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

Brazilian Journal of Microbiology

Cristina P. Araújo²  Ana Luiza A.r. Osório²  Klaudia S.g. Jorge²  Carlos A.n. Ramos¹  Antonio F. Souza Filho²  Carlos E.s. Vidal¹  Agueda P.c. Vargas¹  Eliana Roxo¹  Adalgiza S. Rocha¹  Philip N. Suffys¹  Antônio A. Fonseca Júnior¹  Marcio R. Silva¹  José D. Barbosa Neto¹  Valíria D. Cerqueira¹  Flábio R. Araújo¹ 

[1] ,Universidade Federal de Mato Grosso do Sul Faculdade de Medicina Veterinária e Zootecnia Campo Grande MS ,Brazil

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

DOI  :  10.1590/S1517-83822014000200035

来源: SciELO

【 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.

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