title: Rapid detection and non-subjective characterisation of infectious bronchitis virus isolates using high-resolution melt curve analysis and a mathematical model.
abstract: Infectious bronchitis virus (IBV) is a coronavirus that causes upper respiratory, renal and/or reproductive diseases with high morbidity in poultry. Classification of IBV is important for implementation of vaccination strategies to control the disease in commercial poultry. Currently, the lengthy process of sequence analysis of the IBV S1 gene is considered the gold standard for IBV strain identification, with a high nucleotide identity (e.g. > or =95%) indicating related strains. However, this gene has a high propensity to mutate and/or undergo recombination, and alone it may not be reliable for strain identification. A real-time polymerase chain reaction (RT-PCR) combined with high-resolution melt (HRM) curve analysis was developed based on the 3'UTR of IBV for rapid detection and classification of IBV from commercial poultry. HRM curves generated from 230 to 435-bp PCR products of several IBV strains were subjected to further analysis using a mathematical model also developed during this study. It was shown that a combination of HRM curve analysis and the mathematical model could reliably group 189 out of 190 comparisons of pairs of IBV strains in accordance with their 3'UTR and S1 gene identities. The newly developed RT-PCR/HRM curve analysis model could detect and rapidly identify novel and vaccine-related IBV strains, as confirmed by S1 gene and 3'UTR nucleotide sequences. This model is a rapid, reliable, accurate and non-subjective system for detection of IBVs in poultry flocks.
authors.affiliation: Department of Veterinary Science, The University of Melbourne, VIC, Australia. khewson@unimelb.edu.au
authors.forename: Kylie
authors.initials: K
authors.lastname: Hewson
authors.forename: Amir H
authors.initials: AH
authors.lastname: Noormohammadi
authors.forename: Joanne M
authors.initials: JM
authors.lastname: Devlin
authors.forename: Karim
authors.initials: K
authors.lastname: Mardani
authors.forename: Jagoda
authors.initials: J
authors.lastname: Ignjatovic
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D018352:Coronavirus Infections / Q000662:veterinary* / Q000821:virology; D001351:Infectious bronchitis virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D011200:Poultry; D011201:Poultry Diseases / Q000821:virology*; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment; D044366:Transition Temperature
chemical_list: D020413:3' Untranslated Regions; D012367:RNA, Viral