Sarker Subir, Ghorashi Seyed A, Forwood Jade K, Raidal Shane R
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.
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.
J Virol Methods. 2014 Nov;208:47-55. doi: 10.1016/j.jviromet.2014.07.031. Epub 2014 Aug 4.
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.
喙羽病病毒（BFDV）是全球野生和圈养鹦鹉科鸟类的一种重要病原体。BFDV分离株的基因分型对于制定濒危物种保护的有效控制策略以及疾病爆发的流行病学调查至关重要。本研究开发的技术是一种使用PCR及后续高分辨率熔解（HRM）曲线分析的简单、快速且廉价的BFDV基因分型方法。这是通过在荧光DNA嵌入染料（SYTO9）存在的情况下对保守的Rep基因进行PCR扩增来实现的。对所得扩增子进行HRM曲线分析能够轻松区分本研究中使用的参考菌株（92 - SR14）和其他18种BFDV分离株。对每个分离株扩增子的核苷酸序列分析表明，每个熔解曲线图谱都与一个独特的DNA序列相关。还评估了PCR - HRM曲线分析区分极度濒危的橙腹鹦鹉、吸蜜鹦鹉和凤头鹦鹉宿主间遗传变异的潜力。基于本研究中使用的部分Rep基因序列构建的系统发育树拓扑结构表明，BFDV Rep基因序列模式与HRM曲线分析结果相关。本研究结果表明，该技术可用于临床研究和BFDV分离株的区分，无需进一步的核苷酸测序，且能在短时间内完成，为临床病毒学实验室中BFDV的基因筛查提供了一种新方法。
