Molecular Biology Reports

, Volume 38, Issue 6, pp 3929–3934 | Cite as

Sequence of specific mitochondrial 16S rRNA gene fragment from Egyptian buffalo is used as a pattern for discrimination between river buffaloes, cattle, sheep and goats

  • Hassan A. I. RamadanEmail author


Characterization of molecular markers and the development of better assays for precise and rapid detection of domestic species are always in demand. This is particularly due to recent food scares and the crisis of biodiversity resulting from the huge ongoing illegal traffic of endangered species. The aim of this study was to develop a new and easy method for domestic species identification (river buffalo, cattle, sheep and goat) based on the analysis of a specific mitochondrial nucleotide sequence. For this reason, a specific fragment of Egyptian buffalo mitochondrial 16S rRNA gene (422 bp) was amplified by PCR using two universal primers. The sequence of this specific fragment is completely conserved between all tested Egyptian buffaloes and other river buffaloes in different places in the world. Also, the lengths of the homologous fragments were less by one nucleotide (421 bp) in case of goats and two nucleotides (420 bp) in case of both cattle and sheep. The detection of specific variable sites between investigated species within this fragment was sufficient to identify the biological origin of the samples. This was achieved by alignment between the unknown homologous sequence and the reference sequences deposited in GenBank database (accession numbers, FJ748599–FJ748607). Considering multiple alignment results between 16S rRNA homologous sequences obtained from GenBank database with the reference sequence, it was shown that definite nucleotides are specific for each of the four studied species of the family Bovidae. In addition, other nucleotides are detected which can allow discrimination between two groups of animals belonging to two subfamilies of family Bovidae, Group one (closely related species like cattle and buffalo, Subfamily Bovinae) and Group two (closely related species like sheep and goat, Subfamily Caprinae). This 16S DNA barcode character-based approach could be used to complement cytochrome c oxidase I (COI) in DNA barcoding. Also, it is a good tool for identification of unknown sample belonging to one of the four domestic animal species of family Bovidae quickly and easily.


Sequence analysis Bovidae Species identification Mitochondrial DNA 16S rRNA gene 


  1. 1.
    Pascal G, Mahe S (2001) Identity, traceability, acceptability and substantial equivalence of food. Cell Mol Biol 47:1329–1342PubMedGoogle Scholar
  2. 2.
    Skarpeid HJ, Kvaal K, Hildrum KI (1998) Identification of animal species in ground meat mixtures by multivariate analysis of isoelectric focusing protein profiles. Electrophoresis 19:3103–3109PubMedCrossRefGoogle Scholar
  3. 3.
    Hsieh YH, Sheu SC, Bridgman RC (1998) Development of a monoclonal antibody specific to cooked mammalian meats. J Food Prot 61(4):476–487PubMedGoogle Scholar
  4. 4.
    Ashmoor SH, Monte WC, Stiles PG (1998) Liquid chromatographic identification of meats. J Assoc Off Anal Chem 71:397–403Google Scholar
  5. 5.
    Parson W, Pegoraro K, Niederstatter H, Föger M, Steinlechner M (2000) Species identification by means of the cytochrome b gene. Int J Legal Med 114:23–28PubMedCrossRefGoogle Scholar
  6. 6.
    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 122:7–18PubMedCrossRefGoogle Scholar
  7. 7.
    Murray BW, McClymont RA, Strobeck C (1995) Forensic identification of ungulate species using restriction digests of PCR-amplified mitochondrial DNA. J Forensic Sci 40(6):943–951PubMedGoogle Scholar
  8. 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 119:291–294PubMedCrossRefGoogle Scholar
  9. 9.
    Rodríguez MA, García T, González I, Asensio L, Hernández PE, Martín R (2004) PCR identification of beef, sheep, goat, and pork in raw and heat-treated meat mixtures. J Food Prot 67(1):172–177PubMedGoogle Scholar
  10. 10.
    Rodríguez MA, García T, González I et al (2003) Identification of goose, mule, duck, chicken, turkey, and swine in foie gras by species-specific polymerase chain reaction. J Agric Food Chem 51:1524–1529PubMedCrossRefGoogle Scholar
  11. 11.
    Montiel-Sosa JF, Ruiz-Pesini E, Montoya J, Roncalés P, López-Pérez MJ, Pérez-Martos A (2000) Direct and highly species-specific detection of pork meat and fat in meat products by PCR amplification of mitochondrial DNA. J Agric Food Chem 48:2829–2832PubMedCrossRefGoogle Scholar
  12. 12.
    Ramadan HAI, El Hefnawi M (2008) Phylogenetic analysis and comparison between cow and buffalo (including Egyptian buffaloes) mitochondrial displacement-loop regions. Mitochondrial DNA 19(4):401–410PubMedGoogle Scholar
  13. 13.
    Waugh J (2007) DNA barcoding in animal species: progress, potential and pitfalls. BioEssays 29(2):188–197PubMedCrossRefGoogle Scholar
  14. 14.
    Hebert PDN, Cywinska A, Ball SL, de Waard JR (2003) Biological identifications through DNA barcodes. Proc R Soc B 270:313–321PubMedCrossRefGoogle Scholar
  15. 15.
    Hebert PDN, Stoeckle MY, Zemlak TS, Francis CM (2004) Identification of birds through DNA barcodes. PLoS Biol 2:E312PubMedCrossRefGoogle Scholar
  16. 16.
    Ward RD, Zemlak TS, Innes BH, Last PR, Hebert PDN (2005) DNA barcoding Australia’s fish species. Philos Trans R Soc Lond B 360:1847–1857CrossRefGoogle Scholar
  17. 17.
    Hajibabaei M, Janzen DH, Burns JM, Hallwachs W, Hebert PDN (2006) DNA barcodes distinguish species of tropical Lepidoptera. Proc Natl Acad Sci USA 103:968–971PubMedCrossRefGoogle Scholar
  18. 18.
    Vences M, Thomas M, Meijden AVD, Chiari Y, Vieites DR (2005) Comparative performance of the 16S rRNA gene in DNA barcoding of amphibians. Front Zool 2:5. doi: 10.1186/1742-9994-2-5 PubMedCrossRefGoogle Scholar
  19. 19.
    Bradley RD, Baker RJ (2001) A test of the genetic species concept: cytochrome b sequences and mammals. J Mammal 82:960–973CrossRefGoogle Scholar
  20. 20.
    Lemer S, Aurelle D, Vigliola L, Durand JD, Borsa P (2007) Cytochrome b barcoding, molecular systematics and geographic differentiation in rabbitfishes (Siganidae). C R Biol 330:86–94PubMedCrossRefGoogle Scholar
  21. 21.
    De Salle R, Egan MG, Siddall M (2005) The unholy trinity: taxonomy, species delimitation and DNA barcoding. Philos Trans R Soc Lond B 360(1462):1905–1916CrossRefGoogle Scholar
  22. 22.
    Hajibabaei M, Singer GAC, Clare EL, Hebert PDN (2007) Design and applicability of DNA arrays and DNA barcodes in biodiversity monitoring. BMC Biol 5:24. doi: 10.1186/1741-7007-5-24) PubMedCrossRefGoogle Scholar
  23. 23.
    Ramadan HAI, Mahfouz ER (2009) Sequence of specific mitochondrial 12S rRNA fragment of Egyptian buffalo as a reference for discrimination between buffalo, cattle, sheep and goat. J Appl Biosci 21:1258–1264Google Scholar
  24. 24.
    Ramadan HAI (2009) Direct submission of Egyptian buffalo 16S rRNA sequence data to National Center for Biotechnology Information (NCBI) GenBank Database. Accession numbers FJ748599–FJ748607Google Scholar
  25. 25.
    Altschul S, Madden T, Schaffer A, Zhang J, Zhang Z, Miller W, Lipman D (1997) Gapped BLAST and PSI- BLAST: a new generation of protein database search programs. Nucl Acids Res 25:3389–3402PubMedCrossRefGoogle Scholar
  26. 26.
    Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucl Acids Res 32:1792–1797PubMedCrossRefGoogle Scholar
  27. 27.
    Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucl Acids Res 22:4673–4680PubMedCrossRefGoogle Scholar
  28. 28.
    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 86:6196–6200PubMedCrossRefGoogle Scholar
  29. 29.
    Branicki W, Kupiec T, Pawlowski R (2003) Validation of cytochrome b sequence analysis as a method of species identification. J Forensic Sci 48:83–87PubMedGoogle Scholar
  30. 30.
    Pfeiffer I, Burger J, Brenig B (2004) Diagnostic polymorphisms in the mitochondrial cytochrome b gene allow discrimination between cattle, sheep, goat, roe buck and deer by PCR–RFLP. BMC Genet 5:30. doi: 10.1186/1471-2156-5-30 PubMedCrossRefGoogle Scholar
  31. 31.
    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 9:225–252CrossRefGoogle Scholar
  32. 32.
    Allard WM, Honeycutt LR (1992) Nucleotide sequence variation in the mitochondrial 12S rRNA gene and the phylogeny of African mole-rats (Rodentia: Bathyergidae). Mol Biol Evol 9:27–40PubMedGoogle Scholar
  33. 33.
    Whitfield JB, Cameron AS (1998) Hierarchical analysis of variation in the mitochondrial 16S rRNA gene among Hymenoptera. Mol Biol Evol 15:1728–1743PubMedGoogle Scholar
  34. 34.
    Arctander P, Johansen C, Vreto M (1999) Phylogeography of three closely related African Bovids. Mol Biol Evol 16:1724–1739PubMedGoogle Scholar
  35. 35.
    Carrera E, Garcia T, Cespedes A, Gonzalez I, Fernandez A, Hernandez PE, Martin R (1999) Salmon and trout analysis by PCR–RFLP for identity authentication. J Food Sci 64:410–413CrossRefGoogle Scholar
  36. 36.
    Parma P, Erra-pujadab M, Feliginic M, Greppid G, Ennee G (2004) Water buffalo (Bubalus bubalis): complete nucleotide mitochondrial genome sequence. DNA Seq 15(5/6):369–373PubMedGoogle Scholar
  37. 37.
    Kierstein G, Vallinoto M, Silva A, Schneider MP, Iannuzzi L, Brenig B (2004) Analysis of mitochondrial D-loop region casts new light on domestic water buffalo (Bubalus bubalis) phylogeny. Mol Phylogenet Evol 30(2):308–324PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  1. 1.Genetic Engineering and Biotechnology Division, Department of Cell BiologyNational Research CenterGizaEgypt
  2. 2.Department of Biological Sciences, Faculty of ScienceKing Abdul Aziz UniversityJeddahKingdom of Saudi Arabia

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