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http://dx.doi.org/10.7853/kjvs.2011.34.4.321

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

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)
Publication Information
Korean Journal of Veterinary Service / v.34, no.4, 2011 , pp. 321-331 More about this Journal
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
Real-time PCR; Mycobacterium bovis; M. tuberculosis complex; IS1081; 12.7-kb; fragment;
Citations & Related Records
Times Cited By KSCI : 5  (Citation Analysis)
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