鸭瘟病毒微滴数字PCR方法的建立与优化  

Establishment and Optimization of Droplet Digital PCR for Duck Enteritis Virus

作　　者：邱艳红 廖维连 蒋文明[3] 杨得胜[4] 宋迟 洪瑾芳 刘道泉 Qiu Yanhong;Liao Weilian;Jiang Wenming;Yang Desheng;Song Chi;Hong Jinfang;Liu Daoquan(Fuzhou Center for Animal Disease Prevention and Control,Fuzhou 350025,Fujian,China;Jiangle Center for Animal Disease Prevention and Control,Jiangle 365001,Fujian,China;China Animal Health and Epidemiology Center,Qingdao 266032,Shandong,China;Fujian Center for Animal Disease Prevention and Control,Fuzhou 350003,Fujian,China;Yongtai Golden Egg Development Co.,Ltd.,Yongtai 350714,Fujian,China;Yongtai Animal Husbandry and Fisheries Service Center,Yongtai 350700,Fujian,China)

机构地区：[1]福州市动物疫病预防控制中心,福建福州350025 [2]将乐县动物疫病预防控制中心,福建将乐365001 [3]中国动物卫生与流行病学中心,山东青岛266032 [4]福建省动物疫病预防控制中心,福建福州350003 [5]永泰县金蛋发展有限公司,福建永泰350714 [6]永泰县畜牧水产服务中心,福建永泰350700

出　　处：《中国动物检疫》2024年第10期91-95,共5页China Animal Health Inspection

基　　金：福建省科学技术厅星火项目(2022S0005)。

摘　　要：为快速、准确检测鸭瘟病毒(duck enteritis virus,DEV),根据GenBank中DEV UL6基因序列,设计了微滴数字PCR(ddPCR)的引物和探针,通过条件优化,建立了检测DEV的ddPCR方法,并对该方法的灵敏度、特异性和重复性进行了验证。结果显示:在57.1℃退火温度下,当引物浓度为1.50µmol/L,探针浓度为0.45µmol/L时,病毒拷贝数浓度达到最大,为332 copies/µL;该方法的最低检测限为0.7 copies/µL,且对非鸭瘟病原均仅扩增出阴性微滴,无交叉反应;组内和组间重复试验变异系数均小于10%。结果表明,本研究建立的DEV ddPCR方法具有灵敏度高、特异性强、重复性好和稳定性高的优点,可用于低DEV含量样品的检测及其感染的早期诊断和流行病学调查。In order to quickly and accurately detect duck enteritis virus(DEV),primers and probes for droplet digital PCR(ddPCR)were designed based on DEV UL6 gene sequence registered in GenBank,and a ddPCR method for detection of DEV was established after optimization of reaction conditions,followed by the verification of its sensitivity(332 copies/µL,specificity and reproducibility.The results showed that the virus concentration reached to the maximum)when the concentrations of primers and probes were 1.50µmol/L and 0.45µmol/L,respectively,at an annealing temperature of 57.1℃;the lowest detection limit was 0.7 copies/µL,and it couldn't generate positive microtitres for non-DEV pathogens,without cross-reactivity;the variable coefficients of both the intra-and inter-group repeated tests were less than 10%.In conclusion,the DEV ddPCR method established in this study was characterized by the advantages of high sensitivity,strong specificity,good reproducibility and high stability,and could be used for the detection of samples with low DEV content and early diagnosis and epidemiological investigation of the disease.

关 键 词：鸭瘟病毒 微滴数字PCR 方法建立 优化 

分 类 号：S851.34[农业科学—预防兽医学]