Duplex PCR-Based Molecular Diagnosis of Bovine Tuberculosis and Paratuberculosis: Development and Field Assessment

Zoonotic pathogens (Mycobacterium bovis and Mycobacterium avium subsp. paratuberculosis, or MAP) cause bovine tuberculosis and Johne's disease, respectively, both of which severely affect livestock health and productivity and pose risks to human health. Accurate diagnosis is challenging due to overlapping clinical signs and genetic similarities. In the present study, we developed and preliminarily evaluated a duplex PCR assay for simultaneous detection of MTBC-associated M. bovis and MAP. Based on in silico primer screening and gradient PCR optimization, IS1081 and IS900 were selected as target sequences for the detection of MTBC-associated M. bovis and MAP, respectively, with an optimal annealing temperature of 60 °C. The assay was subsequently assessed using 114 blood and fecal specimens from 57 clinically suspected weak cattle under pilot field conditions, providing a preliminary proof-of-concept evaluation. Sensitivity analysis using spiked fecal samples (10¹-10⁶ CFU/g) demonstrated a detection limit of 10² colony-forming units per gram (CFU/g) for both pathogens with no cross-reactivity; amplification at 10 CFU/g was inconsistent, indicating the lower sensitivity threshold of conventional PCR. When compared with single-target IS900 and IS1081 PCRs, the duplex assay achieved 63.6-100% sensitivity and 95.8-100% specificity, respectively, with substantial Cohen's kappa agreement and statistically significant p-values. The developed assay showed potential as a molecular diagnostic tool for pathogen detection, with future application in herd-level screening warranting multicenter validation.