Detection of Schistosoma mansoni and Schistosoma haematobium DNA by loop-mediated isothermal amplification: identification of infected snails from early prepatency

Abstract

Monitoring post-control transmission of schistosomes by examining humans becomes less effective as infection rates among humans decrease. Molecular monitoring of prepatent schistosome infection in snails by the polymerase chain reaction (PCR) has been used for studying human-to-snail transmission, and snail prepatent infection rates were found to correspond to infection prevalence and average intensity in human populations contacting the sites studied. We have now developed loop-mediated isothermal amplification (LAMP) assays for identifying Schistosoma mansoni and S. haematobium to facilitate large-scale evaluation of post-intervention transmission potential. LAMP primers were designed based on the Sm1-7 and DraI repeated sequences of the corresponding schistosomes, and amplification by LAMP of these 121-basepair highly abundant sequences provided a detection sensitivity of 0.1 fg of genomic DNA. When these LAMP assays were applied for examining infected laboratory snails, it was possible to identify infection from the first day after exposure to miracidia. The potential advantages of these assays are discussed.

Figure 1.

Location of the loop-mediated isothermal amplification forward (F3) and backward (B3) external primers and of internal forward (FIP) and backward (BIP) primers within the respective sequences DraI of Schistosoma haematobium (A) and Sm1-7 of S. mansoni (B).

Figure 2.

A, Agarose gel electrophoresis of loop isothermal amplified products (LAMP) from different concentrations of Schistosoma haematobium genomic DNA. Ten-fold serial dilutions starting from 10 ng genomic DNA (lane 1) down to 0.001 fg (lane 11) were tested. Lane M, molecular mass marker. B, Fluorescence of LAMP products as indicated in A using SYBR Green I staining followed by ultraviolet illumination at 320 nm. Values on the right are in basepairs

Figure 3.

Agarose gel electrophoresis of loop-mediated isothermal amplification products after targeting DraI S. haematobium repetitive sequence within infected snails. Lanes 1 and 8, uninfected snails; lanes 2–4, snails after one day of infection; lanes 5–7, snails after seven days of infection; lanes 9–11, snails after 14 days of infection; lane 12, negative control (no DNA); lane M, molecular mass marker. Values on the right are in basepairs.

Figure 4.

A, Agarose gel electrophoresis of loop-mediated isothermal amplification (LAMP) products from different concentrations of Schistosoma mansoni genomic DNA. Ten-fold serial dilutions starting with 10 ng of genomic DNA (lane 1) down to 0.001 fg (lane 11) were tested. Lane 12, negative control (no DNA); lane M molecular mass marker. B, Fluorescence of LAMP products as indicated in A using SYBR Green I staining followed by ultraviolet illumination at 320 nm. Values on the right are in basepairs.

Figure 5.

Agarose gel electrophoresis of loop-mediated isothermal amplification products after targeting Sm1-7 Schistosoma mansoni repetitive sequence from infected snails. Lanes 1–3, uninfected snails; lanes 4–6, snails after one day of infection; lanes 7–9, snails after seven days of infection; lanes 10 and 11, negative control (no DNA); lane M: molecular mass marker. Values on the right are in basepairs.