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

Development of real-time PCR for rapid detection of Mycobacterium bovis DNA in cattle lymph nodes and differentiation of M. bovis and M. tuberculosis

김용환|조호성|문용운|배성열|나호명|박성도|고바라다|구복경|장영부
한국동물위생학회 (THE KOREAN SOCIETY OF VETERINARY SERVICE)|2011.12|한국동물위생학회지|제34권 제4호|pp. 321-331

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키워드

Real-time PCRMycobacterium bovisM. tuberculosis complexIS108112.7-kb fragment
주제분류수의학

초록

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.

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.

참고문헌 (41)

  1. 고바라다, "PCR 기법을 이용한 도축소의 결핵병 신속진단", 한국가축위생학회지, 30(3), pp.393~406 (2007), 한국가축위생학회
  2. 공신국, "PCR 기법을 이용한 젖소 결핵균 검색 분리 조사", 한국가축위생학회지, 25, pp.135~140 (2002)
  3. 변현섭, "도축한우에서 발견된 결핵병", 한국가축위생학회지, 30(3), pp.407~414 (2007), 한국가축위생학회
  4. 이종진, "ELISA를 이용한 우결핵검사 결과에 대한 PPD 접종법 결과 분석 비교", 한국가축위생학회지, 33(4), pp.335~340 (2010), 한국가축위생학회
  5. Bakshi CS, "Rapid differentiation of Mycobacterium bovis and Mycobacterium tuberculosis based on a 12.7-kb fragment by a single tube multiplex-PCR", Vet Microbiol, 109, pp.211~216 (2005)
  6. Barouni AS, "A pncA polymorphism to differentiate between Mycobacterium bovis and Mycobacterium tuberculosis", Mol Cell Probes, 18(3), pp.167~170 (2004)
  7. Brosch R, "A new evolutionary scenario for the Mycobacterium tuberculosis complex", Proc Natl Acad Sci USA, 99, pp.3684~3689 (2002)
  8. Chen R, "Development of a fluorescent microsphere-based multiplex assay for simultaneous rapid detection of Mycobacterium tuberculosis complex and differentiation of M. tuberculosis and M. bovis in clinical samples", Diagn Mol Pathol, 19, pp.172~179 (2010)
  9. Collins DM, "DNA fingerprinting of Mycobacterium bovis strains by restriction fragment analysis and hybridization with insertion elements IS1081 and IS6110", J Clin Microbiol, 31, pp.1143~1147 (1993)
  10. Collins DM, "Identification of an insertion sequence, IS1081, in Mycobacterium bovis", FEMS Microbiol Lett, 67, pp.11~15 (1991)
  11. Cosivi O, "Zoonotic tuberculosis due to Mycobacterium bovis in developing countries", Emerg Infect Dis, 4, pp.59~70 (1998)
  12. Ereqat S, "Rapid differentiation of Mycobacterium tuberculosis and M. bovis by high-resolution melt curve analysis", J Clin Microbiol, 48, pp.4269~4272 (2010)
  13. Espinosa de los Monteros LE, "Allele-specific PCR method based on pncA and oxyR sequences for distinguishing Mycobacterium bovis from Mycobacterium tuberculosis: intraspecific M. bovis pncA sequence polymorphism", J Clin Microbiol, 36, pp.239~242 (1998)
  14. Fleiss JL, "The equivalence of weighted kappa and the intraclass correlation coefficient as measures of reliability", Educational and Psychological Measurement, 33, pp.613~619 (1973)
  15. Garnier T, "The complete genome sequence of Mycobacterium bovis", Proc Natl Acad Sci USA, 100, pp.7877~7882 (2003)
  16. Heid CA, "Real time quantitative PCR", Genome Res, 6, pp.986~994 (1996)
  17. Klein D, "Quantification using real-time PCR technology: applications and limitations", Trends Mol Med, 8, pp.257~260 (2002)
  18. Kurabachew M, "A multiplex polymerase chain reaction assay for genus-, group- and species-specific detection of mycobacteria", Diagn Microbiol Infect Dis, 49, pp.99~104 (2004)
  19. Miller MB, "Basic concepts of microarrays and potential applications in clinical microbiology", Clin Microbiol Rev, 22, pp.611~633 (2009)
  20. Mostowy S, "Revisiting the evolution of Mycobacterium bovis", J Bacteriol, 187, pp.6386~6395 (2005)
  21. Niemann S, "Differentiation of clinical Mycobacterium tuberculosis complex isolates by gyrB DNA sequence polymorphism analysis", J Clin Microbiol, 38, pp.3231~3234 (2000)
  22. Niemann S, "Differentiation among members of the Mycobacterium tuberculosis complex by molecular and biochemical features: evidence for two pyrazinamide-susceptible subtypes of M. bovis", J Clin Microbiol, 38, pp.152~157 (2000)
  23. Park H, "Detection and genotyping of Mycobacterium species from clinical isolates and specimens by oligonucleotide array", J Clin Microbiol, 43, pp.1782~1788 (2005)
  24. Parra A, "Development of a molecular diagnostic test applied to experimental abattoir surveillance on bovine tuberculosis", Vet Microbiol, 127, pp.315~324 (2008)
  25. Pinsky BA, "Multiplex real-time PCR assay for rapid identification of Mycobacterium tuberculosis complex members to the species level", J Clin Microbiol, 46, pp.2241~2246 (2008)
  26. Pounder JI, "Comparison of real- time polymerase chain reaction using the Smart Cycler and the Gen-Probe amplified Mycobacterium tuberculosis direct test for detection of M. tuberculosis complex in clinical specimens", Diagn Microbiol Infect Dis, 54, pp.217~222 (2006)
  27. Sambrook J, "Molecular cloning: a laboratory manual. 3rd ed" (2001), Cold spring harbor laboratory press
  28. Savelkoul PH, "Detection of Mycobacterium tuberculosis complex with Real Time PCR: comparison of different primer-probe sets based on the IS6110 element", J Microbiol Methods, 66, pp.177~180 (2006)
  29. Shah DH, "A multiplex- PCR for the differentiation of Mycobacterium bovis and Mycobacterium tuberculosis", FEMS Microbiol Lett, 214, pp.39~43 (2002)
  30. Soini H, "Molecular diagnosis of mycobacteria", Clin Chem, 47, pp.809~814 (2001)
  31. Sorlozano Antonio, "Comparative Evaluation of Three Culture Methods for the Isolation of Mycobacteria from Clinical Samples", Journal of Microbiology and Biotechnology, 19(10), pp.1259~1264 (2009), 한국미생물·생명공학회
  32. Taylor GM, "Rapid detection of Mycobacterium bovis DNA in cattle lymph nodes with visible lesions using PCR", BMC Vet Res, 3, pp.12~ (2007)
  33. Thacker TC, "Improved specificity for detection of Mycobacterium bovis in fresh tissues using IS6110 real-time PCR", BMC Vet Res, 7, pp.50~ (2011)
  34. Thierry D, "IS6110, an IS-like element of Mycobacterium tuberculosis complex", Nucleic Acids Res, 18, pp.188~ (1990)
  35. Thoen C, "The importance of Mycobacterium bovis as a zoonosis", Vet Microbiol, 112, pp.339~345 (2006)
  36. Thone CO, "Mycobacterium, In Pathogenesis of bacterial infectious in animals. 2nd ed", pp.44~56 (1993), Iowa State University Press
  37. van Soolingen D, "Insertion element IS1081-associated restricon fragment length polymorphisms in Mycobacterium tuberculosis complex species: a reliable tool for recognizing Mycobacterium bovis BCG", J Clin Microbiol, 30, pp.1772~1777 (1992)
  38. Wards BJ, "Detection of Mycobacterium bovis in tissues by polymerase chain reaction", Vet Microbiol, 43, pp.227~240 (1995)
  39. Yeboah-Manu D, "Application of a simple multiplex PCR to aid in routine work of the mycobacterium reference laboratory", J Clin Microbiol, 39, pp.4166~4168 (2001)
  40. Zhu RY, "Use of visual loop-mediated isotheral amplification of rimM sequence for rapid detection of Mycobacterium tuberculosis and Mycobacterium bovis", J Microbiol Methods, 78, pp.339~343 (2009)
  41. Zumárraga M, "A 12.7 kb fragment of the Mycobacterium tuberculosis genome is not present in Mycobacterium bovis", Microbiology, 145(4), pp.893~897 (1999)

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