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

Goat Diseases Sheep Suppuration Base Sequence Corynebacterium Infections Corynebacterium pseudotuberculosis Goats Molecular Sequence Data Sheep Diseases 04 agricultural and veterinary sciences Polymerase Chain Reaction Sensitivity and Specificity 3. Good health 0403 veterinary science RNA, Bacterial Species Specificity Animals Lymph Nodes
DOI: 10.1099/jmm.0.46997-0 Publication Date: 2007-03-21T00:27:12Z
ABSTRACT
Corynebacterium pseudotuberculosisis 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 facilitateC. pseudotuberculosisdetection, a multiplex PCR (mPCR) assay was developed targeting three genes of this bacterium: the 16S rRNA gene,rpoBandpld. 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 theC. pseudotuberculosismPCR amplification profile, thereby demonstrating the assay's specificity. As little as 1 pg ofC. pseudotuberculosisgenomic 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. pseudotuberculosiscould 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 rapidC. pseudotuberculosisdetection and could supersede bacteriological culture for microbiological and epidemiological diagnosis of CLA.
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