Approaches to genotyping individual miracidia of Schistosoma japonicum

Abstract

Molecular genetic tools are needed to address questions as to the source and dynamics of transmission of the human blood fluke Schistosoma japonicum in regions where human infections have reemerged, and to characterize infrapopulations in individual hosts. The life stage that interests us as a target for collecting genotypic data is the miracidium, a very small larval stage that consequently yields very little DNA for analysis. Here, we report the successful development of a multiplex format permitting genotyping of 17 microsatellite loci in four sequential multiplex reactions using a single miracidium held on a Whatman Classic FTA indicating card. This approach was successful after short storage periods, but after long storage (>4 years), considerable difficulty was encountered in multiplex genotyping, necessitating the use of whole genome amplification (WGA) methods. WGA applied to cards stored for long periods of time resulted in sufficient DNA for accurate and repeatable genotyping. Trials and tests of these methods, as well as application to some field-collected samples, are reported, along with the discussion of the potential insights to be gained from such techniques. These include recognition of sibships among miracidia from a single host, and inference of the minimum number of worm pairs that might be present in a host.

Figure 1

Demonstration of repetitive use for PCR of punched the Whatman FTA card disks holding a single miracidium stored for 11 months. A-F: Images of 1.5% agarose gels on which 5 ml PCR product was run after each successive PCR using a different primer pair. An arrow shows the location of the faint band in F. Image A shows the results (for two disks) amplified using primers 3S/A28 that span the nuclear ribosomal ITS2 region. Remaining images (B-F) show results of re-use of the disk represented in the right-hand lane of A up to 11 times, each time using primers for a different locus. Note that one primer pair (for locus Sj25) used in this test was not used in the final multiplex format, so is not listed in Table 1. 100bp DNA ladder was used as molecular size marker. The blank lane to the right of the molecular size marker is the negative control in each case.