FADV-4环介导等温扩增-HNB可视化检测方法的建立和应用  

Establishment and application of visual loop-mediated isothermal amplification-HNB for FADV-4

作　　者：姜朕元 赵艳 王婷 张静 周海洋 李佳康 黄雪琳 黄勇[1,2] 韩新锋 JIANG Zhenyuan;ZHAO Yan;WANG Ting;ZHANG Jing;ZHOU Haiyang;LI Jiakang;HUANG Xuelin;HUANG Yong;HAN Xinfeng(College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,China;Key Laboratory of Animal Diseases and Human Health of Sichuan Province,Chengdu 611130,China)

机构地区：[1]四川农业大学动物医学院,成都611130 [2]动物疫病与人类健康四川省重点实验室,成都611130

出　　处：《黑龙江畜牧兽医》2020年第24期65-69,76,175,共7页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家级大学生创新训练计划项目(201710626005);教育部《长江学者和创新团队发展计划》创新团队项目(IRTO848)。

摘　　要：为了建立简便、快速的FADV-4环介导等温扩增(LAMP)可视化检测方法,试验以FADV-4高度保守的基因序列hexon基因为靶基因设计了6条引物,对反应体系的Mg^2+浓度、Bst DNA聚合酶用量、反应温度、反应持续时间进行优化,考察了优化体系的灵敏度及特异性。同时用建立的FADV-4 LAMP及LAMP-HNB可视化的检测方法对临床模拟样品进行检测。结果表明:当Mg^2+浓度为6 mmol/L、Bst DNA聚合酶浓度为320 U/mL、反应温度为65℃、反应时间为60 min、HNB使用浓度为120μmol/L时反应最优。LAMP最低DNA检测浓度为1.46 pg/μL,灵敏度是PCR的100倍,临床模拟样品LAMP、LAMP-HNB检测结果与普通PCR检测结果完全符合。说明所建立的FADV-4 LAMP-HNB可视化检测方法适用于临床的快速诊断。In order to develop a convenient and rapid visual loop-mediated isothermal amplification(LAMP)assay for detection of fowl adenovirus serotype 4(FADV-4),six primers were designed based on a highly specific and conserved hexon gene of FADV-4.The concentration of Mg^2+,Bst DNA polymerase,together with reaction temperature and reaction time were optimized accordingly.Moreover,the sensitivity and specificity of the optimized system were tested.At the same time,the established FADV-4 LAMP and LAMP-HNB visual detection methods were used to detect clinical simulation samples.The results showed that the reaction was optimal when the concentration of Mg^2+was 6 mmol/L,the concentration of Bst DNA polymerase was 320 U/mL,the reaction temperature was 65℃,the reaction time was 60 min,and the concentration of HNB was 120μmol/L.The minimum DNA detection concentration was 1.46 pg/μL,and the sensitivity was 100 times higher than that of PCR.LAMP and LAMP-HNB had a high coincidence with the traditional PCR in detection of clinical simulation samples,which indicated that the visual LAMP-HNB is reliable and suitable for rapid diagnosis of FADV-4 in clinical practice.

关 键 词：Ⅰ群禽腺病毒血清4型 六邻体蛋白基因 环介导等温扩增 羟基萘酚蓝 可视化 检测 

分 类 号：S852.65[农业科学—基础兽医学] Q78[农业科学—兽医学]