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

作　　者：雷晨莹 陈亚娜 张洁[1] 崔灿 马岩 原霖 田克恭 LEI Chenying;CHEN Yana;ZHAN Jie;CUI Can;MA Yan;YUAN Lin;TIAN Kegong(College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China;Beijing Zhongke Gene Technology Co.,Ltd.,Beijing 102600,China;Pudao(Beijing)Standard Technology Co.,Ltd.,Beijing 102600,China;National Research Center for Veterinary Medicine,Luoyang 471000,China;PULIKE Biological Engineering,INC.,Luoyang 471000,China)

机构地区：[1]河南农业大学动物医学院,河南郑州450046 [2]北京中科基因技术股份有限公司,北京102600 [3]普道(北京)标准技术有限公司,北京102600 [4]国家兽用药品工程技术研究中心,河南洛阳471000 [5]普莱柯生物工程股份有限公司,河南洛阳471000

出　　处：《中国兽医科学》2025年第3期354-361,共8页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2022YFD1800605)。

摘　　要：建立一种A、B、J、K亚群禽白血病病毒(ALV)微滴式数字PCR(ddPCR)检测方法。根据ALV pol基因序列设计特异性引物探针,优化ddPCR方法的反应条件,并评估其敏感性、特异性和重复性。结果显示:引物和探针浓度分别为900和250 nmol/L、退火温度为55℃时ddPCR反应效率最高,该方法检出限和定量限分别为1.999和6.664 copies/μL,通过梯度稀释得到的标准曲线的相关系数为0.994 1,不与常见的禽病毒发生交叉反应,重复性好,ddPCR方法的最低检测限约为1 copies/μL,相同测试条件下,qPCR方法的最低检测限约为100 copies/μL,ELISA方法的检测限约为1×10^(4)copies/μL。结果表明:建立的ddPCR方法的灵敏度高于qPCR和ELISA方法,可用于A、B、J、K亚群ALV的临床检测以及定量检测。To develop a detection method for avian leukosis virus(ALV) subtypes A,B,J and K utilizing droplet digital PCR(dd PCR),specific primers and probes were designed based on the pol gene sequence of ALV.The reaction conditions for the dd PCR method were optimized,and its sensitivity,specificity,and repeatability were evaluated.The findings indicated that the dd PCR 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 were1.999 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 dd PCR method exhibited a minimum detection limit of approximately 1 copies/μL.In contrast,under identical experimental conditions,the quantitative PCR method demonstrated a minimum detection limit of approximately 100copies/μL,while the enzyme-linked immunosorbent assay method showed a detection limit of approximately 1×10^(4)copies/μL.These findings suggest that the dd PCR method offers greater sensitivity compared to quantitative PCR and enzyme-linked immunosorbent assay,making it suitable for clinical detection 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 

分 类 号：S852.659.6[农业科学—基础兽医学]