Rapid and accurate diagnostic tools, such as Real-Time PCR (qPCR), need to be implemented as a confirmatory test in the framework of bovine tuberculosis (bTB) surveillance and control programs, shortening the turnaround time to confirm bTB infection. The present study aimed to evaluate a direct qPCR from fresh tissue samples targeting the insertion sequence IS6110 using individually homogenized bovine lymph nodes compared with microbiological culture. Retropharyngeal, tracheobronchial, and mesenteric lymph nodes fresh tissue samples (n = 687) were collected from 230 different cattle carcasses at the slaughterhouse. Only 23 of the 230 examined animals showed tuberculosis-like lesions, with 62 of 230 considered as positive. Among these 62 animals, 61 resulted as culture-positive, whereas 48 were qPCR-positive. Thus, this qPCR targeting IS6110 showed an apparent diagnostic sensitivity and specificity values of 77.1% [95% confidence interval (CI): 66.5–87.6%] and 99.4% (95% CI: 98.3–100.6%), respectively, and a positive predictive value of 97.9% (95% CI: 93.9–102.0%) and negative predictive value of 92.3% (95% CI: 88.4–96.2%). Positive and negative likelihood ratios were 130.2 and 0.2, respectively, and the agreement between microbiological culture and this qPCR was almost perfect (κ = 0.82). These results highlight this qPCR targeting IS6110 as a suitable complementary method to confirm bTB in animals with either tuberculosis-like lesions or non-tuberculosis-like lesions, decreasing the number of samples subjected to microbiological culture and, hence, its overall associated costs and the turnaround time (under 48 h) to confirm bTB infection. Besides, sampling mesenteric lymph node, which is uncommonly sampled, together with tracheobronchial and retropharyngeal ones, is advisable during postmortem inspection in bTB surveillance programs at the slaughterhouse, especially in areas with a low bTB prevalence scenario.

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