A rapid multiplex PCR assay for presumptive species identification of rhinoceros horns and its implementation in Vietnam

Abstract

Rhinoceros (rhinos) have suffered a dramatic increase in poaching over the past decade due to the growing demand for rhino horn products in Asia. One way to reverse this trend is to enhance enforcement and intelligence gathering tools used for species identification of horns, in particular making them fast, inexpensive and accurate. Traditionally, species identification tests are based on DNA sequence data, which, depending on laboratory resources, can be either time or cost prohibitive. This study presents a rapid rhino species identification test, utilizing species-specific primers within the cytochrome b gene multiplexed in a single reaction, with a presumptive species identification based on the length of the resultant amplicon. This multiplex PCR assay can provide a presumptive species identification result in less than 24 hours. Sequence-based definitive testing can be conducted if/when required (e.g. court purposes). This work also presents an actual casework scenario in which the presumptive test was successfully utlitised, in concert with sequence-based definitive testing. The test was carried out on seized suspected rhino horns tested at the Institute of Ecology and Biological Resources, the CITES mandated laboratory in Vietnam, a country that is known to be a major source of demand for rhino horns. This test represents the basis for which future ‘rapid species identification tests’ can be trialed.

Humans Are Animals, Too: Critical Commonalities and Differences Between Human and Wildlife Forensic Genetics

“…In cases where there is a well‐defined investigational question (i.e., a designated set of well‐characterized species), an assay using probes, primers, or restriction enzymes to target SNPs can be a fast and low‐cost alternative or precursor to sequencing . These assays are usually designed to detect the target species without a sequencer, which may be beyond the budget of laboratories in developing‐world countries that are often the source of organisms targeted by the illegal wildlife trade.…”

“…In general, validation techniques are very similar to those of human forensic science, though wildlife presents unique challenges, detailed below (also see Ref. ). Table highlights the differences between validation in human and wildlife forensics.…”

“…If published markers do not exist or are not suitable for forensic use, laboratories must develop and validate their own sex identification markers, STRs, and/or SNP loci for each species of interest.…”

“…Protocols are written to reflect the validated range, as different species have different sequences at primer annealing locations, and may require varying reagent concentrations or thermal cycling parameters to produce good PCR products. A factor likely of lesser concern in human forensics is validation of markers to determine cross‐reactivity with other species ; in wildlife forensics, these tests should include common species which might be found in conjunction with the evidence (human, companion, and domesticated animals), common substitutes and look‐alike species, and close relatives of the target taxon .…”

Wildlife forensic science in Hong Kong

“…Current discussions in the global community of wildlife forensics are focusing on new methods for data‐sharing. Suggestions include publication embargos for practitioners using data developed by other laboratories, acknowledgments for those laboratories, as well as formal multi‐laboratory collaborations to prevent duplicated efforts (Ewart et al, 2018; Moore & Frazier, 2019). Designating government resources for the development of reference databases containing genetic, chemical, and morphological data for strategically important species in Hong Kong's illegal trade would be a step forward.…”