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

• DOI: 10.1099/jmm.0.46997-0
• Publication Date: 2007-03-21T00:27:12Z
• Authors: Luis G. C. Pacheco; Roberta R. Pena; Thiago L. P. Castro; Fernanda A. Dorella; Robson C. Bahia; Renato Carminati; Marcílio N. L. Frota; Sérgio C. Oliveira; Roberto Meyer; Francisco S. F. A...; Anderson Miyoshi; Vasco Azevedo
• Keywords: 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

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.