流感疫苗毒种中3种外源性禽病毒荧光定量PCR检测方法的实验室间验证  

Interlaboratory verification of fluorescent quantitative PCR for detection of three extraneous avian viruses in influenza vaccine virus strains

作　　者：王淑菁[1] 付瑞[1] 秦骁 刘朝阳[2] 胡雅灵 王增 韩斌 王魁 岳秉飞[1] WANG Shu-jing;FU Rui;QIN Xiao;LIU Chao-yang;HU Ya-ling;WANG Zeng;HAN Bin;WANG Kui;YUE Bing-fei(National Institute for Food and Drug Control,Beijing 100050,China)

机构地区：[1]中国食品药品检定研究院,北京100050 [2]上海生物制品研究所,上海201403 [3]北京科兴生物制品有限公司,北京100085 [4]华兰生物疫苗有限公司,河南新乡453000

出　　处：《中国生物制品学杂志》2021年第4期453-459,共7页Chinese Journal of Biologicals

基　　金：北京市科技计划课题(D181100000518003)。

摘　　要：目的实验室间验证流感疫苗毒种中3种外源性禽病毒的荧光定量PCR(Q-PCR)检测方法。方法联合4家实验室对已建立的流感疫苗毒种中禽腺病毒Ⅰ型[鸡胚致死孤儿病毒(chicken embryo lethal orphan virus,CELO)]、禽腺病毒Ⅲ型[减蛋综合征病毒(egg drop syndrome virus,EDS)]及禽白血病病毒(avian leukosis virus,ALV)Q-PCR检测方法,从灵敏度、特异性、重复性、病毒污染模拟试验及盲样检测方面,开展实验室间方法学验证。结果4家实验室间验证结果显示,Q-PCR检测方法的标准曲线R2值均大于0.99,扩增效率为93.263%~103.105%;灵敏度均达1×101copies/μL;特异性为仅检出阳性目的病毒,其他病毒均无扩增曲线出现;试验内及试验间循环阈值(Ct)和拷贝数的CV均小于15%;病毒污染模拟试验中CELO、EDS和ALV最低检测限分别为1×10^(-7)、1×10^(-4)、1×10^(-3)病毒掺入;盲样检测中4家实验室结果均一致。结论外源性禽病毒Q-PCR检测方法得到了一致性结果,均具有良好的灵敏度、特异性、重复性,可用于流感疫苗毒种外源性禽病毒的快速检测。Objective To verify the fluorescent quantitative PCR(Q-PCR)for detection of three extraneous avian viruses in influenza vaccine virus strains among various laboratories. Methods The Q-PCR method for detection of avian adenovirus typesⅠ(chicken embryo lethal orphan virus,CELO)and Ⅲ(egg drop syndrome virus,EDS)as well as avian leukemia virus(ALV) in influenza vaccine virus strains were subjected to interlaboratory verification for sensitivity,specificity,reproducibility,virus contamination mimic experiments and blind sample detection by four laboratories.Results The verification results showed that the R2 values of standard curves of Q-PCR methods by various laboratories were more than 0. 99,while the amplification efficiencies were 93. 263% ~ 103. 105%,and the sensitivities reached 1×101 copies/μL. Only the positive target viruses were detected,while no amplification curves of other viruses appeared.All the coefficients of variation(CVs)of the cycle thresholds and copies in intra-and inter-assays were less than 15%. The minimum detection limits of CELO,EDS and ALV in virus contamination experiments were 1 × 10^(-7),1 ×10^(-4) and 1 ×10^(-3) virus incorporation respectively,and the detection results of blind samples by four laboratories were in agreement.Conclusion The verification results of Q-PCR method for detection of extraneous avian viruses by various laboratories were in agreement. The method showed high sensitivity,specificity and reproducibility,which might be used for the rapid detection of extraneous avian virus in influenza vaccine virus strains.

关 键 词：流感疫苗 外源性禽病毒 荧光定量PCR 实验室间验证 

分 类 号：Q939.47[生物学—微生物学]