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

作者：Das Shubhagata; Sarker Subir; Ghorashi Seyed Ali; Forwood Jade K; Raidal Shane R^*

来源：Journal of Virological Methods, 2016, 237: 47-57.

DOI：10.1016/j.jviromet.2016.08.015

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

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 x 10(-5) ng/reaction or 48 viral copies) than that for both HRM-Rep and conventional BFDV PCR which had similar sensitivity (2 x 10(-6) ng 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%).

• 出版日期2016-11