Journal Article

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

SILVA, Marcio Roberto, ARAÚJO, Flábio Ribeiro de, VARGAS, Agueda Palmira Castagna de, CERQUEIRA, Valíria Duarte, BARBOSA NETO, José Diomedes, OSÓRIO, Ana Luiza Alves Rosa, ROXO, Eliana, VIDAL, Carlos Eugênio Soto, SUFFYS, Philip Noel, ROCHA, Adalgiza da Silva, ARAÚJO, Cristina Pires de, RAMOS, Carlos Alberto do Nascimento, SOUZA FILHO, Antonio Francisco de, JORGE, Klaudia dos Santos Gonçalves, FONSECA JÚNIOR, Antônio Augusto

2014

Overview

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.

• Publisher: Springer Nature B.V,Sociedade Brasileira de Microbiologia
• Subject: Animals / Bacteria / Biochemical tests / Buffaloes / Cattle / Deoxyribonucleic acid / DNA / MICROBIOLOGY / Molecular Diagnostic Techniques - methods / Mycobacterium bovis - genetics / Mycobacterium bovis - isolation & purification / Mycobacterium tuberculosis - genetics / Mycobacterium tuberculosis - isolation & purification / Pathology, Molecular - methods / Polymerase Chain Reaction - methods / Sensitivity and Specificity / Time Factors / Tissues / Tuberculosis / Tuberculosis, Bovine - diagnosis / Tuberculosis, Bovine - microbiology / Veterinary Medicine - methods / Veterinary Microbiology