Development of an HRM-qRT-PCR platform for fast and cost-effective genotyping of infectious bronchitis virus in Egypt

Avian infectious bronchitis virus (IBV) is a highly transmissible virus that causes respiratory, reproductive, and renal syndromes, resulting in significant economic losses for the global poultry industry. This study aimed to evaluate the application of high-resolution melting analysis (HRM) as a novel, efficient, and cost-effective tool for the rapid diagnosis and genotyping of Egyptian IBV strains. The investigation focused on the N gene and the 3’ untranslated region (3’ UTR). HRM curves were generated from 435 bp PCR products obtained from 22 IBV-positive avian samples. The purified PCR products, which amplified the 3’ UTR, were sequenced and subsequently compared with analogous S1 gene sequences to differentiate between vaccine strains and field strains, specifically the Egyptian strain 4/91 (GI-13), variant II (GI-23), and the classic strain (GI-1). The high-resolution melting curve analysis (HRM) successfully distinguished vaccine reference strains (Ma5, IB-Primer, 4/91, and Variant II), while the IBV field strains were categorized into three distinct pattern clusters based on the similarity of their melting curves, which were confirmed by S1 gene sequences. Both strains 4/91 and variant II exhibited a deletion of about 15 nucleotides at the terminus of the N-gene compared to classic strains. Closed-tube PCR combined with HRM-curve analysis provides a fast and reliable technique for genotyping IBV strains, including IBV vaccine strains and IBV field strains.