Korean Journal of Veterinary Service (한국동물위생학회지)

The Korean Society of Veterinary Service (한국동물위생학회)
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Development of real-time PCR for rapid detection of Mycobacterium bovis DNA in cattle lymph nodes and differentiation of M. bovis and M. tuberculosis

소 림프절에서 Mycobacterium bovis DNA의 신속 검출과 M. bovis와 M. tuberculosis 감별을 위한 real-time PCR 개발

  • Koh, Ba-Ra-Da (Gwangju Metropolitan Health & Environment Research Institute) ;
  • Jang, Young-Boo (Bacteriology Disease Division, Animal, Plant and Fisheries Quarantine and Inspection Agency) ;
  • Ku, Bok-Kyung (Bacteriology Disease Division, Animal, Plant and Fisheries Quarantine and Inspection Agency) ;
  • Cho, Ho-Seong (College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University) ;
  • Bae, Seong-Yeol (Gwangju Metropolitan Health & Environment Research Institute) ;
  • Na, Ho-Myung (Gwangju Metropolitan Health & Environment Research Institute) ;
  • Park, Seong-Do (Gwangju Metropolitan Health & Environment Research Institute) ;
  • Kim, Yong-Hwan (Gwangju Metropolitan Health & Environment Research Institute) ;
  • Mun, Yong-Un (Gwangju Metropolitan Health & Environment Research Institute)
  • 고바라다 (광주광역시보건환경연구원) ;
  • 장영부 (농림수산검역검사본부 동식물위생연구부 세균질병과) ;
  • 구복경 (농림수산검역검사본부 동식물위생연구부 세균질병과) ;
  • 조호성 (전북대학교 수의과대학) ;
  • 배성열 (광주광역시보건환경연구원) ;
  • 나호명 (광주광역시보건환경연구원) ;
  • 박성도 (광주광역시보건환경연구원) ;
  • 김용환 (광주광역시보건환경연구원) ;
  • 문용운 (광주광역시보건환경연구원)
  • Received : 2011.11.24
  • Accepted : 2011.12.16
  • Published : 2011.12.30
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Abstract

Mycobacterium bovis, a member of the M. tuberculosis complex (MTC), is the causative agent of bovine tuberculosis. Detection of M. bovis and M. tuberculosis using conventional culture- and biochemical-based assays is time-consuming. Therefore, a simple and sensitive molecular assay for rapid detection would be of great help in specific situations such as faster diagnosis of bovine tuberculosis (bTB) infection in the abattoirs. We developed a novel multiplex real-time PCR assay which was applied directly to biological samples with evidence of bTB and it was allowed to differentiate between M. bovis and M. tuberculosis. The primers and TaqMan probes were designed to target the IS1081 gene, the multi-copy insertion element in the MTC and the 12.7-kb fragment which presents in M. tuberculosis, not in the M. bovis genome. The assay was optimized and validated by testing 10 species of mycobacteria including M. bovis and M. tuberculosis, and 10 other bacterial species such as Escherichia coli, and cattle lymph nodes (n=113). The tests identified 96.4% (27/28) as M. bovis from the MTC-positive bTB samples using conventional PCR for specific insertion elements IS1081. And MTC-negative bTB samples (n=85) were tested using conventional PCR and the real-time PCR. When comparative analyses were conducted on all bovine samples, using conventional PCR as the gold standard, the relative accuracy of real-time PCR was 99.1%, the relative specificity was 100%, and the agreement quotient (kappa) was 0.976. The detection limits of the real-time PCR assays for M. bovis and M. tuberculosis genomic DNA were 10 fg and 0.1 pg per PCR reaction, respectively. Consequently, this multiplex real-time PCR assay is a useful diagnotic tool for the identification of MTC and differentiation of M. bovis and M. tuberculosis, as well as the epidemiologic surveillance of animals slaughtered in abattoir.

Keywords

References

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