title: A comparison of PCR assays for beak and feather disease virus and high resolution melt (HRM) curve analysis of replicase associated protein and capsid genes.
abstract: Beak and feather disease virus (BFDV) threatens a wide range of endangered psittacine birds worldwide. In this study, we assessed a novel PCR assay and genetic screening method using high-resolution melt (HRM) curve analysis for BFDV targeting the capsid (Cap) gene (HRM-Cap) alongside conventional PCR detection as well as a PCR method that targets a much smaller fragment of the virus genome in the replicase initiator protein (Rep) gene (HRM-Rep). Limits of detection, sensitivity, specificity and discriminatory power for differentiating BFDV sequences were compared. HRM-Cap had a high positive predictive value and could readily differentiate between a reference genotype and 17 other diverse BFDV genomes with more discriminatory power (genotype confidence percentage) than HRM-Rep. Melt curve profiles generated by HRM-Cap correlated with unique DNA sequence profiles for each individual test genome. The limit of detection of HRM-Cap was lower (2×10-5ng/reaction or 48 viral copies) than that for both HRM-Rep and conventional BFDV PCR which had similar sensitivity (2×10-6ng or 13 viral copies/reaction). However, when used in a diagnostic setting with 348 clinical samples there was strong agreement between HRM-Cap and conventional PCR (kappa=0.87, P<0.01, 98% specificity) and HRM-Cap demonstrated higher specificity (99.9%) than HRM-Rep (80.3%).
authors: School of Animal and Veterinary Sciences, Faculty of Science, Charles Sturt University, NSW 2678, Australia. Electronic address: sdas@csu.edu.au.
authors: Shubhagata
authors: S
authors: Das
authors: School of Animal and Veterinary Sciences, Faculty of Science, Charles Sturt University, NSW 2678, Australia. Electronic address: ssarker@csu.edu.au.
authors: Subir
authors: S
authors: Sarker
authors: School of Animal and Veterinary Sciences, Faculty of Science, Charles Sturt University, NSW 2678, Australia. Electronic address: aghorashi@csu.edu.au.
authors: Seyed Ali
authors: SA
authors: Ghorashi
authors: School of Biomedical Sciences, Faculty of Science, Charles Sturt University, Wagga Wagga, NSW 2650, Australia. Electronic address: jforwood@csu.edu.au.
authors: Jade K
authors: JK
authors: Forwood
authors: School of Animal and Veterinary Sciences, Faculty of Science, Charles Sturt University, NSW 2678, Australia. Electronic address: shraidal@csu.edu.au.
authors: Shane R
authors: SR
authors: Raidal
mesh_terms: D000818:Animals; D001715:Bird Diseases / Q000175:diagnosis* / Q000821:virology; D036022:Capsid Proteins / Q000235:genetics* / Q000302:isolation & purification; D018173:Circoviridae Infections / Q000662:veterinary* / Q000821:virology; D017925:Circovirus / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D005838:Genotype; D009691:Nucleic Acid Denaturation; D010311:Parrots / Q000821:virology; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D044366:Transition Temperature
chemical_list: D036022:Capsid Proteins; D004279:DNA, Viral
keywords: Genotyping; High resolution melt analysis; Psittacine beak and feather disease