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

Daniel O Ouso,Joel L Bargul,Joseph W Oundo,Moses Y Otiende,Maamun M Jeneby,Lillian Wambua,Jandouwe Villinger,Scott E Miller

doi:10.1038/s41598-020-61600-3

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 sequencing of the mitochondrial cytochrome oxidase I (COI) ‘barcode’ gene, which remains costly for purposes of screening large numbers of unknown samples during routine surveillance. Here, we adapted 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.

Highlights

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
Accurate identification of suspect samples forms the basis of forensic evidence, which currently relies widely on sequencing of the barcode cytochrome c oxidase subunit I (COI) gene[9,10], the gold-standard for vertebrate species determination implemented by initiatives such as the International Barcode of Life project[11,12]
By systematically comparing high-resolution melting (HRM) profiles generated by cytochrome oxidase I (COI), cytochrome b oxidase, and 16S ribosomal (r)RNA gene PCR products, we show that this approach can robustly differentiate domestic vertebrate species from wildlife species and can be used to triage forensic barcode sequencing confirmations such that they are limited to only wildlife specimens

Summary

Introduction

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. As only a small proportion of potential samples sequenced are of illegally traded wildlife products, the cost of surveillance by mass-barcode sequencing is high and not sustainable in the long term. While emerging technologies, such as MinION sequencing (Oxford Nanopore Technologies, Oxford, UK), can facilitate rapid on-site DNA metabarcoding of environmental samples[18], costs of library preparation and adaptors required to associate specific sequences with individual samples remain high and are not amenable to on-going screening of smaller sample sizes compared to standard PCR. Restriction fragment length polymorphism[19], random amplification of polymorphic DNA20, and amplified fragment length polymorphism[21] have been used for vertebrate species identification, but still require post-PCR processing and are limited to detecting very specific wildlife species and/ or suffer from poor reproducibility, and thereby limit the development of reference databases[22]

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Discussion

Conclusion