This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mishra, A. Articles by Prasad, H. K. Search for Related Content PubMed PubMed Citation Articles by Mishra, A. Articles by Prasad, H. K.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, November 2005, p. 5670-5678, Vol. 43, No. 11
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.11.5670-5678.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Direct Detection and Identification of Mycobacterium tuberculosis and Mycobacterium bovis in Bovine Samples by a Novel Nested PCR Assay: Correlation with Conventional Techniques

A. Mishra ,^1,, A. Singhal,^1, D. S. Chauhan,² V. M. Katoch,² K. Srivastava,² S. S. Thakral,³ S. S. Bharadwaj,³ V. Sreenivas,⁴ and H. K. Prasad^1*

Department of Biotechnology,¹ Department of Biostatistics, All India Institute of Medical Sciences, New Delhi 110029, India,⁴ Central JALMA Institute for Leprosy and other Mycobacterial Diseases, Taj Ganj, Agra 282001, India,² Central Military Veterinary Laboratory, Meerut Cantt 250001, India³

Received 22 March 2005/ Returned for modification 22 May 2005/ Accepted 5 September 2005

Mycobacterium tuberculosis and M. bovis infect animals and humans. Their epidemiologies in developed and developing countries differ, owing to differences in the implementation of preventive measures (World Health Organization, 1999). Identification and differentiation of these closely related mycobacterial species would help to determine the source, reservoirs of infection, and disease burden due to diverse mycobacterial pathogens. The utility of the hupB gene (Rv2986c in M. tuberculosis, or Mb3010c in M. bovis) to differentiate M. tuberculosis and M. bovis was evaluated by a PCR-restriction fragment length polymorphism (RFLP) assay with 56 characterized bovine isolates (S. Prabhakar et al., J. Clin. Microbiol. 42:2724-2732, 2004). The degree of concordance between the PCR-RFLP assay and the microbiological characterization was 99.0% (P < 0.001). A nested PCR (N-PCR) assay was developed, replacing the PCR-RFLP assay for direct detection of M. tuberculosis and M. bovis in bovine samples. The N-PCR products of M. tuberculosis and M. bovis corresponded to 116 and 89 bp, respectively. The detection limit of mycobacterial DNA by N-PCR was 50 fg, equivalent to five tubercle bacilli. M. tuberculosis and/or M. bovis was detected in 55.5% (105/189) of the samples by N-PCR, compared to 9.4% (18/189) by culture. The sensitivities of N-PCR and culture were 97.3 and 29.7, respectively, and their specificities were 22.2 and 77.7%, respectively. The percentages of animals or samples identified as infected with M. tuberculosis or M. bovis by N-PCR and culture reflected the clinical categorizations of the cattle (P of <0.05 to <0.01). Mixed infection by N-PCR was detected in 22 animals, whereas by culture mixed infection was detected in 1 animal.

Present address: Section of Experimental Haematology/Oncology, Department of Paediatric Haematology/Oncology, Medical School Hanover, Carl Neuberg Strasse 1, D-30625 Hanover, Germany.

A.M. and A.S. contributed equally to the manuscript.

This article has been cited by other articles:

• Ritacco, V., Kantor, I. N. d. (2007). Simultaneous Detection of Mycobacterium bovis and Mycobacterium tuberculosis in Human Cerebrospinal Fluid. J. Clin. Microbiol. 45: 684-684 [Full Text]  
• Shah, N. P., Singhal, A., Jain, A., Kumar, P., Uppal, S. S., Srivatsava, M. V. P., Prasad, H. K. (2006). Occurrence of Overlooked Zoonotic Tuberculosis: Detection of Mycobacterium bovis in Human Cerebrospinal Fluid. J. Clin. Microbiol. 44: 1352-1358 [Abstract] [Full Text]