Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR

Revista de microbiologia

Roxo, Eliana¹  Vidal, Carlos E.S.²  Ramos, Carlos A.N.³  Jorge, Klaudia S.G.⁴  Souza Filho, Antonio F.⁵  Araújo, Cristina P.⁶  Osório, Ana Luiza A.R.⁷  Vargas, Agueda P.C.⁸ 

[1] Embrapa Gado de Corte, Campo Grande, Brazil;Embrapa Gado de Leite, Juiz de Fora, Brazil;Fundação Oswaldo Cruz, Rio de Janeiro, Brazil;Instituto Biológico de São Paulo, São Paulo, Brazil;Laboratório Nacional Agropecuário, Pedro Leopoldo, Brazil;Universidade Federal de Mato Grosso do Sul, Campo Grande, Brazil;Universidade Federal de Santa Maria, Santa Maria, Brazil;Universidade Federal do Pará, Castanhal, Brazil

关键词: bovine and bubaline tuberculosis;    nested-PCR;    real-time PCR;    tissue;    sanitary inspection.;   

DOI  :  10.1590/S1517-83822014000200035

学科分类：农业科学（综合）

来源: Sociedade Brasileira de Microbiologia / Brazilian Society for Microbiology

【 摘 要 】

Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

【 授权许可】

CC BY-NC