1种检测新城疫病毒的微滴式数字PCR方法  

A Droplet Digital PCR Method for Detection of Newcastle Disease Virus

作　　者：梅力 王英超 吴迪 李月[2] 宋彦军 韦海涛 王林 高晓龙 冯小宇 MEI Li;WANG Yingchao;WU Di;LI Yue;SONG Yanjun;WEI Haitao;WANG Lin;GAO Xiaolong;FENG Xiaoyu(Beijing Animal Disease Control Center,Beijing 102629,China;Beijing Veterinary Drug Control Institution,Beijing 102629,China)

机构地区：[1]北京市动物疫病预防控制中心,北京102629 [2]北京市兽药监察所,北京102629

出　　处：《畜牧兽医学报》2020年第12期3187-3192,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：北京市科委重大专项首都食品质量安全保障课题(Z171100001317013)。

摘　　要：本研究旨在构建一种新城疫病毒(Newcastle disease virus,NDV)的微滴式数字PCR方法(droplet digital PCR,ddPCR)。以NDV F基因为靶基因,选取保守区域设计引物和探针,构建了NDV ddPCR。对ddPCR反应引物和探针浓度、退火温度进行了优化,并对该方法的灵敏度、特异性和重复性进行测定。试验结果表明,ddPCR的最佳引物和探针浓度分别为900和250 nmol·L-1,最佳的退火温度为55℃。ddPCR的线性良好,最低检测下限为1.8 copies·μL-1,与禽传染性支气管炎病毒等其他6种病毒无交叉反应,样本重复的变异系数为2.4%。结果提示,本研究建立的ddPCR方法灵敏度高、特异性强,可对新城疫病毒感染的临床样品进行定量检测。This experiment was conducted to establish a droplet digital PCR(ddPCR)method for the detection of Newcastle disease virus(NDV).A ddPCR method was developed,which the primers and probes were designed based on the conservative regions of F gene of NDV.The concentration of primer and probe,the annealing temperature in ddPCR reaction were optimized.The sensitivity,specificity and reproducibility of ddPCR method were evaluated.In results,the optimal primer concentration and the probe concentration were 900 and 250 nmol·L-1,the optimum annealing temperature was 55℃.The detection limit of ddPCR method was 1.8 copies·μL-1 with a good linear response,it had no cross reaction with other six viruses(include IBV),the coefficient of variation of sample repetition was 2.4%.All the results showed that NDV ddPCR was sensitive and specific,it was suitable for quantitative detection of clinical samples infected with NDV.

关 键 词：新城疫病毒 数字PCR 检测方法 

分 类 号：S855.3[农业科学—临床兽医学]