Three-gene PCR and high-resolution melting analysis for differentiating vertebrate species mitochondrial DNA for forensic and biodiversity research pipelines

Reliable molecular identification of vertebrate species from morphologically unidentifiable tissue is critical for prosecution of illegally traded wildlife products, biodiversity research for conservation purposes, and identification of blood-meal hosts of hematophagous invertebrates. However, forensic identification of vertebrate tissue relies on sequencing of mitochondrial cytochrome oxidase I (COI) barcode genes, which remains costly for purposes of screening large numbers of unknown samples during routine surveillance. Here, we adopted a rapid, low-cost approach to differentiate 10 domestic and 24 wildlife species that are common in the East African illegal wildlife products trade based on their unique high-resolution melting profiles from COI, cytochrome b, and 16S ribosomal RNA gene PCR products. Using the approach, we identified giraffe among covertly sampled meat from Kenyan butcheries and savannah elephant reference samples with forest elephant mitochondrial sequences. This approach is being adopted for high-throughput pre-screening of potential bushmeat samples in East African forensic science pipelines.