Format

Send to

Choose Destination
J Virol Methods. 2014 Nov;208:47-55. doi: 10.1016/j.jviromet.2014.07.031. Epub 2014 Aug 4.

Rapid genotyping of beak and feather disease virus using high-resolution DNA melt curve analysis.

Author information

1
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.
2
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.
3
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.
4
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.

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.

KEYWORDS:

Beak and feather disease virus; Circovirus; Genotyping; HRM curve analysis; PCR

PMID:
25102431
DOI:
10.1016/j.jviromet.2014.07.031
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center