Isothermal Recombinase Polymerase amplification (RPA) of Schistosoma haematobium DNA and oligochromatographic lateral flow detection.
BACKGROUND
Accurate diagnosis of urogenital schistosomiasis is vital for surveillance/control programs. Amplification of schistosome DNA in urine by PCR is sensitive and specific but requires infrastructure, financial resources and skilled personnel, often not available in endemic areas. Recombinase Polymerase Amplification (RPA) is an isothermal DNA amplification/detection technology that is simple, rapid, portable and needs few resources.


RESULTS
Here a Schistosoma haematobium RPA assay was developed and adapted so that DNA amplicons could be detected using oligochromatographic Lateral Flow (LF) strips. The assay successfully amplified S. haematobium DNA at 30-45 °C in 10 mins and was sensitive to a lower limit of 100 fg of DNA. The assay was also successful with the addition of crude urine, up to 5% of the total reaction volume. Cross amplification occurred with other schistosome species but not with other common urine microorganisms.


CONCLUSIONS
The LF-RPA assay developed here can amplify and detect low levels of S. haematobium DNA. Reactions are rapid, require low temperatures and positive reactions are interpreted using lateral flow strips, reducing the need for infrastructure and resources. This together with an ability to withstand inhibitors within urine makes RPA a promising technology for further development as a molecular diagnostic tool for urogenital schistosomiasis.
Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK. andrew.rosser@gmail.com.
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A
Rosser
Department of Life Sciences, Natural History Museum, London, UK. d.rollinson@nhm.ac.uk.
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D
Rollinson
TwistDX Ltd, Cambridge, UK. M.Forrest@TWISTDx.co.uk.
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M
Forrest
Department of Life Sciences, Natural History Museum, London, UK. b.webster@nhm.ac.uk.
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BL
Webster
D000818:Animals; D002845:Chromatography / Q000379:methods*; D003429:Cross Reactions; D018509:DNA, Helminth / Q000235:genetics*; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010865:Pilot Projects; D012548:Schistosoma haematobium / Q000235:genetics*; D012680:Sensitivity and Specificity
D018509:DNA, Helminth