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J Med Microbiol 56 (2007), 480-486; DOI: 10.1099/jmm.0.46997-0
© 2007 Society for General Microbiology
ISSN 1473-5644

Multiplex PCR assay for identification of Corynebacterium pseudotuberculosis from pure cultures and for rapid detection of this pathogen in clinical samples

Luis G. C. Pacheco1,2, Roberta R. Pena1, Thiago L. P. Castro1, Fernanda A. Dorella1, Robson C. Bahia3, Renato Carminati3, Marcílio N. L. Frota4, Sérgio C. Oliveira2, Roberto Meyer3, Francisco S. F. Alves5, Anderson Miyoshi1 and Vasco Azevedo1

1 Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte – MG, Brazil

2 Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte – MG, Brazil

3 Departamento de Bio-Interação, Universidade Federal da Bahia, Salvador – BA, Brazil

4 Universidade Estadual Vale do Acaraú, Sobral – CE, Brazil

5 Centro Nacional de Pesquisa de Caprinos, Empresa Brasileira de Pesquisa Agropecuária, Sobral – CE, Brazil

Correspondence
Vasco Azevedo
vasco{at}icb.ufmg.br

Received 11 October 2006
Accepted 23 November 2006

Corynebacterium pseudotuberculosis is the aetiological agent of caseous lymphadenitis (CLA), a debilitating disease of sheep and goats. Accurate diagnosis of CLA primarily relies on microbiological examination, followed by biochemical identification of isolates. In an effort to facilitate C. pseudotuberculosis detection, a multiplex PCR (mPCR) assay was developed targeting three genes of this bacterium: the 16S rRNA gene, rpoB and pld. This method allowed efficient identification of 40 isolates of this bacterium that had been identified previously by biochemical testing. Analysis of taxonomically related species did not generate the C. pseudotuberculosis mPCR amplification profile, thereby demonstrating the assay's specificity. As little as 1 pg of C. pseudotuberculosis genomic DNA was detected by this mPCR assay, demonstrating the sensitivity of the method. The detection limit in clinical samples was estimated to be 103 c.f.u. C. pseudotuberculosis could be detected directly in pus samples from infected sheep and goats (n=56) with a high diagnostic sensitivity (94.6 %). The developed assay significantly improves rapid C. pseudotuberculosis detection and could supersede bacteriological culture for microbiological and epidemiological diagnosis of CLA.

Abbreviations: CLA, caseous lymphadenitis; mPCR, multiplex PCR; PLD, phospholipase D.

The GenBank/EMBL/DDBJ accession number for the rpoB gene sequence of Arcanobacterium pyogenes is DQ680032.




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