PCR-high Resolution Melting Analysis of the COI, cyt b and 16S rRNA Mini-barcode Genes: A Tool for Species Identification and Discrimination in Illegal Bushmeat Trade

Abstract

Reliable molecular identification of vertebrate species from morphologically unidentifiable tissue is critical to the prosecution of illegally traded wildlife products to limit their trade, as well us for surveillance to inform conservation policies and identification of blood-meal hosts. Currently, this is mainly dependent on sequencing of the cytochrome c oxidase subunit I (COI) ‘barcode’ genes, which remains costly for purposes of screening large numbers of unknown samples and routine surveillance. High-resolution melting (HRM) analysis was optimized as a cheaper supplement to conventional sequencing. Here, we adopted HRM analysis of COI, cytochrome b and 16S ribosomal RNA mini-barcode genes Polymerase chain reaction products. The process uses DNA intercalating dye to report the unique melting pattern of duplex DNA. We analysed 107 samples using the optimised approach and robustly differentiate 10 domestic species from 24 wildlife species that are common in the East African illegal wildlife trade. To validate the tool, we assessed whether bushmeat was sold in township butcheries by covert field surveillance sampling in Naivasha, Kenya. We identified one out of 90 samples as bushmeat (giraffe). This approach is being adopted for high-throughput pre-screening of potential bushmeat samples for exclusion of non-bushmeat samples from downstream processing in forensic species identification pipelines in Kenya and Tanzania. It is also useful as a sustainable surveillance and monitoring tool for illegal bushmeat trade, as well as for studies on hematophagous invertebrate vector-host interactions at wildlife-human interfaces in disease epidemiology