Research Article| Volume 16, P64-70, May 2015

Molecular identification of python species: Development and validation of a novel assay for forensic investigations

Published:December 11, 2014DOI:


      • A novel primer set to identify python species in forensic inquiries.
      • Phylogenetic reconstruction accurately identifies species with statistical support.
      • Primer pair shows high sensitivity, amplifying 30 fg or 9 mitochondrial DNA copies.
      • The assay has demonstrated suitability to samples of poor quality.
      • Validation tests demonstrate this assay as suitable for forensic investigations.


      Python snake species are often encountered in illegal activities and the question of species identity can be pertinent to such criminal investigations. Morphological identification of species of pythons can be confounded by many issues and molecular examination by DNA analysis can provide an alternative and objective means of identification. Our paper reports on the development and validation of a PCR primer pair that amplifies a segment of the mitochondrial cytochrome b gene that has been suggested previously as a good candidate locus for differentiating python species. We used this DNA region to perform species identification of pythons, even when the template DNA was of poor quality, as might be the case with forensic evidentiary items. Validation tests are presented to demonstrate the characteristics of the assay. Tests involved the cross-species amplification of this marker in non-target species, minimum amount of DNA template required, effects of degradation on product amplification and a blind trial to simulate a casework scenario that provided 100% correct identity. Our results demonstrate that this assay performs reliably and robustly on pythons and can be applied directly to forensic investigations where the presence of a species of python is in question.


      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Forensic Science International: Genetics
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Auliya M.
        Hot Trade in Cool Creatures–A Review of the Live Reptile Trade in the European Union in the 1990 with a focus on Germany. TRAFFIC Europe.
        Brussels, Belgium2003
      1. L. Edwards, Silent victims of a cruel trade. 2007 (accessed 12/01/2014); available from:

        • Barry C.
        Rogue traders.
        Aust. Geogr. Mag. 2011; : 124-128
      2. CITES. Appendices I, II and III. 2013 (accessed 26/01/2014); available from:

      3. B. Halstead, Traffic in flora and fauna. Trends and issues in crime and criminal justice - No. 41 1992; available from:

        • Linacre A.
        • Tobe S.
        An overview to the investigative approach to species testing in wildlife forensic science.
        Investig. Genet. 2011; 2: 2
        • Irwin D.
        • Kocher T.
        • Wilson A.
        Evolution of the cytochrome b gene of mammals.
        J. Mol. Evol. 1991; 32: 128-144
        • Kocher T.
        • Thomas W.
        • Meyer A.
        • Edwards S.
        • Pääbo S.
        • Villablanca A.
        • Wilson F.
        Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers.
        Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6196-6200
        • Hsieh H.
        • Chiang H.
        • Tsai L.
        • Lai S.
        • Huang N.
        • Linacre J.
        • Lee A.
        Cytochrome b gene for species identification of the conservation animals.
        Forensic Sci. Int. 2001; 122: 7-18
        • Bartlett S.
        • Davidson W.
        FINS (forensically informative nucleotide sequencing): a procedure for identifying the animal origin of biological specimens.
        Biotechniques. 1992; 12: 408-411
        • Hebert P.
        • Cywinska A.
        • Ball J.
        • DeWaard S.
        Biological identifications through DNA barcodes.
        Proc. R. Soc. Lond. B Biol. Sci. 2003; 270: 313-321
        • Wong K.
        • Wang J.
        • But P.
        • Shaw P.
        Application of cytochrome b DNA sequences for the authentication of endangered snake species.
        Forensic Sci. Int. 2004; 139: 49-55
        • Dubey B.
        • Meganathan I.
        • Haque P.
        Molecular identification of three Indian snake species using simple PCR-RFLP method.
        J. Forensic Sci. 2010; 55: 1065-1067
        • Dubey B.
        • Meganathan I.
        • Haque P.
        Multiplex PCR assay for rapid identification of three endangered snake species of India.
        Conserv. Genet. 2009; 10: 1861-1864
        • Dubey B.
        • Meganathan I.
        • Haque P.
        DNA mini-barcoding: an approach for forensic identification of some endangered Indian snake species.
        Forensic Sci. Int. Genet. 2011; 5: 181-184
        • An J.
        • Lee M.
        • Min M.
        • Lee M.
        • Lee H.
        A molecular genetic approach for species identification of mammals and sex determination of birds in a forensic case of poaching from South Korea.
        Forensic Sci. Int. 2007; 167: 59-61
        • Verma L.
        • Singh S.
        Novel Universal primers establish identity of an enormous number of animal species for forensic application.
        Mol. Ecol. Notes. 2003; 3: 28-31
        • Baker S.
        • Palumbi C.
        Which whales are hunted? A molecular genetic approach to monitoring whaling.
        Science (New York, N.Y.). 1994; 265: 1538-1539
      4. SWGDAM. Revised Validation Guidelines: Scientific Working Group on DNA Analysis Methods (SWGDAM). 2004 (accessed 09/08/2014); available from:

        • Budowle B.
        • Garofano P.
        • Hellmann A.
        • Ketchum M.
        • Kanthaswamy S.
        • Parson W.
        • van Haeringen W.
        • Fain S.
        • Broad T.
        Recommendations for animal DNA forensic and identity testing.
        Int. J. Legal Med. 2005; 119: 295-302
        • Linacre A.
        • Gusamo L.
        • Hecht W.
        • Hellmann A.
        • Mayr W.
        • Parson W.
        • Prinz M.
        • Schneider P.
        • Morling N.
        ISFG: recommendations regarding the use of non-human (animal) DNA in forensic genetic investigations.
        Forensic Sci. Int. Genet. 2011; 5: 501-505
      5. SWGWILD. SWGWILD Standards and Guidelines 2013 (accessed 09/08/2014); available from:–0_12192012pdf.

        • Ciavaglia S.
        • Donnellan S.
        • Henry A.
        • Linacre J.
        Species identification of protected carpet pythons suitable for degraded forensic samples.
        Forensic Sci. Med. Pathol. 2014; 10: 295-305
        • Wiens J.
        • Hutter C.
        • Mulcahy D.
        • Noonan B.
        • Townsend T.
        • Sites T.
        • Reeder J.
        Resolving the phylogeny of lizards and snakes (Squamata) with extensive sampling of genes and species.
        Biol. Lett. 2012; 8: 1043-1046
        • Rozen S.
        • Skaletsky H.
        Primer3 on the WWW for general users and for biologist programmers.
        in: Misener S. Krawetz S. Bioinformatics Methods and Protocols. Humana Press, 1999: 365-386
        • Tamura K.
        • Peterson D.
        • Peterson N.
        • Stecher G.
        • Nei S.
        • Kumar M.
        MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance and maximum parsimony methods.
        Mol. Biol. Evol. 2011; 28: 2731-2739
      6. M. Miller, W. Pfeiffer and T. Schwartz. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In Gateway Computing Environments Workshop (GCE), 2010. 2010.

        • Lanfear R.
        • Calcott B.
        • Ho S.
        • Guindon S.
        Partition finder: combined selection of partitioning schemes and substitution models for phylogenetic analyses.
        Mol. Biol. Evol. 2012; 29: 1695-1701
        • Castoe T.
        • de Koning A.
        • Hall K.
        • Card D.
        • Schield D.
        • Fujita M.
        • Ruggiero R.
        • Degner J.
        • Daza J.
        • Gu W.
        • Reyes-Velasco J.
        • Shaney K.
        • Castoe J.
        • Fox S.
        • Poole A.
        • Polanco D.
        • Dobry J.
        • Vandewege M.
        • Li Q.
        • Schott R.
        • Kapusta A.
        • Minx P.
        • Feschotte C.
        • Uetz P.
        • Ray D.
        • Hoffmann F.
        • Bogden R.
        • Smith E.
        • Chang B.
        • Vonk F.
        • Casewell N.
        • Henkel C.
        • Richardson M.
        • Mackessy S.
        • Bronikowski A.
        • Yandell M.
        • Warren W.
        • Secor S.
        • Pollock D.
        The Burmese python genome reveals the molecular basis for extreme adaptation in snakes.
        Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 20645-20650
        • Satoh T.
        • Kuroiwa M.
        Organization of multiple nucleoids and DNA molecules in mitochondria of a human cell.
        Exp. Cell Res. 1991; 196: 137-140
        • Rawlings S.
        • Donnellan L.
        Phylogeographic analysis of the green python, Morelia viridis, reveals cryptic diversity.
        Mol. Phylogenet. Evol. 2003; 27: 36-44