Detection of Mycobacterium bovis in bovine and bubaline tissues using nested?PCR for TbD1

ARAUJO, CRISTINA; OSORIO, ANA LUIZA; JORGE, KLAUDIA; RAMOS, CARLOS ALBERTO; SOUZA FILHO, ANTONIO FRANCISCO; VIDAL, CARLOS EUGENIO; ROXO, ELIANA; NISHIBE, CHRISTIANE ; ALMEIDA, NALVO F. ; FONSECA JUNIOR, ANTONIO ; SILVA, MARCIO; NETO, JOSÉ DIOMEDES ; CERQUEIRA, VALÍRIA D. ; ZUMÁRRAGA, MARTÍN JOSÉ; ARAUJO, FLABIO

PLOS ONE

PUBLIC LIBRARY SCIENCE

Lugar: San Francisco; Año: 2014 vol. 9 p. 1 - 6

1932-6203

In the present study, a nested-PCR system, targeting the TbD1 region, involving the performance of conventional PCRfollowed by real-time PCR, was developed to detect Mycobacterium bovis in bovine/bubaline tissue homogenates. Thesensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculousmycobacteria, as well as other actinomycetales species and DNA samples extracted directly from bovine and bubaline tissuehomogenates. In terms of analytical sensitivity, the DNA of M. bovis AN5 was detected up to 1.56 ng with conventional PCR,97.6 pg with real-time PCR, and 1.53 pg with nested-PCR in the reaction mixture. The nested-PCR exhibited 100% analyticalspecificity for M. bovis when tested with the DNA of reference strains of environmental mycobacteria and closely-relatedActinomycetales. A clinical sensitivity value of 76.0% was detected with tissue samples from animals that exhibited positiveresults in the comparative intradermal tuberculin test (CITT), as well as from those with lesions compatible with tuberculosis(LCT) that rendered positive cultures. A clinical specificity value of 100% was detected with tissue samples from animals withCITT- results, with no visible lesions (NVL) and negative cultures. No significant differences were found between the nested-PCR and culture in terms of detecting CITT+ animals with LCT or with NVL. No significant differences were recorded in thedetection of CITT- animals with NVL. However, nested-PCR detected a significantly higher number of positive animals thanthe culture in the group of animals exhibiting LCT with no previous records of CITT. The use of the nested-PCR assay todetect M. bovis in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.