Establishment and application of EmDEA, a rapid on-site nucleic acid detection method for parrot bornavirus type 4

Parrot bornavirus (PaBV) is a neurotropic virus that causes chronic infection in parrots, affecting their nervous and gastrointestinal systems and often resulting in high mortality in captive populations. It is a major threat to the parrot breeding industry and the ornamental bird trade. We used Enzyme-Mediated Dual Exponential Amplification (EmDEA) rapid nucleic acid detection technology to create a novel, simple, and highly sensitive method for detecting parrot bornavirus type 4 (PaBV-4). Primers and probes specific to the M gene of PaBV-4 were designed. After two rounds of screening and optimization, the optimal primer pair was identified as F4R7RNA1. The assay was tested for specificity, sensitivity, and clinical usefulness. The test showed no cross-reactivity with H5N2, H7N9, H9N2, NDV, IBV, or IBDV. It had a detection limit of 5 copies/µL and a repeatability coefficient of variation of less than 5%. Among 270 clinical tissue samples from parrots, the assay achieved a 100% positive concordance rate and an overall agreement of 97.03% with conventional RT-PCR results. The entire detection process takes only 30 min and allows for direct RNA detection of PaBV-4. The method is simple to use, fast, sensitive, and accurate, making it an invaluable tool for on-site detection of PaBV-4.