Duplex real-time PCR assay targeting insertion elements IS1081 and IS6110 for detection of Mycobacterium bovis in lymph nodes of cattle

Abstract

The development of a reliable and rapid screening test for detection of Mycobacterium bovis (M. bovis) helps in control of bovine tuberculosis. The aim of this study was evaluate a sensitive and specific assay for detecting M. bovis DNA in lymph nodes with lesions suggestive to tuberculosis taken from slaughtered cattle. A duplex real-time PCR assay was developed for the identification of M. bovis targeting insertion elements (IS) IS1081 and IS6110 in one internally controlled reaction. M. bovis DNA extraction protocols from tissue samples were evaluated. The specificity and sensitivity of the assay were evaluated for detecting serial dilutions of reference Mycobacteria strains as well as from spiked lymph node homogenate. Results revealed that microscopical examination of 600 lymph nodes with tuberculous-like lesion for detection of Acid-fast bacilli (AFB) showed a detection rate of 96.6%, compared to 98% M. bovis with duplex real-time PCR. The reproducible detection limit of the IS1081-PCR was 10 M. bovis cells/ml and the IS6110-PCR was 100 M. bovis cells/ml. Besides, both primer set of PCR protocol could detect 20 M. bovis cells/ml in spiked lymph node tissue. The assay was evaluated on 19 bacterial strains and was determined to be 100% specific for M. bovis. We suggest that the IS1081-PCR is a good candidate assay for routine screening of cattle lymph nodes and other tissue for M. bovis infection.nema