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 tuberculous-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 |
