Development and validation of a one-tube, nested real-time PCR method suitable for routine detection ofMycobacterium bovisin animal tissue

Abstract Aims Development and validation of a real-time PCR test for high-throughput routine screening of animal tissue for Mycobacterium bovis and other Mycobacterium tuberculosis complex (MTBC) members. Methods and results A preliminary study compared the results of a combination of five tissue preparation/DNA extraction methods and nine PCR assays on a panel of 92 cattle tissue samples of known M. bovis culture status (55 positive and 37 negative). The combination of DNA extraction and PCR was found to be important in achieving optimal detection of M. bovis. The optimal combination of a simple tissue preparation/DNA extraction method and a one-tube, nested real-time PCR to maximize the sensitivity of detection of an M. bovis-specific RD4 deletion and an IS1081 MTBC-specific target was selected for further evaluation. In total, tissue samples collected from 981 cattle and 366 non-bovine animals and submitted for routine TB culture were parallel tested with the selected method, as well as tissue samples obtained from 156 animals in certified TB-free cattle herds. Conclusion For cattle, the optimized RD4-IS1081 PCR test exhibited a diagnostic sensitivity of 96% (95% CI: 94–97%) and specificity of 97% (95% CI: 95–98%) compared to culture. Specificity was 100% when testing the 156 samples from known TB-free cattle. For non-bovine species, the PCR had a diagnostic sensitivity of 93% (95% CI: 83–98%) and a specificity of 99% (95% CI: 97–100%).