Skip to main content
Log in

Two universal primer sets for species identification among vertebrates

  • Short Communication
  • Published:
International Journal of Legal Medicine Aims and scope Submit manuscript


The aim of this study was to develop a simple method using universal primers for species identification based on direct PCR sequencing. Two primer sets were designed based on the conserved regions of the 12S and 16S rRNA loci detected by the comprehensive sequence comparison among 30 mammalian whole mitochondrial genomes. In humans, the expected sizes of PCR products of the 12S and 16S rRNAs were 215 and 244  bp, respectively. Both primer sets successfully amplified the expected PCR products from various kinds of vertebrates including mammals, birds, reptiles, amphibians, and fish, and the sequenced segments contained sufficient nucleotide differences to identify each animal species. A case example of the identification of a piece of buried bone of unknown species is presented, and the species was identified as a pig by this method.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2


  1. Zehner R, Zimmermann S, Mebs D (1998) RFLP and sequence analysis of the cytochrome b gene of selected animals and man: methodology and forensic application. Int J Legal Med 111:323–327

    Article  PubMed  CAS  Google Scholar 

  2. Bravi CM, Liron JP, Mirol PM, Ripoli MV, Peral-Garcia P, Giovambattista G (2004) A simple method for domestic animal identification in Argentina using PCR-RFLP analysis of cytochrome b gene. Leg Med (Tokyo) 6:246–251

    CAS  Google Scholar 

  3. Kocher TD, Thomas WK, Meyer A, Edwards SV, Pääbo S, Villablanca FX, Wilson AC (1989) Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proc Natl Acad Sci USA 86:6196–6200

    Article  PubMed  CAS  Google Scholar 

  4. Parson W, Pegoraro K, Niederstatter H, Föger M, Steinlechner M (2000) Species identification by means of the cytochrome b gene. Int J Legal Med 114:23–28

    Article  PubMed  CAS  Google Scholar 

  5. Hsieh HM, Chiang HL, Tsai LC, Lai SY, Huang NE, Linacre A, Lee JC (2001) Cytochrome b gene for species identification of the conservation animals. Forensic Sci Int 122:7–18

    Article  Google Scholar 

  6. Branicki W, Kupiec T, Pawlowski R (2003) Validation of cytochrome b sequence analysis as a method of species identification. J Forensic Sci 48:83–87

    PubMed  CAS  Google Scholar 

  7. Bellis C, Ashton KJ, Freney L, Blair B, Griffiths LR (2003) A molecular genetic approach for forensic animal species identification. Forensic Sci Int 134:99–108

    Article  PubMed  CAS  Google Scholar 

  8. Balitzki-Korte B, Anslinger K, Bartsch C, Rolf B (2005) Species identification by means of pyrosequencing the mitochondrial 12S rRNA gene. Int J Legal Med 119:291–294

    Article  PubMed  CAS  Google Scholar 

  9. Bataille M, Crainic K, Leterreux M, Durigon M, de Mazancourt P (1999) Multiplex amplification of mitochondrial DNA for human and species identification in forensic evaluation. Forensic Sci Int 99:165–170

    Article  PubMed  CAS  Google Scholar 

  10. Dalmasso A, Fontanella E, Piatti P, Civera T, Rosati S, Bottero MT (2004) A multiplex PCR assay for the identification of animal species in feedstuffs. Mol Cell Probes 18:81–87

    Article  PubMed  CAS  Google Scholar 

  11. Thompson JD, Gibson TJ, Higgins DG (1994) ClustalW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

    Article  PubMed  CAS  Google Scholar 

  12. Nei M (1987) Molecular evolutionary genetics. Columbia University Press, New York, pp 254–286

    Google Scholar 

  13. Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120

    Article  PubMed  CAS  Google Scholar 

  14. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    PubMed  CAS  Google Scholar 

  15. Springer MS, Douzery E (1996) Secondary structure and patterns of evolution among mammalian mitochondrial 12S rRNA molecules. J Mol Evol 43:357–373

    PubMed  CAS  Google Scholar 

  16. Burk A, Douzery EJP, Springer MS (2002) The secondary structure of mammalian mitochondrial 16S rRNA molecules: refinements based on a comparative phylogenetic approach. J Mamm Evol 9:225–252

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations


Corresponding author

Correspondence to Motoki Osawa.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Table

(DOC 71 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kitano, T., Umetsu, K., Tian, W. et al. Two universal primer sets for species identification among vertebrates. Int J Legal Med 121, 423–427 (2007).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: