摘要
建立针对牛腺病毒5型六邻体(Hexon)基因的实时荧光定量TaqMan PCR检测方法。根据牛腺病毒5型Hexon基因高度保守区设计引物和TaqMan探针,绘制标准曲线,对其灵敏度、特异性、重复性进行验证,建立牛腺病毒5型实时荧光定量TaqMan PCR快速检测方法。结果显示,构建的重组质粒pUC57-BAV5,标准曲线在10~2~10~7拷贝数之间有较好的线性关系,相关系数为0.975 2,建立检测方法的最低检测限为32拷贝/μL;应用建立的方法检测牛传染性鼻气管炎病毒、牛轮状病毒、牛呼吸道合胞体病毒无交叉反应;重复性试验表明,同一浓度的20个平行样品的变异系数为1.4%。牛腺病毒5型TaqMan探针荧光定量PCR方法具有灵敏度高、特异性强、重复性好等特点,可以用于牛腺病毒5型的快速、准确的检测。
To establish a detection method of bovine adenovirus type 5 by means of TaqMan probe real time quantitative polymerase chain reaction(PCR).By targeting the gene Hexon of bovine adenovirus type 5,designing a primer and TaqMan probe,making quantitative standard curve,and verifying sensitivity,specificity and repeatability of the method,we established a TaqMan real time quantitative PCR method for detecting bovine adenovirus type 5.The results showed that the constructed recombinant plasmid pUC57-BAV5 had a good linear relationship between 10~2-10~7 copies of the standard curve with a correlation coefficient of 0.975 and a minimum detection limit of 32 copies/μL.The established method was successfully applied to detecting the absence of bovine infectious rhinotracheitis virus,bovine rotavirus and bovine respiratory syncytial virus with no cross reaction.The repeatability test showed that the coefficient of variation of 20 parallel samples at the same concentration was 1.4%.The study indicated that the method of real time quantitative PCR with TaqMan probe was of high sensitivity,specificity and repeatability and it might be recommended for rapid and accurate detection of bovine adenovirus type 5.
作者
安微
陈世界
王史
张婧
杨苗
薛昌华
余姓鸿
林华
AN Wei;CHEN Shijie;WANG Shi;ZHANG Jing;YANG Miao;XUE Changhua;YU Xinghong;LIN Hua(The Inspection and Quarantine Technology Center of Sichuan Province Entry-Exit Inspection and Quarantine Bureau,Chengdu 610041,China;The Inspection and Quarantine Technology Center of Hainan Entry-Exit Inspection and Quarantine Bureau,Haikou 570100,China)
出处
《畜牧与兽医》
北大核心
2019年第8期101-104,共4页
Animal Husbandry & Veterinary Medicine
基金
四川出入境检验检疫局科技项目(SK201703)
