
Accurate diagnosis of urogenital schistosomiasis is vital for surveillance and control programmes. While a number of diagnostic techniques are available there is a need for simple, rapid and highly sensitive point-of-need (PON) tests in areas where infection prevalence and intensity are low. Recombinase Polymerase Amplification (RPA) is a sensitive isothermal molecular diagnostic technology that is rapid, portable and has been used at the PON for several pathogens.
Results
A real time fluorescence RPA assay (RT-ShDra1-RPA) targeting the Schistosoma haematobium Dra1 genomic repeat region was developed and was able to detect 1 fg of S. haematobium gDNA. Results were obtained within 10 minutes using a small portable battery powered tube scanner device that incubated reactions at 40 °C, whilst detecting DNA amplification and fluorescence over time. The assay’s performance was evaluated using 20 urine samples, with varying S. haematobium egg counts, from school children from Pemba Island, Zanzibar Archipelago, Tanzania. Prior to RPA analysis, samples were prepared using a quick crude field DNA extraction method, the Speed Extract Kit (Qiagen, Manchester, UK). Positive assay results were obtained from urine samples with egg counts of 1–926 eggs/10 ml, except for two samples, which had inconclusive results. These two samples had egg counts of two and three eggs/10 ml of urine.
Conclusions
The RT-ShDra1-RPA assay proved robust for S. haematobium gDNA detection and was able to amplify and detect S. haematobium DNA in urine samples from infected patients. The assay’s speed and portability, together with the use of crude sample preparation methods, could advance the rapid molecular diagnosis of urogenital schistosomiasis at the PON within endemic countries.
Citation
Rostron, P., Pennance, T., Bakar, F. et al. Development of a recombinase polymerase amplification (RPA) fluorescence assay for the detection of Schistosoma haematobium. Parasites Vectors 12, 514 (2019). https://doi.org/10.1186/s13071-019-3755-6
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Copyright © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. The attached file is the published version of the article.
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1756-3305
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1756-3305
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openAccess