Development of a multiplex, PCR-based genotyping assay for African and Asian elephants for forensic purposes

  • Margret J. Potoczniak
  • Michael Chermak
  • Lawrence Quarino
  • Shanan S. Tobe
  • Jillian ConteEmail author
Original Article


Wildlife crimes and the threats they present to elephant populations raise the need to develop and implement DNA-based methodology as an aid for wildlife forensic investigations and conservation efforts. This study describes the development of a tetra-nucleotide repeat STR multiplex, genotyping assay that will identify Asian elephant (Elephas maximus) and African elephant (Loxodonta africana) DNA. The assay targets six tetra-nucleotide STRs and two sex-typing markers simultaneously in both genera of elephants, a first for elephant genotyping assays. The developed assay has potential application in wildlife investigations to associate a biological sample to a particular individual elephant and additionally in conservation science for population management.


Wildlife forensics Elephant Validation Genotyping Loxodonta africana Elephas maximus 



This work would not be possible without DNA sources from Six Flags: Safari Off Road Adventure, the Cincinnati Zoo, the St. Louis Zoo, the Northeast Wildlife DNA Laboratory, Quakertown Veterinary Clinic, and the Brandywine Zoo. Thank you for your contributions.

Funding information

Arcadia University’s Master of Forensic Science Program, the University of the Sciences Biological Sciences Department, and the Department of Biological and Physical Sciences at Keystone College provided funds towards this project.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

The authors did not come into contact nor were they responsible for the care of any animals in the course of this research; thus, ethical approval is not needed. Animal samples were collected by their caretakers.

Research involving human subjects

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee (Keystone College IRB Committee IRB ID No. 2017-000673) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Supplementary material

414_2019_2097_MOESM1_ESM.docx (29 kb)
ESM 1 (DOCX 29 kb)


  1. 1.
    Wasser SK, Brown L, Mailand C, Mondol S, Clark W, Laurie C, Weir BS (2015) Genetic assignment of large seizures of elephant ivory reveals Africa’s major poaching hotspots. Science 349:84–87. CrossRefGoogle Scholar
  2. 2.
    Hughes CR, Queller DC (1993) Detection of highly polymorphic microsatellite loci in a species with little allozyme polymorphism. Mol Ecol 2:131–137. CrossRefGoogle Scholar
  3. 3.
    Rassmann K, Schlötterer C, Tautz D (1991) Isolation of simple-sequence loci for use in polymerase chain reaction-based DNA fingerprinting. Electrophoresis 12:113–118. CrossRefGoogle Scholar
  4. 4.
    Butler JM (2005) Forensic DNA typing: biology, technology, and genetics of STR markers. Burlington, MA: Elsevier Academic PressGoogle Scholar
  5. 5.
    Archie EA, Moss CJ, Alberts SC (2003) Characterization of tetranucleotide microsatellite loci in the African Savannah elephant (Loxodonta africana africana). Mol Ecol Notes 3:244–246. CrossRefGoogle Scholar
  6. 6.
    Fernando P, Vidya TNC, Melnick DJ (2001) Isolation and characterization of tri- and tetranucleotide microsatellite loci in the Asian elephant, Elephas maximus. Mol Ecol Notes 1:232–233. CrossRefGoogle Scholar
  7. 7.
    Wasser SK, Joseph Clark W, Drori O et al (2008) Combating the illegal trade in African elephant ivory with DNA forensics. Conserv Biol 22:1065–1071. CrossRefGoogle Scholar
  8. 8.
    Wasser SK, Shedlock AM, Comstock K, Ostrander EA, Mutayoba B, Stephens M (2004) Assigning African elephant DNA to geographic region of origin: applications to the ivory trade. Proc Natl Acad Sci U S A 101:14847–14852. CrossRefGoogle Scholar
  9. 9.
    Ahlering MA, Hailer F, Roberts MT, Foley C (2011) A simple and accurate method to sex savannah, forest and Asian elephants using noninvasive sampling techniques. Mol Ecol Resour 11:831–834. CrossRefGoogle Scholar
  10. 10.
    Vallone PM, Butler JM (2004) AutoDimer: a screening tool for primer-dimer and hairpin structures. Biotechniques 37:226–231.
  11. 11.
    Conte J, Potoczniak MJ, Tobe SS (2018) Using synthetic oligonucleotides as standards in probe-based qPCR. BioTechniques 64:177–179. CrossRefGoogle Scholar
  12. 12.
    Conte J, Potoczniak MJ, Mower C, Tobe SS (2019) ELEquant: a developmental framework and validation of forensic and conservation real-time PCR assays. Mol Biol Rep 46:2093–2100. CrossRefGoogle Scholar
  13. 13.
    Bustin, SA (2004). AZ of quantitative PCR. La Jolla, CA: International University LineGoogle Scholar
  14. 14.
    Henegariu O, Heerema NA, Dlouhy SR, Vance GH, Vogt PH (1997) Multiplex PCR: critical parameters and step-by-step protocol. Biotechniques 23:504–511. CrossRefGoogle Scholar
  15. 15.
    Bregu J, Conklin D, Coronado E, Terrill M, Cotton RW, Grgicak CM (2013) Analytical thresholds and sensitivity: establishing RFU thresholds for forensic DNA analysis. J Forensic Sci 58:120–129. CrossRefGoogle Scholar
  16. 16.
    Ishida Y, Demeke Y, de GPJ v C et al (2012) Short amplicon microsatellite markers for low quality elephant DNA. Conserv Genet Resour 4:491–494. CrossRefGoogle Scholar
  17. 17.
    Nyakaana S, Okello JBA, Muwanika V, Siegismund HR (2005) Six new polymorphic microsatellite loci isolated and characterized from the African savannah elephant genome. Mol Ecol Notes 5:223–225. CrossRefGoogle Scholar
  18. 18.
    Wozney KM, Wilson PJ (2012) Real-time PCR detection and quantification of elephantid DNA: species identification for highly processed samples associated with the ivory trade. Forensic Sci Int 219:106–112. CrossRefGoogle Scholar
  19. 19.
    Gugala NA, Ishida Y, Georgiadis NJ, Roca AL (2016) Development and characterization of microsatellite markers in the African forest elephant (Loxodonta cyclotis). BMC Res Notes 9(364):364. CrossRefGoogle Scholar
  20. 20.
    Suwattana D, Jirasupphachok J, Kanchanapangka S, Koykul W (2010) Tetranucleotide microsatellite markers for molecular testing in Thai domestic elephants (Elephas maximus indicus). Thai J Vet Med 40:405–409Google Scholar
  21. 21.
    Kinuthia J, Harper C, Muya S, Kimwele C, Alakonya A, Muigai A, Gakuya F, Mwaniki M, Gatebe E (2015) The selection of a standard STR panel for DNA profiling of the African elephant. Conserv Genet Resour 7:305–307. CrossRefGoogle Scholar
  22. 22.
    Chakraborty R, Kimmel M, Stivers DN, Davison LJ, Deka R (1997) Relative mutation rates at di-, tri-, and tetranucleotide microsatellite loci. Proc Natl Acad Sci 94:1041–1046.
  23. 23.
    Ellegren H (2004) Microsatellites: simple sequences with complex evolution. Nat Rev Genet 5:435–445CrossRefGoogle Scholar
  24. 24.
    Linacre A, Tobe S (2013) Wildlife DNA analysis: applications in forensic science. Boca Raton, FL: CRC PressGoogle Scholar
  25. 25.
    Gupta SK, Thangaraj K, Singh L (2006) A simple and inexpensive molecular method for sexing and identification of the forensic samples of elephant origin. J Forensic Sci 51:805–807. CrossRefGoogle Scholar
  26. 26.
    Linacre A, Gusmao L, Hecht W et al (2011) ISFG: recommendations regarding the use of non-human (animal) DNA in forensic genetic investigations. Forensic Sci Int Genet 5:501–505. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Chemistry, Physics & Forensic ScienceArcadia UniversityGlensideUSA
  2. 2.Department of Biological & Physical SciencesKeystone CollegeLa PlumeUSA
  3. 3.Department of Chemical and Physical SciencesCedar Crest CollegeAllentownUSA
  4. 4.College of Science, Health, Engineering and EducationMurdoch UniversityMurdochAustralia
  5. 5.Department of Biological SciencesUniversity of the SciencesPhiladelphiaUSA

Personalised recommendations