Development and performance of recombinase-aided amplification (RAA) assay for detecting Schistosoma haematobium DNA in urine samples

Abstract

Rapid diagnosis of urogenital schistosomiasis caused by Schistosoma haematobium requires an accurate and timely assay, especially for low-intensity S. haematobium infection cases and in non-endemic areas. The mitochondrial cytochrome c oxidase 1 (cox1) gene fragment of S. haematobium was selected as detection target as this short fragment, which can be rapidly sequenced and yet possess good diagnostic resolution. A pair of primers and a fluorescent probe were designed according to the principle of recombinase-aided amplification (RAA), which was subsequently optimized and applied as an S. haematobium-specific RAA assay. Its diagnostic performance was validated for sensitivity and specificity in comparison to microscopy-based egg counting after urine filtration. The RAA assay could detect as little as 10 copies/μL of S. haematobium recombinant plasmid, and no cross-reactions were observed with S. mansoni, S. japonicum, Ancylostoma duodenale, Clonorchis sinensis, Echinococcus granulosus, or Ascaris lumbricoides. This test can be conducted at 39 °C and the whole RAA reaction can be completed within 20 min. The validation of the RAA assay showed that it had 100 % consistency with urine-egg microscopy, as it does not require an elaborate reading tool, is simple to use, and should be useful for field diagnostics and point-of-care applications.

Keywords

• Schistosoma haematobium
• Field diagnosis
• Recombinase-aided amplification (RAA)
• Cytochrome c oxidase subunit 1 (cox1)
• Urine