Development of a time-resolved fluorescent lateral flow immunochromatographic strip for detection of Mycoplasma bovis antibodies.

Mycoplasma bovis (M. bovis), a significant etiological agent of Bovine Respiratory Disease (BRD), has exhibited a marked increase in global prevalence, manifesting as multi-systemic diseases including pneumonia, mastitis, and arthritis. These infections are characterized by protracted courses, antibiotic resistance, and high mortality, posing substantial threats to livestock health and causing severe economic losses. To address the current lack of rapid and accurate diagnostic methods, this study developed a time-resolved fluorescent microsphere-based immunochromatographic test strip (TRFM-ICTS) for M. bovis antibody detection. Using carboxyl-modified Eu(III) chelate-doped polystyrene nano-fluorescent microspheres as probes, and coupled with a portable fluorescence intensity analyzer, this system enables rapid on-site serological diagnosis. This method demonstrated fourfold higher sensitivity compared to a commercial ELISA kit. Specificity testing revealed no cross-reactivity with positive sera against other bovine pathogens (e.g., Pasteurella multocida, Mycobacterium bovis, Brucella abortus, Mycoplasma bovirhinis, Mycoplasma agalactiae), confirming exclusive reactivity to M. bovis-positive serum. The test strips exhibited excellent reproducibility, with coefficients of variation below 10%. Evaluation using clinical samples showed a total concordance rate of 94.58% between TRFM-ICTS and commercial ELISA. Notably, detection requires minimal manual intervention and can be completed at the point-of-care within 15 min. The TRFM-ICTS developed herein, with its operational simplicity, high sensitivity, and specificity, presents a promising tool for rapid on-site detection of M. bovis antibodies.