牛呼吸道主要病原体多重PCR检测方法的建立与应用  

Establishment and Application of Multiplex PCR Detection Method for Major Pathogens in Bovine Respiratory Tract

作　　者：沈思思 江波涛 冯万宇 秦平伟 兰世捷 苗艳 李丹 张蕾 刘雪松 张国华 王欢 李青莹 薛沾枚 王岩 南景东 刘文 陈亮 SHEN Si-si;JIANG Bo-tao;FENG Wan-yu;QIN Ping-wei;LAN Shi-jie;MIAO Yan;LI Dan;ZHANG Lei;LIU Xue-song;ZHANG Guo-hua;WANG Huan;LI Qing-ying;XUE Zhan-mei;WANG Yan;NAN Jing-dong;LIU Wen;CHEN Liang(Branch of Animal Husbandry and Veterinary Medicine,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：《动物医学进展》2025年第2期61-66,共6页Progress In Veterinary Medicine

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,ZNKT-2022-ZD03)。

摘　　要：为建立可同时检测牛腺病毒3型(BAdV-3)、牛呼吸道合胞体病毒(BRSV)、牛支原体(MB)、牛传染性鼻气管炎病毒(IBRV)和牛病毒性腹泻病毒(BVDV)的五重PCR方法,基于病原体的保守序列设计5对特异性引物。对退火温度和引物浓度筛选,验证方法的特异性、敏感性和重复性,建立可同时检测BAdV-3、BRSV、MB、IBRV和BVDV的五重PCR方法,并初步应用于临床。结果表明,该方法特异性强,仅对BAdV-3、BRSV、MB、IBRV和BVDV有特异性扩增,最适退火温度54.5℃,最适引物浓度0.6μmol/L;对5种病原体最低检出限分别为BAdV-31.67×10^(4)拷贝/μL、BRSV 1.58×10^(4)拷贝/μL、MB 1.75×10^(3)拷贝/μL、IBRV 1.44×10^(3)拷贝/μL、BVDV 1.25×10^(4)拷贝/μL。用该五重PCR和单项PCR分别检测268份临床样本,显示五重PCR阳性检出率分别为BAdV-39.33%(25/268)、BRSV 7.09%(19/268)、MB 28.73%(77/268)、IBRV 11.94%(32/268)、BVDV 25.75%(69/268)。混合感染主要以MB和BVDV为主,感染率为12.69%(34/268)。五重PCR扩增结果与单项PCR扩增结果符合率为93.36%,表明建立的五重PCR方法为临床快速检测及流行病学调查提供了技术支持。To establish a quintuplex PCR method that can simultaneously detect bovine adenovirus type 3(BAdV-3),bovine respiratory syncytial virus(BRSV),Mycoplasma bovis(MB),infectious bovine rhinotracheitis virus(IBRV),and bovine viral diarrhea virus(BVDV),five pairs of specific primers were designed based on the conserved sequences of the above pathogens.By screening for annealing temperature and primer concentration,the specificity,sensitivity,and repeatability of the method were verified.A quintuplex PCR method was established to simultaneously detect BAdV-3,BRSV,MB,IBRV,and BVDV,and was preliminarily applied in clinical practice.The results showed that the method had strong specificity and only showed specific amplification for BAdV-3,BRSV,MB,IBRV,and BVDV.The optimal annealing temperature for the method was 54.5℃,and the optimal primer concentration was 0.6μmol/L;the minimum detection limits for 5 pathogens were BAdV-31.67×10^(4) copies/μL,BRSV 1.58×10^(4) copies/μL,MB 1.75×10^(3) copies/μL,IBRV 1.44×10^(3) copies/μL,BVDV 1.25×10^(4) copies/μL.Using the quintuplex PCR and single PCR,268 clinical samples from Heilongjiang province were detected,and the positive detection rates of the quintuplex PCR were BAdV-39.33%(25/268),BRSV 7.09%(19/268),MB 28.73%(77/268),IBRV 11.94%(32/268),BVDV 25.75%(69/268).Mixed infections were mainly caused by MB and BVDV,with a positive detection rate of 12.69%(34/268),followed by MB and BVDV,with a infection rate of 12.69%(34/268).The total positive coincidence rate of quintuplex PCR and single PCR was 93.36%.The results indicated that the established quintuplex PCR provides technical support for the clinical rapid detection and epidemiological investigation of BAdV-3,BRSV,MB,IBRV,and BVDV infections.

关 键 词：牛 呼吸道疾病 多重聚合酶链反应 

分 类 号：S852.653[农业科学—基础兽医学]