Development and validation of a rapid triple-target gold nanoparticle lateral flow immunoassay for detection of three avian mycoplasmas immunochromatographic strip test for avian mycoplasma detection

Avian mycoplasmas, including Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), and Mycoplasma meleagridis (MM) are major pathogens of poultry causing respiratory and reproductive disorders that result in considerable economic losses. The existing diagnostic methods such as culture, ELISA, and PCR are accurate and reliable but labour-intensive, costly, and impractical for routine field screening. This study aimed to develop a rapid and cost-effective colloidal gold–based immunochromatographic strip for the simultaneous serological detection of antibodies against MG, MS, and MM in poultry, reflecting exposure or infection status.

Recombinant proteins pMGA (MG), VlhA (MS), and Mm19 (MM) were expressed in Escherichia coli, purified, and immobilized on nitrocellulose membranes as test lines. Monoclonal and polyclonal antibodies were conjugated to 20 nm gold nanoparticles and reaction parameters (pH, antibody concentration, conjugate ratio) were optimized. The diagnostic performance of the colloidal gold–based immunochromatographic strip was evaluated using a panel of well-characterized clinical samples. ELISA assays were used as the reference standard. The panel comprised samples confirmed positive for Mycoplasma infections as well as samples positive for other common avian infectious agents, to assess diagnostic sensitivity, specificity, and potential cross-reactivity.

The developed strip test reacted exclusively with MG, MS, and MM with no cross-reactivity to other avian Mycoplasma species or common respiratory pathogens. The visual detection limit was 31.25 ng/mL, with results obtained in 15 min without any specialized equipment. Compared to ELISA, the diagnostic sensitivity of the strip reached 96.7% (95% CI: 82.8–99.9%) and specificity 98% (95% CI: 89.4–99.9%), with excellent agreement (Cohen’s κ = 0.96). The inter-assay coefficient of variation was < 8%, and strips retained full reactivity for at least 6 months upon storage at 4 °C.

This study presents the first validated multiplex serological platform for the simultaneous detection of antibodies against avian mycoplasmas. The developed triple-target immunochromatographic strip provides a rapid, accurate, and cost-effective tool for on-site serological screening of MG, MS, and MM infections. Its simplicity, rapid turnaround time, and reliability make it a promising alternative for large-scale surveillance and early monitoring programs in poultry health management.