Abstract
BackgroundThe specific identification of animals through the analysis of faecal DNA is important in many areas of scientific endeavour, particularly in the field of parasitology.MethodsHere, 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.ResultsThese 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.ConclusionsWe 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.
Highlights
The specific identification of animals through the analysis of faecal DNA is important in many areas of scientific endeavour, in the field of parasitology
Control DNA was extracted from muscle samples from each common wombat, eastern grey kangaroo, swamp wallaby, fallow deer, red deer and sambar deer (‘target’ species), goat and rabbit using the same kit with the following alteration to the protocol: ~ 0.25 g of muscle was placed in 400 μl of extraction buffer (20 mM Tris-HCl, 100 mM ethylenediaminetetraacetic acid (EDTA), and 1% sodium dodecyl sulfate (SDS)) and 20 μl of proteinase K (20 mg/ml; Promega, Fitchburg, Wisconsin, USA) and heated to 56 °C overnight
Our goal was to design forward and reverse primers that would produce specific polymerase chain reaction (PCR) amplicons of < 500 bp that would differ in length by 40–50 bp among species, so that amplicons representing individual animal species were unique in size and differentiable from one another on an agarose gel
Summary
The specific identification of animals through the analysis of faecal DNA is important in many areas of scientific endeavour, in the field of parasitology. One strategy relies on the use of a common reverse primer in conjunction with unique (specific) forward primers, resulting in amplicons of varying sizes [2], which (if primers are designed and positioned well) are readily discernible on agarose gels [19]. We propose that this or a similar approach could be applicable to the identification and differentiation of faeces from species of wildlife in water catchments for the purpose of being able to match parasite species/genotypes with animal host species
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