• Donnelly, Owain
• Mesquita, Silvia
• Archer, John
• Bartonicek, Zikmund
• Ali, Said M.
• Lugli, Elena B.
• Webster, Bonnie L.

Abstract

<jats:title>Abstract</jats:title><jats:sec><jats:title>Background</jats:title><jats:p>Urogenital schistosomiasis is caused by the parasitic trematode <jats:italic>Schistosoma haematobium</jats:italic>. Sensitive and specific point-of-care diagnostics are needed for elimination of this disease. Recombinase polymerase amplification (RPA) assays meet these criteria, and an assay to diagnose <jats:italic>S. haematobium</jats:italic> has been developed (Sh-RPA). However, false-positive results can occur, and optimisation of reaction conditions to mitigate these is needed. Ease of use and compatibility of DNA extraction methods must also be considered.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>Using synthetic DNA, <jats:italic>S. haematobium</jats:italic> genomic DNA (gDNA), and urine samples from clinical cases, Sh-RPA reactions incorporating different betaine concentrations (0 M, 1 M, 2.5 M, 12.5 M) and the sample-to-water ratios were tested to determine effects on assay specificity and sensitivity. In addition, five commercial DNA extraction kits suitable for use in resource-limited settings were used to obtain gDNA from single <jats:italic>S. haematobium</jats:italic> eggs and evaluated in terms of DNA quality, quantity, and compatibility with the Sh-RPA assay. All samples were also evaluated by quantitative polymerase chain reaction (qPCR) to confirm DNA acquisition.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>The analytical sensitivity of the Sh-RPA with all betaine concentrations was ≥ 10 copies of the synthetic <jats:italic>Dra1</jats:italic> standard and 0.1 pg of <jats:italic>S. haematobium</jats:italic> gDNA. The addition of betaine improved Sh-RPA assay specificity in all reaction conditions, and the addition of 2.5 M of betaine together with the maximal possible sample volume of 12.7 µl proved to be the optimum reaction conditions. DNA was successfully isolated from a single <jats:italic>S. haematobium</jats:italic> egg using all five commercial DNA extraction kits, but the Sh-RPA performance of these kits varied, with one proving to be incompatible with RPA reactions.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>The addition of 2.5 M of betaine to Sh-RPA reactions improved reaction specificity whilst having no detrimental effect on sensitivity. This increases the robustness of the assay, advancing the feasibility of using the Sh-RPA assay in resource-limited settings. The testing of commercial extraction kits proved that crude, rapid, and simple methods are sufficient for obtaining DNA from single <jats:italic>S. haematobium</jats:italic> eggs, and that these extracts can be used with Sh-RPA in most cases. However, the observed incompatibility of specific kits with Sh-RPA highlights the need for each stage of a molecular diagnostic platform to be robustly tested prior to implementation.</jats:p></jats:sec><jats:sec><jats:title>Graphical Abstract</jats:title></jats:sec>

Topics

• impedance spectroscopy
• extraction
• size-exclusion chromatography