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J Virol Methods. 2016 Nov;237:47-57. doi: 10.1016/j.jviromet.2016.08.015. Epub 2016 Aug 23.

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.

Author information

1
School of Animal and Veterinary Sciences, Faculty of Science, Charles Sturt University, NSW 2678, Australia. Electronic address: sdas@csu.edu.au.
2
School of Animal and Veterinary Sciences, Faculty of Science, Charles Sturt University, NSW 2678, Australia. Electronic address: ssarker@csu.edu.au.
3
School of Animal and Veterinary Sciences, Faculty of Science, Charles Sturt University, NSW 2678, Australia. Electronic address: aghorashi@csu.edu.au.
4
School of Biomedical Sciences, Faculty of Science, Charles Sturt University, Wagga Wagga, NSW 2650, Australia. Electronic address: jforwood@csu.edu.au.
5
School of Animal and Veterinary Sciences, Faculty of Science, Charles Sturt University, NSW 2678, Australia. Electronic address: shraidal@csu.edu.au.

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%).

KEYWORDS:

Genotyping; High resolution melt analysis; Psittacine beak and feather disease

PMID:
27565820
DOI:
10.1016/j.jviromet.2016.08.015
[Indexed for MEDLINE]

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