Development and validation of a direct real-time PCR assay for Mycobacterium bovis and implementation into the United States national surveillance program

Abattoir surveillance for bovine tuberculosis, which consists of identifying and submitting granulomas for histopathology and mycobacterial culture was the primary means for detecting new cases in the United States. Mycobacterial culture is expensive, labor intensive and identifies cases weeks after slaughter, hampering trace back efforts. To address this inefficiency, the United States Department of Agriculture replaced culture with real-time PCR for screening granulomas. The objectives of this paper were to describe the development and validation of this PCR as well as the performance of the assay during the first year of implementation. Using archived culture and histologically positive tissue, the sensitivity was 0.96 (95% CI: 0.89, 0.99) for the Mycobacterium tuberculosis complex primer-probe set and 0.89 (95% CI: 0.80, 0.95) for the Mycobacterium bovis specific primer-probe set. Specificity, estimated during by side by side testing was 0.998 (95% CI: 0.994, 1.000). After implementation, 6124 samples over 54 weeks were tested and all 36 histopathology positive samples were detected including 2 additional cases initially misclassified by histopathology. It appeared that specificity may have declined during post validation testing with 47/6086 signaling positive but not confirmed by either histopathology or culture. While PCR implementation has significantly improved the efficiency of the US slaughter surveillance program, careful attention must be paid to prevent and address cross contamination in the laboratory.