A new multiplex PCR assay to distinguish among three cryptic Galba species, intermediate hosts of Fasciola hepatica

Abstract A molecular tool described here allows in one step for specific discrimination among three cryptic freshwater snailspecies (genus Galba) involved in fasciolosistransmission, a worldwide infectious disease of humans and livestock. The multiplex PCR approach taken targets for each species a distinctive, known microsatellite locus which is amplified using specific primers designed to generate an ampliconof a distinctive size that can be readily separated from the ampliconsof the other two species on an agarose gel. In this way, the three Galbaspecies ( G. cubensis , G. schirazensis , and G. truncatula ) can be differentiated from one another, including even if DNA from all three were present in the same reaction. The accuracy of this new molecular tool was tested and validated by comparing multiplex PCR results with species identification based on sequences at mitochondrial and nuclear markers. This new method is accurate, inexpensive, simple, rapid, and can be adapted to handle large sample sizes. It will be helpful for monitoring invasion of Galbaspecies and for developing strategies to limit the snail species involved in the emergence or re-emergence of fasciolosis.