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

Abstract

Reliable molecular identification of vertebrate species from morphologically unidentifiable tissue is critical for the prosecution of illegally-traded wildlife products, conservation-based biodiversity research, and identification of blood-meal hosts of hematophagous invertebrates. However, forensic identification of vertebrate tissue relies on the 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 (i) giraffe among covertly sampled meat from Kenyan butcheries, and (ii) forest elephant mitochondrial sequences among savannah elephant reference samples. This approach is being adopted for high-throughput pre-screening of potential bushmeat samples in East African forensic science pipelines.

Tsetse blood-meal sources, endosymbionts and trypanosome-associations in the Maasai Mara National Reserve, a wildlife-human-livestock interface

“…We were able to identify blood-meals from wild-caught non-engorged flies and detect mixed blood-meals that were confirmed by DNA sequencing. Unlike serological and other PCR-based techniques for blood-meal identification [ 66 , 80 , 81 ], the use of HRM to detect sequence variants is fast, cost-effective, accurate, easy-to-use, and sensitive, making it a more economical tool for blood-meal analysis [ 44 , 46 , 82 , 83 ].…”

“…Blood-meal sources were determined by PCR coupled with high-resolution melting (HRM) analysis of vertebrate cytochrome c oxidase subunit I (COI) and cytochrome b (cyt b) mitochondrial genes as previously described [44][45][46]. We analyzed 760 flies, representing 65% of the sampled population, including all engorged flies (n = 39), trypanosome-positive flies (n = 28), and randomly selected non-engorged flies.…”

“…This was followed by a final extension at 72˚C for 7 minutes. Thereafter, HRM analysis of PCR products was conducted as described by [44][45][46]. HRM profiles were analyzed using the Rotor-Gene Q software version 2.1 with normalized regions between 76.0-78.0˚C and 89.50-90.0˚C.…”

Tsetse blood-meal sources, endosymbionts, and trypanosome infections provide insight into African trypanosomiasis transmission in the Maasai Mara National Reserve, a wildlife-human-livestock interface

“…Blood-meal sources were determined by PCR coupled with high-resolution melting (HRM) analysis of vertebrate cytochrome c oxidase subunit I (COI) and cytochrome b (cyt b) mitochondrial genes as previously described [30–32]. We analyzed 760 flies, representing 65% of the sampled population, which included all engorged flies (n = 39) and 721 randomly selected non-engorged flies.…”

“…This was followed by a final extension at 72°C for 7 minutes. Thereafter, HRM analysis of PCR products was conducted as described by [30–32]. HRM profiles were analysed using the Rotor-Gene Q software v2.1 with normalized regions between 76.0-78.0°C and 89.50-90.0°C.…”