中国兽医科学2025，Vol.55Issue(3)：354-361,8.DOI:10.16656/j.issn.1673-4696.2025.0063
四个亚群禽白血病病毒微滴式数字RT-PCR检测方法的建立
Development of droplet digital RT-PCR detection method for subgroups A,B,J,and K avian leukosis viruses
摘要
Abstract
To develop a detection method for avian leukosis virus(ALV)subtypes A,B,J and K utiliz-ing droplet digital PCR(ddPCR),specific primers and probes were designed based on the pol gene se-quence of ALV.The reaction conditions for the ddPCR method were optimized,and its sensitivity,speci-ficity,and repeatability were evaluated.The findings indicated that the ddPCR reaction efficiency was maximized when the primer and probe concentrations were set at 900 nmol/L and 250 nmol/L,respectively,with an annealing temperature of 55℃.The limits of detection and quantification of the method were 1.999 copies/μL and 6.664 copies/μL,respectively.The standard curve,derived from gradient dilution,exhibited a correlation coefficient of 0.994 1.Additionally,the method showed no cross-reactivity with common avian viruses and demonstrated excellent repeatability.The ddPCR method exhibited a mini-mum detection limit of approximately 1 copies/μL.In contrast,under identical experimental condi-tions,the quantitative PCR method demonstrated a minimum detection limit of approximately 100 copies/μL,while the enzyme-linked immunosorbent assay method showed a detection limit of approxi-mately 1 × 104 copies/µL.These findings suggest that the ddPCR method offers greater sensitivity com-pared to quantitative PCR and enzyme-linked immunosorbent assay,making it suitable for clinical de-tection and quantitative analysis of ALV subtypes A,B,J and K.关键词
禽白血病病毒/微滴式数字PCR/定量检测Key words
avian leukosis virus/droplet digital PCR/quantitative detection分类
农业科技引用本文复制引用
雷晨莹,陈亚娜,张洁,崔灿,马岩,原霖,田克恭..四个亚群禽白血病病毒微滴式数字RT-PCR检测方法的建立[J].中国兽医科学,2025,55(3):354-361,8.基金项目
国家重点研发计划项目(2022YFD1800605) （2022YFD1800605）
