F亚群禽白血病病毒SYBR GreenⅠ荧光定量PCR方法的建立  

Development of Quantitative PCR Method based on SYBR GreenⅠFluorescence for Detecting Subgroup F Avian Leukosis Virus

作　　者：潘洪艳 李锦群 李琪 袁伊琳 曹伟胜[1,2] PAN Honyan;LI Jinqun;LI Qi;YUAN Yilin;CAO Weisheng(College of Veterinary Medicine,South China Agricultural University,Guangzhou Guangdong 510642;Key Laboratory of Zoonosis Prevention and Control of Guangdong Province,National and Regional Joint Engineering Laboratory for Medicament of Zoonosis Prevention and Contro,Key Laboratory of Veterinary Vaccine Innovation of the Ministry of Agriculture and Rural Affairs,Key Laboratory of Zoonosis of Ministry of Agriculture and Rural Affairs,Guangzhou Guangdong 510642)

机构地区：[1]华南农业大学兽医学院,广东广州510642 [2]广东省动物源性人兽共患病预防与控制重点实验室/人兽共患病防控制剂国家地方联合工程实验室/农业农村部兽用疫苗创制重点实验室/农业农村部人兽共患病重点实验室,广东广州510642

出　　处：《广东畜牧兽医科技》2025年第2期23-29,共7页Guangdong Journal of Animal and Veterinary Science

基　　金：广东省重点领域研发计划项目(2020B020222001);广东省家禽产业技术体系(2023KJ128);国家肉鸡产业技术体系(CARS⁃41⁃G10)。

摘　　要：为精准检测F亚群禽白血病病毒(ALV⁃F),该研究针对ALV⁃F流行毒株env基因设计了一对特异性引物F1/F2,建立了ALV⁃F的SYBR GreenⅠ荧光定量PCR检测方法。条件优化试验结果显示,该方法最适退火温度为64.5℃,最适引物浓度为0.4μM。利用最适反应条件建立的标准曲线方程为y=-3.303x+42.525,R2=0.997,扩增效率E=100.8%,重组质粒标准品拷贝数与Ct值具有良好的线性关系。特异性试验结果表明,该方法仅能特异性检测ALV⁃F,对ALV⁃A、ALV⁃B、ALV⁃J、ALV⁃K、禽流感病毒(AIV)、马立克病毒(MDV)、网状内皮组织增生病病毒(REV)和鸡传染性贫血病病毒(CIAV)无交叉反应;敏感试验结果表明,该方法最低检测限度为4.67×10^(2)拷贝/μL,敏感性是普通PCR的1000倍;稳定性试验结果表明,批间和批内稳定性试验的变异系数均小于2%。利用该方法、ALV p27 ELISA和普通PCR方法分别对23份七彩山鸡胚所制备的原代细胞(PEF)样品进行检测,结果显示该方法和普通PCR方法阳性检出率均为95.65%(22/23),而ALV p27 ELISA阳性检出率仅为39.13%(9/23),该方法与普通PCR总符合率为100%,与ALV p27 ELISA总符合率为43.48%;进一步利用该方法、病毒分离法(结合ALV p27 ELISA)和普通PCR方法分别对20份七彩山鸡抗凝血样品进行检测,结果显示该方法和普通PCR方法阳性检出率分别为100.00%(20/20)和75.00%(15/20),两者总符合率为75.00%,而病毒分离法阳性检出率为0.00%(0/20)。本研究建立的方法具有良好的特异性、敏感性和稳定性,能够有效检测临床样品,可以为ALV⁃F的精准检测提供技术支持。To accurately detect subgroup F avian leukosis virus(ALV⁃F),a pair of specific primers F1/F2 were designed according to the env gene of the ALV⁃F epidemic strains,and a SYBR GreenⅠfluorescence quantitative PCR detection method for ALV⁃F was established.The condition optimization experiment results showed that the optimal annealing temperature was 64.5℃,and the optimal primer concentration was 0.4μM.The standard curve equation established using the optimal reaction conditions was y=-3.303x+42.525,R2=0.997,amplification efficiency E=100.8%,and the copy number of the standard plasmid had a good linear relationship with the Ct value.The specificity results showed that this method can only specifically detect ALV⁃F,and has no cross⁃reaction to ALV⁃A,ALV⁃B,ALV⁃J,ALV⁃K,avian influenza virus(AIV),Marek's virus(MDV),reticuloendotheliosis virus(REV)and chicken infectious anemia virus(CIAV).The sensitivity results showed that the minimum detection limit of standard plasmid was 4.67×102 copies/μL,and the sensitivity was 1000 times that of routine PCR.The reproducibility results showed that the coefficient of variation of intra⁃assay and inter⁃assay reproducibility was less than 2%.23 primary cell(PEF)samples prepared from pheasant embryos were detected by using this method,ALV p27 ELISA and routine PCR respectively,and the results showed that the positive detection rates of this method and PCR method were both 95.65%(22/23),while the positive detection rate of ALV p27 ELISA was only 39.13%(9/23).The total consistency rate of this method with routine PCR was 100%,while the total consistency rate with ALV p27 ELISA was 43.48%.20 anticoagulated blood samples of pheasant were further detected by using this method,virus isolation method through ALV p27 ELISA and routine PCR method,and the results showed that the positive detection rates of this method and ordinary PCR method were 100.00%(20/20)and 75.00%(15/20),respectively,with an overall compliance rate of 75.00%,while the positive detection rate of

关 键 词：禽白血病 ALV⁃F SYBR GreenⅠ荧光定量PCR 

分 类 号：S852.657[农业科学—基础兽医学]