Multiplex PCR assay for identification of Corynebacterium pseudotuberculosis from pure cultures and for rapid detection of this pathogen in clinical samples.
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.
Pacheco
Luis G C
LGC
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.
Pena
Roberta R
RR
Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte - MG, Brazil.
Castro
Thiago L P
TLP
Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte - MG, Brazil.
Dorella
Fernanda A
FA
Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte - MG, Brazil.
Bahia
Robson C
RC
Departamento de Bio-Interação, Universidade Federal da Bahia, Salvador - BA, Brazil.
Carminati
Renato
R
Departamento de Bio-Interação, Universidade Federal da Bahia, Salvador - BA, Brazil.
Frota
Marcílio N L
MNL
Universidade Estadual Vale do Acaraú, Sobral - CE, Brazil.
Oliveira
Sérgio C
SC
Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte - MG, Brazil.
Meyer
Roberto
R
Departamento de Bio-Interação, Universidade Federal da Bahia, Salvador - BA, Brazil.
Alves
Francisco S F
FSF
Centro Nacional de Pesquisa de Caprinos, Empresa Brasileira de Pesquisa Agropecuária, Sobral - CE, Brazil.
Miyoshi
Anderson
A
Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte - MG, Brazil.
Azevedo
Vasco
V
Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte - MG, Brazil.
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
D012329:RNA, Bacterial