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Journal of Clinical Microbiology, June 2004, p. 2724-2732, Vol. 42, No. 6
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.6.2724-2732.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Use of the hupB Gene Encoding a Histone-Like Protein of Mycobacterium tuberculosis as a Target for Detection and Differentiation of M. tuberculosis and M. bovis

S. Prabhakar,1 A. Mishra,1,{dagger} A. Singhal,1 V. M. Katoch,2 S. S. Thakral,3 J. S. Tyagi,1 and H. K. Prasad1*

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

Received 23 July 2003/ Returned for modification 11 September 2003/ Accepted 11 January 2004

The gene for histone-like protein (hupB [Rv2986c]) of Mycobacterium tuberculosis has been identified as a singular target which allows differentiation of two closely related mycobacterial species, namely, M. tuberculosis and M. bovis of the MTB complex, by a PCR assay. The N and S primer-generated PCR amplicons differed in M. tuberculosis and M. bovis; these amplicons were determined to be 645 and 618 bp, respectively. This difference was localized to the C-terminal part of the gene by using primers M and S. The C-terminal PCR amplicons of M. tuberculosis and M. bovis were determined to be 318 and 291 bp, respectively. The differences in the C-terminal portion of the gene were confirmed by restriction fragment length polymorphism analysis and sequencing. Sequence analysis indicated that in M. bovis there was a deletion of 27 bp (9 amino acids) in frame after codon 128 in the C-terminal part of the hupB gene. In the present study 104 mycobacterial strains and 11 nonmycobacterial species were analyzed for hupB gene sequences. Of the 104 mycobacterial strains included, 62 belonged to the MTB complex and 42 were non-MTB complex strains and species. Neither the hupB gene-specific primers (N and S) nor the C-terminal primers (M and S) amplify DNA from any other mycobacteria, making the assay suitable for distinguishing members of the MTB complex from other mycobacterial species, as well as for differentiating between members of the MTB complex, namely, M. tuberculosis and M. bovis.

* Corresponding author. Mailing address: Department of Biotechnology, All India Institute of Medical Sciences, New Delhi 110029, India. Phone: 91-11-6594994. Fax: 91-11-6852286. E-mail: hk_prasad{at}hotmail.com.

{dagger} Present address: Public Health Research Institute, New York, NY 10016.

Journal of Clinical Microbiology, June 2004, p. 2724-2732, Vol. 42, No. 6
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.6.2724-2732.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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