Comparison of qPCR methods for the diagnosis of bovine tuberculosis from granulomas collected at slaughterhouses.

To improve the direct diagnosis of bovine tuberculosis (bTB) in lesions from carcasses condemned at slaughterhouses - the primary method for identifying infected farms within the Surveillance System - two qPCR assays were evaluated against the gold standard: culture and identification of Mycobacterium bovis. A total of 167 lesion samples were collected by inspection services in Mato Grosso and Santa Catarina. Samples were homogenized and analyzed using culture and qPCR targeting the IS1081 sequence (for the M. tuberculosis complex) and the RD4 region (specific to M. bovis). Both qPCR assays demonstrated acceptable sensitivity and specificity, confirming the diagnostic value of these targets. The IS1081 qPCR showed a sensitivity of 0.80 [95% CI: 0.69-0.89] and specificity of 0.79 [95% CI: 0.70-0.87]. The RD4 qPCR yielded a sensitivity of 0.74 [95% CI: 0.61-0.84] and specificity of 0.84 [95% CI: 0.76-0.91]. Agreement between the two qPCR assays was high (K = 0.89 [95% CI: 0.82-0.96]). When using the parallel results of culture and IS1081 qPCR as gold standard, the RD4 assay achieved a sensitivity of 0.74 [95% CI: 0.64-0.83] and specificity of 1.00 [95% CI: 0.96-1.00]. In conclusion, both assays produced comparable results. The RD4 qPCR, due to its specificity for M. bovis, shows promise as a replacement for classical bacteriology and conventional PCR in bTB surveillance, offering operational advantages.