Abstract: 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 Green Ⅰ fluorescence 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 0.4 μM. 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 the virus isolation method was 0.00%（0/20）. All results indicated that the method established in this study has good specificity，sensitivity and stability to provide technical support for the accurate detection of ALV⁃F.

Key words: Avian leukosis, ALV?F, SYBR Green Ⅰ fluorescence quantitative PCR

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 Green Ⅰ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0.4 μM。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4.67×102拷贝/μL，敏感性是普通PCR 的1 000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。

关键词: 禽白血病, ALV?F, SYBR GreenⅠ荧光定量PCR