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Journal of Clinical Microbiology, August 2000, p. 3048-3054, Vol. 38, No. 8
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Duplex PCR for Differential Identification of Mycobacterium bovis, M. avium, and M. avium subsp. paratuberculosis in Formalin- Fixed Paraffin-Embedded Tissues from Cattle

Christophe Coetsier,1 Pascal Vannuffel,2 Nathalie Blondeel,1 Jean-Francois Denef,1 Carlo Cocito,1 and Jean-Luc Gala2,3,*

Histology Unit1 and Laboratory of Applied Molecular Technology,2 Medical Faculty, Université catholique de Louvain, and Section MSW, Operational Epidemiology and Infectious Diseases, Queen Astrid Military Hospital,3 Brussels, Belgium

Received 8 December 1999/Returned for modification 26 February 2000/Accepted 30 April 2000

We previously isolated and sequenced two genomic segments of Mycobacterium avium subsp. paratuberculosis, namely, f57, a species-specific sequence, and the p34 gene, coding for a 34-kDa antigenic protein. Comparison of sequences upstream of the p34 open reading frame (us-p34) from M. avium subsp. paratuberculosis and M. tuberculosis showed a 79-base deletion in M. tuberculosis. Sequence analysis of the p34 genes in another two species, M. bovis (strain BCG) and M. avium (strain D4), confirmed the differences observed between tuberculous and nontuberculous species. A duplex diagnostic PCR strategy based on coamplification of nonhomologous us-p34 and species-specific f57 sequences was therefore developed. Duplex PCR yielded three different patterns, specific either for tuberculous bacilli (M. tuberculosis, M. bovis, and M. africanum), for both nontuberculous mycobacteria M. avium and M. intracellulare, or for M. avium subsp. paratuberculosis. The specificity of this single-step DNA-based assay was assessed on DNA from cultured mycobacterial strains, as well as on a panel of formalin-fixed and paraffin-embedded tissues from cattle. Molecular assay results from tissular DNA were compared to conventional bacteriological and histological test results, including those obtained by Ziehl-Neelsen staining on tissue biopsy specimens. Molecular discrimination was successful and confirmed the value of duplex us-p34 and f57 sequence amplification for differential diagnosis of tuberculosis, paratuberculosis, or infections caused by other members of the M. avium complex.

* Corresponding author. Mailing address: Laboratory of Applied Molecular Technology, Clos Chapelle-aux-Champs, 30-UCL/30.46, B-1200 Brussels, Belgium. Phone: 32-2-764 3165. Fax: 32-2-764 3166. E-mail: gala{at}lbcm.ucl.ac.be.

Journal of Clinical Microbiology, August 2000, p. 3048-3054, Vol. 38, No. 8
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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