Differentiation of Schistosoma haematobium from related schistosomes by PCR amplifying an inter-repeat sequence

Abstract

Schistosoma haematobium infects nearly 150 million people, primarily in Africa, and is transmitted by select species of local bulinid snails. These snails can host other related trematode species as well, so that effective detection and monitoring of snails infected with S. haematobium requires a successful differentiation between S. haematobium and any closely related schistosome species. To enable differential detection of S. haematobium DNA by simple polymerase chain reaction (PCR), we designed and tested primer pairs from numerous newly identified Schistosoma DNA repeat sequences. However, all pairs tested were found unsuitable for this purpose. Differentiation of S. haematobium from S. bovis, S. mattheei, S. curassoni, and S. intercalatum (but not from S. margrebowiei) was ultimately accomplished by PCR using one primer from a newly identified repeat, Sh110, and a second primer from a known schistosomal splice-leader sequence. For evaluation of residual S. haematobium transmission after control interventions, this differentiation tool will enable accurate monitoring of infected snails in areas where S. haematobium is sympatric with the most prevalent other schistosome species.

Figure 1

A, The newly identified S. haematobium repeated sequence clone Sh110. B, The S. mansoni splice leader sequence from S. mansoni—Sm-S1. Primers designed from both (in bold) were used in PCR assays for species-specific discrimination between S. haematobium DNA and the DNA of other related schistosome species.

Figure 2

The 525-bp DNA sequence of the amplification product of PCR using the Sm-S1 primer and the reverse Sh110 primer. The primers and their positions are marked in bold.

Figure 3

The distribution of the DNA segment amplified by the Sh110 and Sm-S1 primers. Total S. haematobium DNA digested by RsaI (1) or AluI (2) underwent Southern blot and probing with labeled SmS1/Sh110 PCR product.

Figure 4

Sensitivity of detection by SmS1/Sh110 PCR. Different concentrations of S. haematobium genomic DNA were used: lane 1, 10 ng; lane 2, 1 ng; lane 3, 0.1 ng; lane 4, 10 pg; lane 5, 1 pg; l;ane 6, 0.1 pg; lane 7, no DNA. M, DNA size marker.

Figure 5

Specificity of detection of S. haematobium by SmS1/Sh110 PCR. Varying concentrations of genomic DNA from different species were tested. Lanes 1 and 2: 0.1 ng and 10 pg of S. haematobium DNA; lanes 3 and 4: 10 and 1 ng of S. bovis DNA; lanes 5 and 6: 10 and 1 ng of S. mattheei DNA; lanes 7 and 8: 10 and 1 ng of S. intercalatum DNA; lanes 9 and 10: 10 and 1 ng of S. curassoni DNA; lanes 11 and 12: 10 and 1 ng of S. margrebowiei DNA; lane 13: S. haematobium DNA 0.1 ng (positive control); lane 14: no DNA. M, DNA molecular size markers.

Figure 6

Detection of S. haematobium–infected snails by SmS1/Sh110 PCR. Lanes 1–6: PCR reaction run with infected snails; lanes 7–13: reaction run with uninfected snails; lane 7 only: 1 ng of S. haematobium DNA added to uninfected snail extract; lane 8: no DNA added; lanes 9–13: additional reactions run with uninfected snails. M, DNA size marker.