Isothermal Recombinase Polymerase amplification (RPA) of Schistosoma haematobium DNA and oligochromatographic lateral flow detection

Abstract

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.

Findings: 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.

Conclusion: 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.

Fig. 1

a Agarose gel image of the Dra1 RPA amplicons. Low limit of detection can be seen at 100 fg of S. haematobium gDNA. b LF strips showing the detection of the Dra LF-RPA amplicons. Lower limit of detection can be seen at 100 fg of S. haematobium gDNA. NC = Negative control. The LF-RPA oligonucleotides consisted of a forward primer, a specialised 6-FAM labelled oligonucleotide probe and a reverse biotin labelled primer. Upon successful binding to the complementary gDNA target, amplification ensues resulting in the formation of a double-labelled amplicon. When run on an oligochromatographic LF strip, the amplicon binds to anti-FAM antibodies and antibody labelled gold colloid nanoparticles in the running buffer bind to the biotin antigen resulting in a semi quantitative colour change. The LF strip also has a control line to test for reaction failure

Fig. 2

LF strips showing Dra1 LF-RPA amplicon detection at different reaction temperatures (a) and times (b)