Development and validation of a one-tube, nested real-time PCR method suitable for routine detection of Mycobacterium bovis in animal tissue.

This article focuses on the development and validation of a one-tube, nested real-time PCR test targeting the RD4 deletion and IS1081 insertion sequence for high-throughput detection of *Mycobacterium bovis* and other members of the *Mycobacterium tuberculosis* complex (MTBC) in animal tissue samples. The optimized PCR method demonstrated diagnostic sensitivities of 96% in cattle and 93% in non-bovine species, with specificities of 97–100% compared to the gold standard culture method. The assay incorporates a simple tissue preparation and DNA extraction protocol, uses robotics for processing, and includes a β-actin internal control to ensure DNA quality, enabling rapid and reliable detection suitable for routine diagnostic use. This PCR approach has been formally adopted by the UK Animal and Plant Health Agency (APHA) for bovine tuberculosis surveillance, offering results within days versus the 6–12 weeks required for culture, thus supporting more effective disease control and management.