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DNA-based identification of big cats and traditional Chinese medicine artifacts in the Czech Republic

  • Lenka Vankova
    Affiliations
    Forensic DNA Service, Budinova 2, 180 81 Prague 8, Czech Republic

    Charles Univ Prague, Institute for Environmental Sciences, Prague, Czech Republic
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  • Daniel Vanek
    Correspondence
    Corresponding author at: Forensic DNA Service, Janovskeho 18, 170 00 Prague 7, Czech Republic.
    Affiliations
    Forensic DNA Service, Budinova 2, 180 81 Prague 8, Czech Republic

    Bulovka University Hospital, Prague, Czech Republic

    Charles Univ Prague, Institute for Environmental Sciences, Prague, Czech Republic

    Department of Forensic Medicine, Second Faculty of Medicine, Charles University, Czech Republic
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Published:October 06, 2022DOI:https://doi.org/10.1016/j.fsigss.2022.10.005

      Abstract

      The aim of this study is to provide an overview of ongoing research on and the development of identification tools for big cats (Panthera tigris, Panthera leo, Panthera pardus, …). The set of tools includes a species-specific RT PCR quantitation system (nuclear and mitochondrial), STR multiplexes, a rapid system for big cat species determination, and a database solution.

      Keywords

      1. Introduction

      Illegal trade of parts and products from big cats (Panthera tigris, Panthera leo, Panthera onca, Panthera pardus, …) is extensive not only in Asia but also in Europe due to high demand in Asian communities living outside Asia. There are many big cats housed in various facilities, and some of those animals are probably used as a source of products for the black market and are involved in illicit trade. There has been a significant increase in product seizures, including TCM (traditional Chinese medicine), in the Czech Republic in recent years [
      • Votrubova J.
      • Rihova P.
      • Saskova L.
      • Vanek D.
      Operation Tiger’s Eye: DNA testing of traditional Chinese medicine artifacts in the Czech Republic.
      ]. The traded items include not only bones, claws, teeth and skins (as described by enforcement authorities worldwide) but also processed products such as broth, paste, powder, and wine, which are difficult to identify using standard methodologies. Therefore, our labs focused on the research and development of protocols and tools for DNA-based species and individual identification from TCM products and other artifacts.

      2. Materials and methods

      2.1 Species identification

      For species identification, we used the following approaches:
      • 1)
        mtDNA sequencing of at least 2 targets (COI, cytB, and 12S rRNA) with subsequent database comparison (NCBI, Boldsystems) [
        • Harris D.J.
        Can you bank on GenBank?.
        ,
        • Dawnay N.
        • Ogden R.
        • McEwing R.
        • Carvalho G.R.
        • Thorpe R.S.
        Validation of the barcoding gene COI for use in forensic genetic species identification.
        ].
      • 2)
        tRNA-proline length polymorphism (SeqStudio 3200) with subsequent species library comparison [
        • Pun K.M.
        • Albrecht C.
        • Castella V.
        • Fumagalli L.
        Species identification in mammals from mixed biological samples based on mitochondrial DNA control region length polymorphism.
        ] (Fig. 1). The tRNA-proline length polymorphism system does not require special instruments. It is sufficiently sensitive and able to discriminate all species of big cats that are commonly found in traditional Chinese medicine (TCM) artifacts.
        Fig. 1
        Fig. 1tRNA-proline length polymorphism (SeqStudio 3200) with subsequent species library comparison.

      2.2 DNA-based individual identification

      We have developed an in-house Panthera tigris-specific RT PCR quantification system (Ptig Qplex) [
      • Vaněk D.
      • Ehler E.
      • Vaňková L.
      Development of DNA quantitation and STR typing systems for Panthera tigris species determination and individual identification in forensic casework.
      ] (Fig. 2), STR multiplexes for Pantheridae (Ptig STRplex) [
      • Vaněk D.
      • Ehler E.
      • Vaňková L.
      Development of DNA quantitation and STR typing systems for Panthera tigris species determination and individual identification in forensic casework.
      ] (Fig. 3) and an MS Access database-based SW environment for the comparison of DNA profiles. The resulting database matches were confirmed by statistical evaluation using species-specific STR allelic frequencies.
      Fig. 2
      Fig. 2RT PCR quantitation system Ptig Qplex targeting mtDNA (red), nuclear DNA (blue) and IPC (green) on P.tigris, P.leo and Tigon samples.
      Fig. 3
      Fig. 3STR-multiplexed Ptig STRplex for DNA-based identification of big cats.
      RT PCR quantitation of P. tigris DNA (Fig. 2) utilizes TaqMan probes and targets the following sequences: mtDNA (D-loop Panthera tigris), nuclear DNA (STR locus Pati01, repeat (TGC)n P. tigris altaica), and the internal positive amplification control IPC (artificial DNA sequence; measures the efficiency of PCR + presence of inhibitors). Candidate loci (4 bp repeats preferred, as recommended by ISFG [
      • Linacre A.
      • Gusmão L.
      • Hecht W.
      • Hellmann A.
      • Mayr W.
      • Parson W.
      • et al.
      ISFG: recommendations regarding the use of non-human (animal) DNA in forensic genetic investigations.
      ]) for STR multiplexes were selected using bioinformatic analyses of Pantheridae genome sequences. STR loci were tested on numerous reference samples, and highly polymorphic loci were combined into multiplexes (Fig. 3). The comparison of resulting DNA profiles was performed in a DNA database (TigrisID) that is used for the storage and exchange of animal DNA typing results. The database uses MS Access/SQL/ASP.NET.

      3. Results

      Species-specific quantitation and STR typing assays (Ptig Qplex, Ptig STRPlex) enable rapid, sensitive and streamlined analysis of forensic samples submitted for examination.
      The TigrisID database contains > 450 records (reference and casework samples). A total of 108 reference samples are from abroad (Slovakia 70, Russia 11, United Kingdom 9, Finland 4, Germany 4, Ireland 3, Spain 3, France 2, Latvia 1, and Italy 1). DNA profiles of 90 unrelated individuals (cross-checked) have been used for the population study. Twelve percent of DNA profiles are related to casework. To date, the database searches revealed 6 cold matches (linking artefacts/animals).

      4. Conclusion

      The illegal trade of CITES protected organisms, including big cats, seems to be more extensive than originally estimated. This trade exists not only in Asia but also in Europe and includes less recognizable types of goods such as bones, claws, teeth, broth, paste, wine, and powder. DNA-based species and individual identification helps fight illicit trade. RT PCR species-specific DNA quantitation systems for other big cats and additional autosomal and gonosomal STR markers are under development.

      Conflict of interest statement

      The authors of this manuscript declare no conflicts of interest. This project was supported by research project VH20182021028 of the Ministry of Interior, Czech Republic. The research was also partially covered by the project, An effective use of forensic methods in wildlife crime investigation“ (Ministry of Interior, Czech Republic, VJ01010026). The reference samples were kindly provided by the Czech Environmental Inspectorate and various zoos.

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