title: Rapid genotyping of beak and feather disease virus using high-resolution DNA melt curve analysis.
abstract: Beak and feather disease virus (BFDV) is a significant pathogen both for wild and captive psittacine birds globally. Genotypic differentiation of BFDV isolates is crucial to establish effective control strategies for the conservation of endangered species and epidemiological investigations of disease outbreaks. The technique developed in this study is a simple, rapid and inexpensive genotyping method for BFDV using PCR and subsequent high-resolution melt (HRM) curve analysis. This was achieved using PCR amplification of the conserved Rep gene in the presence of a fluorescent DNA intercalating dye (SYTO9). HRM curve analysis of the resultant amplicon could readily differentiate between reference strain (92-SR14) and 18 other BFDV isolates used in this study. Analysis of the nucleotide sequences of the amplicon from each isolate revealed that each melt curve profile was related to a unique DNA sequence. The potential of the PCR-HRM curve analysis to differentiate inter-host genetic variation among critically endangered orange-bellied parrots, lorikeets and cockatoos was also evaluated. Phylogenetic tree topology based on partial Rep gene sequences used in this study showed that BFDV Rep gene sequence patterns were correlated with the results of HRM curve analysis. The results presented in this study indicate that this technique could be used in both clinical research and differentiation of BFDV isolates in a fraction of time without further nucleotide sequencing and provides a novel approach for the genetic screening of BFDV in clinical virology laboratories.
authors.affiliation: School of Animal and Veterinary Sciences, Charles Sturt University, Boorooma Street, Wagga Wagga, New South Wales 2678, Australia; Graham Centre for Agricultural Innovation, NSW Department of Primary Industries and Charles Sturt University, Boorooma Street, Wagga Wagga, New South Wales 2678, Australia. Electronic address: ssarker@csu.edu.au.
authors.forename: Subir
authors.initials: S
authors.lastname: Sarker
authors.affiliation: School of Animal and Veterinary Sciences, Charles Sturt University, Boorooma Street, Wagga Wagga, New South Wales 2678, Australia; Graham Centre for Agricultural Innovation, NSW Department of Primary Industries and Charles Sturt University, Boorooma Street, Wagga Wagga, New South Wales 2678, Australia. Electronic address: aghorashi@csu.edu.au.
authors.forename: Seyed A
authors.initials: SA
authors.lastname: Ghorashi
authors.affiliation: School of Biomedical Sciences, Charles Sturt University, Boorooma Street, Wagga Wagga, New South Wales 2678, Australia; Graham Centre for Agricultural Innovation, NSW Department of Primary Industries and Charles Sturt University, Boorooma Street, Wagga Wagga, New South Wales 2678, Australia. Electronic address: jforwood@csu.edu.au.
authors.forename: Jade K
authors.initials: JK
authors.lastname: Forwood
authors.affiliation: School of Animal and Veterinary Sciences, Charles Sturt University, Boorooma Street, Wagga Wagga, New South Wales 2678, Australia; Graham Centre for Agricultural Innovation, NSW Department of Primary Industries and Charles Sturt University, Boorooma Street, Wagga Wagga, New South Wales 2678, Australia. Electronic address: shraidal@csu.edu.au.
authors.forename: Shane R
authors.initials: SR
authors.lastname: Raidal
mesh_terms: D000818:Animals; D001715:Bird Diseases / Q000821:virology*; D001717:Birds; D018173:Circoviridae Infections / Q000662:veterinary* / Q000821:virology; D017925:Circovirus / Q000145:classification* / Q000235:genetics*; D003365:Costs and Cost Analysis; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D058889:Molecular Typing / Q000191:economics / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D013997:Time Factors; D044366:Transition Temperature
chemical_list: D004279:DNA, Viral
keywords: Beak and feather disease virus; Circovirus; Genotyping; HRM curve analysis; PCR