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The development of a reliable and rapid screening test for detection of Mycobacterium bovis (M. bovis) helps to control of bovine tuberculosis and preventing zoonotic infections. This study aimed to 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 IS 6110 in one internally controlled reaction. M. bovis DNA extraction protocols from tissue samples was evaluated. The specificity and sensitivity of the assay were evaluated for detecting serial dilutions of reference Mycobacteria strains as well as form spiked lymph node homogenate.  Results revealed that microscopical examination of 600 lymph nodes with tuberculosis-like lesion for detection of 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. In conclusion, 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. Future validation of this assay for monitoring BTB in humans and animal species will be valuable for guiding public health policy

zenodo (cern european organization for nuclear research)

A Sensitive and Specific Duplex Real-Time PCR Assay Targeting Insertion Elements IS1081 and IS6110 for Detection of Mycobacterium bovis in Lymph Nodes of Cattle