A multiplex PCR for the detection of Fasciola hepatica in the intermediate snail host Galba cubensis

[Display omitted] •The first multiplex PCR to detect Fasciola hepatica in Galba cubensis was developed.•A set of primers was designed to amplify a conserved region of G. cubensis 18S rDNA.•Concentration ratio between both set of primers was crucial in the PCR optimization.•The multiplex PCR detect less than one miracidium even at high amounts of snail DNA.•The assay discriminated between non-infected and experimentally infected snails. Fasciolosis is a snail-borne trematode infection that has re-emerged as a human disease, and is considered a significant problem for veterinary medicine worldwide. The evaluation of the transmission risk of fasciolosis as well as the efficacy of the strategies for its control could be carried out through epidemiological surveillance of the snails that act as intermediate hosts of the parasites. The present study aimed to develop the first multiplex PCR to detect Fasciola hepatica in Galba cubensis, an important intermediate host of the parasite in the Americas and especially in the Caribbean basin. The multiplex PCR was optimized for the amplification of a 340bp fragment of the second internal transcribed spacer (ITS-2) of F. hepatica rDNA, while another set of primers was designed and used to amplify a conserved segment of the nuclear 18S rDNA of the snail (451bp), as an internal control of the reaction. The assay was able to detect up to 100pg of the parasite even at high concentrations of snail DNA, an analytical sensitivity that allows the detection of less than a single miracidium, which is the minimal biological infestation unit. A controlled laboratory-reared G. cubensis – F. hepatica system was used for the evaluation of the developed multiplex PCR, and 100% sensitivity and specificity was achieved. This assay constitutes a novel, useful and suitable technique for the survey of fasciolosis transmission through one of the main intermediate hosts in the Western hemisphere.