Development of a molecular diagnostic test applied to experimental abattoir surveillance on bovine tuberculosis

One of the most essential systems applied to the eradication of bovine tuberculosis by Mycobacterium bovis is the epidemiologic surveillance of animals slaughtered in abattoir by means of inspection and sample taking of lesions compatible with tuberculosis, confirming the existence of the disease through culture and molecular detection, which takes weeks before a result can be obtained. An interesting alternative is to develop high-throughput molecular systems for the direct detection of M. bovis on biological samples. In this sense, our research has developed a molecular detection system by means of a real-time based PCR process which is applied directly to bovine biological samples and it allows to differentiate between Mycobacterium tuberculosis complex, Mycobacterium avium complex and other atypical mycobacteria that are interesting from the veterinary point of view. The sensitivity was analyzed by applying a conventional extraction system based on guanidine thiocyanate and a robotized system based on the selective magnetic capture of mycobacterial DNA. The molecular detection system showed a high specificity and a detection threshold of only two to three genomes. The sensitivity depended on the DNA extraction system being used and on the kind of lesions on which it was used; the sensitivity ranged from 61.11% for samples with non-visible lesions to 80.64% for chronic lesions, with an average sensitivity of 73.87% when using the manual extraction system and between 27.77 and 74.19% (average sensitivity 47.74%) when using the automated robotic system. In conclusion, our multiplex real-time PCR assay represents a fully controlled, high-throughput diagnostic tool for the rapid detection of Myobacterium presence directly in animal clinical specimens, which could be a practical tool in the context of bovine tuberculosis abattoir surveillance programs and granuloma submission programs.