河北省致犊牛腹泻主要病原多重PCR检测方法的建立  

Establishment of a multiple PCR method for detection of main viruses causing calf diarrhea in Hebei Province

作　　者：高亚桃 李妍[1] 刘立元[1] 王猛 郭姚蕊 刘胜丽 马亚宾[2] 蒋桂娥[2] 刘志勇 秦建华[1] 赵月兰[1] GAO Yatao;LI Yan;LIU Liyuan;WANG Meng;GUO Yaorui;LIU Shengli;MA Yabin;JIANG Guie;LIU Zhiyong;QIN Jianhua;ZHAO Yuelan(College of Veterinary Medicine,Agricultural University of HeBei,Baoding,Hebei 071001,China;Animal Husbandry and Breeding Station of Hebei,Shijiazhuang 050061,China;Animal Disease Prevention and Control Center of Tangshan,Tangshan,Hebei 064001,China)

机构地区：[1]河北农业大学动物医学院,河北保定071001 [2]河北省畜牧良种工作站,河北石家庄050061 [3]河北省唐山市动物疫病防控中心,河北唐山064001

出　　处：《中国兽医学报》2021年第10期1930-1936,共7页Chinese Journal of Veterinary Science

基　　金：河北省重点研发计划项目奶牛场犊牛多病原感染性腹泻综合防治技术研究资助项目(20326603D);河北省现代农业产业技术体系奶牛创新团队建设项目奶牛疫病防控资助项目(HBCT2018120205)。

摘　　要：为建立一种能同时检测牛病毒性腹泻病毒(BVDV)、牛轮状病毒(BRV)和牛传染性鼻气管炎病毒(IBRV)的多重PCR检测方法,本研究根据GenBank中登录的BVDV的5′-UTR序列、BRV的VP6基因和IBRV的gB基因设计3对特异性引物,通过对反应条件的优化,建立了一种多重PCR检测方法。特异性试验结果表明,该方法对牛冠状病毒(BCoV)、牛细小病毒(BPV)、牛隐孢子虫没有交叉反应,特异性较好;灵敏性试验结果显示,该方法对BVDV、BRV和IBRV的最低检出限分别为1.44×10^(5),1.38×10^(5),1.2×10^(5)copies/μL。利用该方法对从河北省部分地区采集的50份犊牛腹泻粪便样品进行检测,结果显示,BVDV和BRV混合感染检出率为18%(9/50),BVDV和IBRV混合感染检测率为14%(7/50),BRV和IBRV混合感染检测率为22%(11/50),3种病毒混合感染率为4%(2/50)。多重PCR检测和单一PCR检测方法均能检出被检样品中的病原,两种方法的总符合率分别为BVDV 90%、BRV 84%和IBRV 88%。该方法比单一PCR检测方法检测阳性率高,优于单一PCR检测方法,表明该方法可应用于临床检测。本研究建立的多重PCR检测方法,能快速、有效、准确地检测出BVDV、BRV和IBRV 3种病毒,对于流行病学调查及疾病的诊断等具有重要意义。In order to establish a multiple PCR method for detection of bovine viral diarrhea virus(BVDV),bovine rotavirus(BRV)and infectious bovine rhinotracheitis virus(IBRV)in calves,three pairs of specific primers were designed based on conserved sequence of 5′-UTR of BVDV,VP6 gene of BRV and gB gene of IBRV.After optimizing the reaction conditions,a multiple PCR detection method was established.The results showed that this method had high specificity and no cross-reaction with Cryptosporidium and BPV,and the detection limits of BVDV,BRV and IBRV were 1.44×10^(5),1.38×10^(5)and 1.2×10^(5)copies/μL respectively,indicating that the established method had high specificity and sensitivity.Clinical detection of 50 samples collected from calves with bovine diarrhea in Hebei Province showed that the co-infection rate of BVDV and BRV was 18%(9/50),and the co-infection rate of BVDV and IBRV was 14%(7/50),and the co-infection rate of BVDV and BRV was 22%(11/50),and the co-infection rate of the three viruses was 4%(2/50).The pathogens in the tested samples could be detected with multiple PCR method and single PCR method.The total coincidence rates of the two methods were 90%for BVDV,84%for BRV and 88%for IBRV.The positive rate of this method was higher than that of single PCR method,and it was better than single PCR method,suggesting that this method could be applied to clinical detection.In conclusion,the multiple PCR detection method established here can detect BVDV,BRV and IBRV viruses quickly,effectively and accurately,which is of great significance for epidemiological investigation and disease diagnosis.

关 键 词：牛病毒性腹泻病毒 牛轮状病毒 牛传染性鼻气管炎病毒 多重PCR 

分 类 号：S855.3[农业科学—临床兽医学]