Rapid Identification and Differential Detection of Infectious Bursal Disease Virus by an Innovative ARMS-qRT-PCR Assay.

Infectious bursal disease (IBD), caused by IBD virus (IBDV), is a major threat to the poultry industry, with the cocirculation of multiple genotyping strains complicating the diagnosis and control. Since the differences of antigenicity among different genotyping and their differences with the commonly used vaccine strains, rapid and accurate identification and differential detection are crucial for surveillance and control of IBDV. In this study, a novel amplification refractory mutation system quantitative reverse transcription PCR (ARMS-qRT-PCR) assay, for the rapid differential diagnosis of very virulent IBDV (vvIBDV), novel variant IBDV (nvIBDV), and attenuated IBDV (attIBDV) strains that are now popularly cocirculating in China and some other countries, was developed. By combining ARMS technology with qRT-PCR, this method enables accurate differentiation of these genotypes, including the coinfected samples. The developed ARMS-qRT-PCR assay demonstrated high repeatability and specificity, with no cross-reactivity among the three genotyping or other common avian pathogens. The assay exhibited high sensitivity and excellent linear correlation, with detection limits of 65.6, 57.8, and 70.2 copies/μL for vvIBDV, nvIBDV, and attIBDV, respectively. It also showed results that were completely consistent with the sequencing validation in identifying single and coinfected field samples. Crucially, it successfully identified and quantified individual differential detection in complex dual and triple coinfection cases. This high-throughput, user-friendly method offers significant advantages over traditional techniques, making it an ideal tool for real-time diagnosis, epidemiological surveillance, and outbreak management of IBDV.