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

<div><p>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.</p></div

Multiplex PCR assay protocol: Identifying an unknown rhino specimen.

<p>Multiplex PCR assay protocol: Identifying an unknown rhino specimen.</p

Cytochrome b markers and their corresponding primers.

<p>Cytochrome b markers and their corresponding primers.</p

A 2% agarose gel run at IEBR showing the amplicons for 16 unknown horn samples using the multiplex PCR assay.

<p>The positive controls are known samples from Indian rhino (M.39431.001; 310 bp amplicon) white rhino (M.47191.001; 266 bp amplicon) and black rhino (M.46281.001; 222 bp amplicon). Based on this gel, all of the unknown samples are from white rhino (14 out of 16) and black rhino (2 out of 16) horn. NTC = no template control; IR = Indian rhino amplicon; WR = white rhino amplicon; BR = black rhino amplicon.</p

Identification key to interpret the results of the multiplex PCR assay.

<p>Identification key to interpret the results of the multiplex PCR assay.</p

Additional file 3: Table S7. of Development of a SNP-based assay for measuring genetic diversity in the Tasmanian devil insurance population

SNP genotypes for pedigree determination. (XLSX 279 kb

Probability, random processes, and ergodic properties

In this new edition of this classic text, much of the material has been rearranged and revised for pedagogical reasons. Many classic inequalities and proofs are now incorporated into the text, and many citations have been added

Additional file 1: Table S1. of Developing a 670k genotyping array to tag ~2M SNPs across 24 horse breeds

Whole genome sequencing (WGS) samples. Whole genome sequencing samples with horse identification and read depth. 153 Individuals representing 24 breeds (Twilight is included as 4 entries). Table includes horse identifier, breed, contributing laboratory and coverage statistics for each individual in both the nuclear and mitochondrial genomes. Variant calling groups indicate which individuals were grouped together during variant discovery. (XLSX 21 kb

Additional file 2: Table S2. of Developing a 670k genotyping array to tag ~2M SNPs across 24 horse breeds

SNP50 breed informativeness. Impact of breed on SNP50 informativeness. Table shows informativeness (MAF ≥ 0.05) of the legacy, SNP50 SNP set for light, draft, and pony breeds described by McCue et al. [1, 2]. Mean number of informative SNPs per breed was ~42,000 (~77%). 19,427 SNPs were informative in all 14 breeds. (XLSX 8 kb

Additional file 5: Table S5. of Developing a 670k genotyping array to tag ~2M SNPs across 24 horse breeds

Sample breed and tagging breed groups. Samples (n = 485) from either whole genome sequence (WGS) or from the 2M test array (SNP) were assigned to one of 15 tagging breed groups based on their reported breed. (XLSX 21 kb