title: 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 10(3) 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.
authors: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte - MG, Brazil.|Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte - MG, Brazil.
authors: Luis G C
authors: LGC
authors: Pacheco
authors: Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte - MG, Brazil.
authors: Roberta R
authors: RR
authors: Pena
authors: Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte - MG, Brazil.
authors: Thiago L P
authors: TLP
authors: Castro
authors: Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte - MG, Brazil.
authors: Fernanda A
authors: FA
authors: Dorella
authors: Departamento de Bio-Interação, Universidade Federal da Bahia, Salvador - BA, Brazil.
authors: Robson C
authors: RC
authors: Bahia
authors: Departamento de Bio-Interação, Universidade Federal da Bahia, Salvador - BA, Brazil.
authors: Renato
authors: R
authors: Carminati
authors: Universidade Estadual Vale do Acaraú, Sobral - CE, Brazil.
authors: Marcílio N L
authors: MNL
authors: Frota
authors: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte - MG, Brazil.
authors: Sérgio C
authors: SC
authors: Oliveira
authors: Departamento de Bio-Interação, Universidade Federal da Bahia, Salvador - BA, Brazil.
authors: Roberto
authors: R
authors: Meyer
authors: Centro Nacional de Pesquisa de Caprinos, Empresa Brasileira de Pesquisa Agropecuária, Sobral - CE, Brazil.
authors: Francisco S F
authors: FSF
authors: Alves
authors: Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte - MG, Brazil.
authors: Anderson
authors: A
authors: Miyoshi
authors: Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte - MG, Brazil.
authors: Vasco
authors: V
authors: Azevedo
mesh_terms: D000818:Animals; D001483:Base Sequence; D003354:Corynebacterium Infections / Q000382:microbiology / Q000662:veterinary; D016925:Corynebacterium pseudotuberculosis / Q000302:isolation & purification*; D015511:Goat Diseases / Q000382:microbiology; D006041:Goats / Q000382:microbiology; D008198:Lymph Nodes / Q000473:pathology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012329:RNA, Bacterial; D012680:Sensitivity and Specificity; D012756:Sheep; D012757:Sheep Diseases / Q000382:microbiology; D013045:Species Specificity; D013492:Suppuration / Q000382:microbiology
chemical_list: D012329:RNA, Bacterial