Multiplex PCRs for the specific identification of marsupial and deer species from faecal samples as a basis for non-invasive epidemiological studies of parasites

BACKGROUND: The specific identification of animals through the analysis of faecal DNA is important in many areas of scientific endeavour, particularly in the field of parasitology. METHODS: Here, we designed and assessed two multiplex PCR assays using genetic markers in a mitochondrial cytochrome b (cytb) gene region for the unequivocal identification and discrimination of animal species based on the specific amplification of DNA from faecal samples collected from water catchment areas in Victoria, Australia. One of these assays differentiates three marsupial species (eastern grey kangaroo, swamp wallaby and common wombat) and the other distinguishes three deer species (fallow, red and sambar deer). We tested these two assays using a total of 669 faecal samples, collected as part of an ongoing programme to monitor parasites and microorganisms in these animals. RESULTS: These two PCR assays are entirely specific for these animal species and achieve analytical sensitivities of 0.1-1.0 picogram (pg). We tested 669 faecal samples and found that some previous inferences of species based on faecal morphology were erroneous. We were able to molecularly authenticate all of the 669 samples. CONCLUSIONS: We have established PCR assays that accurately distinguish the faecal samples of some of the prominent large mammalian herbivores found within a water catchment system in the state of Victoria, Australia. The multiplex assays for marsupials and deer produce amplicons that are easily differentiable based on their size on an agarose gel, and can be readily sequenced for definitive species authentication. Although established for marsupials and deer, the methodology used here can be applied to other host-parasite study systems to ensure data integrity