牛呼吸道病原体四重PCR检测方法的建立与应用  

Establishment and Application of a Quadruple PCR Method for Detection of Bovine Respiratory Pathogens

作　　者：沈思思 冯万宇 陈亮 兰世捷 李丹 张蕾 周景明 刘雪松 张国华 王欢 王岩 刘文 SHEN Sisi;FENG Wanyu;CHEN Liang;LAN Shijie;LI Dan;ZHANG Lei;ZHOU Jingming;LIU Xuesong;ZHANG Guohua;WANG Huan;WANG Yan;LIU Wen(Branch of Animal Husbandry and Veterinary,Heilongjiang Academy of Agricultural Sciences,Qiqihar Heilongjiang 161005,China)

机构地区：[1]黑龙江省农业科学院畜牧兽医分院,黑龙江齐齐哈尔161005

出　　处：《现代畜牧科技》2025年第3期5-8,共4页Modern Animal Husbandry Science & Technology

基　　金：黑龙江省农业科学院畜牧兽医分院2022年自拟项目(ZNKT-202213)。

摘　　要：为建立一种可同时检测牛腺病毒3(BAdV-3)、牛呼吸道合胞体病毒(BRSV)、溶血性曼氏杆菌(MH)和牛冠状病毒(BCoV)的四重PCR方法,根据Genbank公布的BAdV-3、BRSV、MH和BCoV基因保守序列设计了特异性引物4对,通过对退火温度和引物浓度的筛选,并验证了其特异性、敏感性和重复性,建立了能够同时检测BAdV-3、BRSV、MH和BCoV的四重PCR方法,初步应用于临床。结果显示,最佳退火温度为54℃,最佳引物浓度为0.5μmol/L;其特异性结果显示四重PCR只能检出BAdV-3、BRSV、MH和BCoV 4种病原体,对BAdV-3、BRSV、MH和BCoV最低检出限分别为1.67×10^(4)、1.58×10^(4)、1.75×10^(3)、1.29×10^(3)copies/μL,建立的四重PCR对40份临床样本进行检测,结果与单一PCR检测结果一致。结果表明,该研究建立的四重PCR方法可用于BAdV-3、BRSV、MH和BCoV的流行病学调查。In order to establish one method for simultaneous detection of Bovine adenovirus virus type 3(BAdV-3),Bovine respiratory syncytial virus(BRSV),Mannheimia haemolytica(MH),and Bovine coronavirus(BCoV),four pairs of specific primers were designed according to the conserved sequences of BAdV-3、BRSV、MH and BCoV genes published by Genbank.The specificity,sensitivity and repeatability of the PCR were verified,and a quadruple PCR method was established to detect BAdV-3、BRSV、MH and BCoV at the same time,preliminary clinical application.The results showed that the optimal annealing temperature was 54℃and the optimal primer concentration was 0.5μmol/L.The specific results showed that the quadruple PCR could only detect BAdV-3、BRSV、MH and BCoV.The minimum detection limits of BAdV-3、BRSV、MH and BCoV were 1.67×10^(4)、1.58×10^(4)、1.75×10^(3)、1.29×10^(3)copies/μL,respectively.The established quadruple PCR was used to detect 40 clinical samples,and the results were consistent with that of single PCR.The results show that the quadruple PCR method established in this study can be used for the epidemiological investigation of BAdV-3、BRSV、MH and BCoV.

关 键 词：腺病毒 呼吸道合胞病毒 支原体 冠状病毒 四重PCR 

分 类 号：S854.4[农业科学—临床兽医学]