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

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

Abstract: 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.

Saved in:
Bibliographic Details
Main Authors: PACHECO, L. G. C., PENA, R. R., CASTRO, T. L. P., DORELLA, F. A., BAHIA, R. C., CARMINATI, R., FROTA, M. N. L., OLIVEIRA, S. C., MEYER, R., ALVES, F. S. F., MIYOSHI, A., AZEVEDO, V.
Other Authors: Luis G. C. Pacheco, Instituto de Ciências Biológicas UFMG; Roberta R. Pena, Instituto de Ciências Biológicas UFMG; Thiago L. P. Castro Instituto de Ciências Biológicas UFMG; Fernanda A. Dorella Instituto de Ciências Biológicas UFMG; Robson C. Bahia, UFBA-Departamento de Bio-Interação; Renato Carminati, UFBA-Departamento de Bio-Interação; Maurício N. L. Frota, UVA - Universidade Estadual Vale do Acaraú; Sérgio C. Oliveira, Instituto de Ciências Biológicas UFMG; Roberto Meyer, UFBA-Departamento de Bio-Interação; FRANCISCO SELMO FERNANDES ALVES, CNPC; Anderson Miyoshi, Instituto de Ciências Biológicas UFMG; Vasco Azevedo, Instituto de Ciências Biológicas UFMG.
Format: Artigo de periódico biblioteca
Language:English
eng
Published: 2008-03-31
Subjects:PCR, CLA., Caprino, Corynebacterium Pseudotuberculosis, Linfadenite Caseosa, Ovino., caseous lymphadenitis.,
Online Access:http://www.alice.cnptia.embrapa.br/alice/handle/doc/533624
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Abstract: 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.

Similar Items