Korean Journal of Veterinary Service (한국동물위생학회지)

The Korean Society of Veterinary Service (한국동물위생학회)
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Development of species-specific multiplex PCR assays of mitochondrial 12S rRNA and 16S rRNA for the identification of animal species

식육감별을 위한 미토콘드리아 12S rRNA와 16S rRNA 유전자의 종 특이적 multiplex PCR 기법 개발

  • Koh, Ba-Ra-Da (Department of Veterinary Research, Gwangju Metropolitan City Institute Health & Environment) ;
  • Kim, Ji-Yeon (Department of Veterinary Research, Gwangju Metropolitan City Institute Health & Environment) ;
  • Na, Ho-Myung (Department of Veterinary Research, Gwangju Metropolitan City Institute Health & Environment) ;
  • Park, Seong-Do (Department of Veterinary Research, Gwangju Metropolitan City Institute Health & Environment) ;
  • Kim, Yong-Hwan (Department of Veterinary Research, Gwangju Metropolitan City Institute Health & Environment)
  • 고바라다 (광주광역시보건환경연구원 동물위생연구부) ;
  • 김지연 (광주광역시보건환경연구원 동물위생연구부) ;
  • 나호명 (광주광역시보건환경연구원 동물위생연구부) ;
  • 박성도 (광주광역시보건환경연구원 동물위생연구부) ;
  • 김용환 (광주광역시보건환경연구원 동물위생연구부)
  • Received : 2011.08.24
  • Accepted : 2011.12.12
  • Published : 2011.12.30
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Abstract

Species-specific PCR assay was developed for detection of cattle, sheep, goat, horse, dog, pig, chicken, duck, goose, and turkey using mitochondrial 12S rRNA and 16S rRNA as target genes. Also, an internal positive control was used to detect possible false negatives by using 18S rRNA gene. We designed species-specific primers with amplicon length of 190, 219, 350, 467, 241, 119, 171, 229, 111 and 268 bp for cattle, sheep, goat, horse, dog, pig, chicken, duck, goose, and turkey respectively. The specificity of the primers was tested against the other 10 non-target animal species and a cross-reaction was not observed. We developed two multiplex PCR assays for the simultaneous identification of Korea's major livestock species (cattle, pig, chicken and duck) and poultry species (chicken, duck, goose and turkey) from analogous samples, retaining the same specificity. The limit of detection of the multiplex PCR assay (cattle, pig, chicken and duck) ranged between 1 pg and 0.1 pg of template DNA extracts from raw meat. Applying multiplex PCR assays to DNA extracts from experimental pork/beef and pork/chicken tested raw and heat-treated ($120^{\circ}C$ for 30 min) mixtures respectively, detection limit was 0.1% level beef in pork, pork in beef and chicken in pork and 1.0% level pork in chicken. In conclusion, this assay using gel-based capillary electrophoresis would be very useful in highly sensitive and rapid identification of animal species or ingredients in minced meat and other meat products.

Keywords

References

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