抄録
Infectious bursal disease (IBD), caused by infectious bursal disease virus (IBDV), remains a major threat to poultry production due to its immunosuppressive effects and economic losses. Rapid and reliable detection of IBDV is essential for disease surveillance and outbreak management, particularly in field and resource-limited laboratory settings. In this study, an isothermal reverse transcription–thermophilic helicase-dependent amplification (RT-tHDA) assay targeting a conserved region of VP1 gene of IBDV was developed and evaluated, amplifying a 99-bp fragment at 65°C within 90 min without the need for a thermocycler. Analytical sensitivity testing using ten-fold serial dilutions of viral RNA demonstrated a detection limit of 440 fg, representing a 100-fold improvement over conventional RT-PCR and sensitivity comparable to real-time RT-PCR. The assay showed high analytical specificity, with no cross-reactivity against other avian viral or bacterial pathogens. Diagnostic evaluation of 180 suspected field bursal samples showed IBDV detection rates of 35.0% by RT-tHDA, 35.6–36.1% by RT-PCR (VP1/VP2), 31.7% by virus isolation, and 45.0% by real-time RT-PCR. Comparing with World Organization for Animal Health (WOAH) reference methods, RT-tHDA demonstrated excellent performance, with 100% sensitivity to virus isolation, ≥ 96.9% sensitivity to RT-PCR, high specificity (≥ 95%), and almost perfect agreement (κ > 0.93). Although real-time RT-PCR detected more positive samples due to its ability to identify very low viral loads, RT-tHDA consistently detected all clinically relevant infections confirmed by reference methods. Overall, RT-tHDA offers high sensitivity, specificity, and strong agreement with WOAH methods, supporting its use as a rapid screening tool for IBDV in field and resource-limited laboratories.
