IDENTIFYING THE SOURCE OF MEAT SEIZED FROM A POACHING
INCIDENT USING DNA ANALYSIS
ABSTRACT
Wildlife crime continues to perpetuate around the world, representing a critical threat to numerous species of flora
and fauna. Despite legislative protections offered by countries like Australia, prosecutions of suspected wildlife
crimes have remained low. To this end, forensic sciences aided by DNA analysis techniques offer a viable tool for
the rapid detection and identification of meat and blood samples obtained at the crime scenes. In this article, we
describe a standardised protocol for DNA isolation, amplification and sequencing which can be utilised to accurately
identify species in a reproducible and cost-effective manner. DNA extraction using QIAmp Spin columns was
carried out from wildlife meat samples obtained from an alleged bushmeat vendor. Subsequently, Cytochrome b
specific primers were used to amplify and sequence a highly conserved locus within the Cytb gene. Comparison of
the obtained sequence against known sequences on the NCBI database using the BLAST algorithm allowed us to
conclude with certainty that the unknown meat sample was obtained from Smutsia Gigantea (The Giant Pangolin).
KEYWORDS: FORENSIC, SPECIES IDENTIFICATION, WILDLIFE CRIME, CYTOCHROME B , PANGOLIN
INTRODUCTION
Wildlife crime is an ever-growing global crisis that has driven countless species of flora and fauna to extinction.
Despite increasing awareness and stringent policing from many countries around the world, wildlife crime and illicit
wildlife trafficking has been estimated to generate over $32 billion dollars in 2019 (Linacre & Ciavaglia, 2015). Yet,
only estimates are available for the scale of this crisis, as experts predict numerous incidents of poaching continue to
remain unreported. The persistent growth of wildlife crime has been attributed to the existence of a ‘hidden
economy’ which hinders the enactment of international laws (Wong, 2020).
Australia, an island with an incredible diversity of reptiles, amphibians, birds and mammals is a prime target for
wildlife crime. Despite being a signee of CITES, an international convention regulating trade in endangered species,
Australia continues to remain a site of export for high profile endangered species. In particular, Australia has been
found to be a hotspot for the illegal killing of animals used in traditional medicine (Byard, 2016). Yet, only a small
percentage of these cases are successfully prosecuted in court (Ciavaglia et al., 2015). However, recent
advancements in DNA analysis that have made sequencing faster, cheaper and more accurate, thus representing a
viable opportunity for forensic identification of wildlife crime. In this article, we aimed to describe a standardized
procedure for DNA analysis that can be used to isolate, amplify, sequence and identify meat samples.
The choice of DNA isolation method in forensic DNA analysis can critically influence the quality, and consequently
the interpretation of results. Furthermore, owing to the minimal amount of sample that is typically available in
forensic incidents, it is vital that the isolation procedure does not contribute to the degradation of sample (S. B. Lee
& Shewale, 2017). Consequently, The QIAmp Mini Spin columns were chosen for this study which have previously
been demonstrated to provide a high-quality, rapid, and reproducible method for DNA extraction (Menu et al.,
2018). Secondly, the choice of gene locus for the species identification of unknown samples is a critical component
of the protocol design. Although whole genome analysis has been made considerably cost-effective due to
advancements in molecular methods, this study chose a singular locus for comparison since it can yield results
rapidly. In contrast to nuclear DNA, mitochondrial DNA offers an advantage of rapid replication and protection with
an additional membrane. To this end, several conserved mitochondrial gene sequences have been explored for use in
forensic science. However, a particularly well-explored locus is the Cytochrome b, found on the mitochondrial
genome owing to its high inter-species and low intra-specific variation. Consequently, the locus is even able to
differentiate between closely related species with recent common ancestors (Lee et al., 2013). Therefore, in this
study, we chose to sequence a small highly conserved locus within the cytochrome b gene.
METHODS
Following a tip-off to the police regarding the sale of bushmeat, a knife utilised in the alleged poaching incident was
seized. Residual tissue present on the knife was then sent to the Murdoch University Wildlife Forensic Research
Laboratory. The Surface of the knife was swabbed and fibres isolated from the swab was dissolved in 180L of
ATL Buffer. Following centrifugation and incubation at 85oC for 10 minutes to dissolve the fibres, 20 μL proteinase
K was added and incubated at 56°C for 45 min. Following protein digestion, the cell lysate was dissolved in 200 μl
of 96% ethanol and DNA extraction was carried out using the QIAmp Mini Spin Column. Finally, the eluted DNA
was dissolved in 150 μl Buffer AE (Qiagen, 2016).
Amplification and sequencing of the isolated DNA was carried out by the Australian Genome Research Facility
(AGRF). The gene locus chosen for identification of the species of meat was a 420bp fragment in the cytochrome b
gene. To this end, primers L14841 and H15149 previously described by Kocher et al., (1989) was utilised as the
forward and reverse primer respectively (Appendix). Subsequently, DNA was amplified for 40 cycles (Appendix) in
the Applied BioSystems 9700 thermal cycler using 50ml reaction mixtures, containing 20mM primers, 2.5 units of
Taq polymerase, 10X Gold ST+R reaction buffer and approximately 1.6 × 10 93 ng of DNA. The amplicons were
analysed using agarose gel and purified using QIAGEN PCR purification kits. Then the amplicons were cleaned up
using a combination of exonuclease-1 and shrimp alkaline phosphatase for 15 minutes at room temperature and
80OC respectively. Finally, sequencing of the amplicon was carried out in the Applied Biosystems ABI automated
DNA analyser using the BigDye Terminator cycle sequencing reaction kit (Gupta et al., 2011). The obtained
sequences were profiled by comparing them against known sequences in the NCBI database using the Basic Local
Alignment Search Tool (BLAST) (Altschul et al., 1990).
RESULTS & DISCUSSION
The primary challenge in the identification and prosecution of wildlife crime has been the inability of authorities to
accurately prove the source of specimens. To this end, the forensic sciences aided by robust DNA analysis methods
have come a long way in controlling the senseless killing and trade of wildlife (Linacre & Tobe, 2013). In this
article, we sought to describe and demonstrate a simple, rapid and cost-effective method for DNA analysis using
meat specimens isolated from an alleged poaching incident.
The BLAST analysis revealed several possible matches between the sequenced unknown sample and cytochrome b
sequences of mammals (Description table, Appendix). In particular, significant similarity was observed between the
unknown query and placentals (Taxonomic table, Appendix). Concurrently, a basic pairwise alignment-based
distance tree was constructed using Fast Alignment displaying the relationship between the sequences of unknown
and matched samples. A high degree of concurrence was revealed between 100% of the unknown sample sequence
and cytochrome b of Smutsia gigantea (distance=1) (Figure 1).
Figure 1: Distance tree of unknown query with aligned samples. Pairwise Alignment based tree constructed by
search and alignment of unknown sample sequence against nucleotide sequences in the NCBI database.
Table 1 Alignment of Cytochrome b Amplicon of unknown meat sample versus Smutsia gigantea
Score
765 bits(414)
Expect
Identities
0.0
418/420(99%)
Gaps
0/420(0%)
Strand
Plus/Plus
Query
1
ATGACAAACATCCGAAAATCCCACCCTCTATTAAAAATTATTAATGACTCTTTCATCGAC
60
Sbjct
14135
............................................................
14194
Query
61
CTCCCAACCCCCTCTAATATCTCAGCATGATGAAATTTCGGATCCCTATTAGGAATTTGC
120
Sbjct
14195
......................................T.....................
14254
Query
121
TTAATCTTACAAATTATAACCGGCCTATTCCTAGCAATACACTACACGGCAGACACCATA
180
Sbjct
14255
............................................................
14314
Query
181
ACCGCATTCTCATCAGTCACACACATTTGCCGAGACGTAAACTACGGCTGAATTATCCGT
240
Sbjct
14315
............................................................
14374
Query
241
TACATACACGCCAACGGCGCATCAATATTCTTTATTTGCCTATTTATCCATATCGGACGA
300
Sbjct
14375
............................................................
14434
Query
301
GGCCTATATTACGGATCCTTCATCTGCAAAGAAACATGAAACATTGGAATTATCCTCTTA
360
Sbjct
14435
.........................A..................................
14494
Query
361
TTTACAGTCATAGCTACAGCCTTCGTAGGATATGTCCTACCATGAGGACAAATATCCTTC
420
Sbjct
14495
............................................................
14554
Furthermore, to confirm the identity of the unknown sequence, a pairwise alignment of the unknown sample
sequence was carried out with the cytochrome b gene of Smutsia gigantea (Table 1), revealing a 99% identity
between the sequences. In 2019, pangolins were the most highly traded species, owing to their use in traditional
medicine and consumption as a delicacy in some Asian countries. As a result, in 2019, all 8 species of pangolin were
classified as endangered for the first time, and placed in appendix I of the CITES database (Heinrich et al., 2016).
Taken together, the findings provide significant support for the proposition that the unknown meat sample was
obtained illegal from Smutsia gigantea (Common Name: Giant Pangolin) ( E Value=0%, Query Length =100%,
Percent Identity 99%). In this study, we provide an easily reproducible protocol which can be utilised to isolate and
sequence DNA from wildlife crime scenes. Going forward, we hope that this protocol can be utilised to prosecute,
and hence control the rates of wildlife crime.
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APPENDICES
PRIMER SEQUENCES
FORWARD PRIMER (L14841)
REVERSE PRIMER (H15149)
5'-AAAAAGCTTCCATCCAACATCTCAGCATGATGAAA-3'
5'-AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA-3'
INITIAL PCR CYCLING CONDITIONS
SEQUENCE DATA
ATGACAAACATCCGAAAATCCCACCCTCTATTAAAAATTATTAATGACTCTTTCATCGACCTCCCAACCCCCTCTAATATCTCAGC
ATGATGAAATTTCGGATCCCTATTAGGAATTTGCTTAATCTTACAAATTATAACCGGCCTATTCCTAGCAATACACTACACGGCAG
ACACCATAACCGCATTCTCATCAGTCACACACATTTGCCGAGACGTAAACTACGGCTGAATTATCCGTTACATACACGCCAACGG
CGCATCAATATTCTTTATTTGCCTATTTATCCATATCGGACGAGGCCTATATTACGGATCCTTCATCTGCAAAGAAACATGAAACA
TTGGAATTATCCTCTTATTTACAGTCATAGCTACAGCCTTCGTAGGATATGTCCTACCATGAGGACAAATATCCTTC
TAXONOMIC CLASSIFICATION OF HITS
DESCRIPTIONS TABLE
Description
Scientific Name
Max Score
Total Score
Query Cover
E value
Per. ident
Manis gigantea isolate CAM011 mitochondrion,
complete genome
Smutsia gigantea
765
765
100%
0.0
99.52
Manis gigantea isolate T2269 mitochondrion,
complete genome
Smutsia gigantea
760
760
100%
0.0
99.29
Manis tricuspis isolate T1855 mitochondrion,
complete genome
Phataginus tricuspis
455
455
100%
1E-123
86.19
Antilocapra americana isolate UAM mitochondrion,
complete genome
Antilocapra americana
427
427
100%
3E-115
85.00
Manis tetradactyla isolate T612 mitochondrion,
complete genome
Phataginus tetradactyla
470
470
99%
5E-128
87.02
726
726
95%
0.0
99.25
Smutsia gigantea isolate Scale-Sg-H006 cytochrome b Smutsia gigantea
(cytb) gene, partial cds; mitochondrial