鸭瘟病毒、鸭坦布苏病毒和鸭疫里默杆菌多重PCR检测方法的建立与初步应用  

Development and preliminary application of multiplex PCR detection methods for duck plague virus,duck Tembusu virus and Riemerella anatipestifer

作　　者：曲哲会[1,2,3] 张喜文 鲁绍芳 陈敏 曾超[1] 李梦莉 焦凤超 何书海[1,2] 赵瑜[1,2] 黄立[1] QU Zhehui;ZHANG Xiwen;LU Shaofang;CHEN Min;ZENG Chao;LI Mengli;JIAO Fengchao;HE Shuhai;ZHAO Yu;HUANG Li(College of Animal Science and Veterinary Medicine,Xinyang Agriculture and Forestry University,Xinyang,Henan 464000,China;Engineering and Technology Research Center for Waterfowl Resources Development and Utilization and Epidemic Disease Prevention and Control of Henan Province,School of Animal Science and Veterinary Medicine,Xinyang Agriculture and Forestry University,Xinyang,Henan 464000,China;Xinyang Key Laboratory of Animal Husbandry and Environmental Control,Xinyang,Henan 464000,China)

机构地区：[1]信阳农林学院动物科技学院,河南信阳464000 [2]信阳农林学院动物科技学院,河南省水禽资源开发利用与疫病防控工程技术研究中心,河南信阳464000 [3]信阳市畜禽养殖与环境控制重点实验室,河南信阳464000

出　　处：《中国兽医学报》2023年第6期1175-1180,共6页Chinese Journal of Veterinary Science

基　　金：河南省科技攻关资助项目(222102110188);信阳农林学院高水平科研孵化器建设资助项目(FCL202004);信阳农林学院重要水禽源病原致病与免疫机制研究科技创新团队建设资助项目(XNKJTD-014);信阳农林学院青年科研基金资助项目(20200113);信阳市创新应用专项资助项目(20200016)。

摘　　要：设计分别检测鸭坦布苏病毒(duck Tembusu virus,DTMUV)、鸭瘟病毒(duck plague virus,DPV)和鸭疫里默杆菌(Riemerella anatipestifer,RA)的特异性引物,且PCR产物大小差异明显;优化多重PCR的引物浓度、退火温度,并分析特异性和敏感性;利用多重PCR方法和单一PCR方法对54份临床采集的鸭源病料进行检测。结果显示,该多重PCR检测方法可用于DTMUV、RA和DPV的单纯或混合感染的诊断,PCR产物大小分别为220,308,551 bp;反应体系中最佳引物终浓度组合为1.0μmol/L的引物DTMUV-S和DTMUV-A、0.5μmol/L的引物RA-S和RA-A、0.5μmol/L的引物DPV-S和DPV-A;该检测方法退火温度在46.5~63.8℃之间均可以检测到3种病原体核酸,其中最佳退火温度为57.0℃。该方法检测DTMUV、DPV和RA的核酸最低量分别为6.570,0.600,0.440μg/L。临床样品检测结果显示,本试验建立的DTMUV、DPV和RA多重PCR检测结果与单一PCR检测结果符合率为98.1%。结果表明,本试验建立的复合PCR方法可适用于3种病原体单纯或混合感染的快速诊断,为流行病学调查及快速、准确制定针对性防控措施提供了技术支持。In order to establish multiplex PCR detection method for duck Tembusu virus(DTMUV),duck plague virus(DPV)and Riemerella anatipestifer(RA),the specific primers were designed to detect DTMUV,DPV,and RA,respectively,and the PCR products were significantly different in size.The primer concentration and annealing temperature of multiplex PCR were optimized,and specificity and sensitivity analysis are performed.The 54 clinically collected duck-derived materials were detected by multiplex PCR method and the single PCR method,respectively.The results showed that multiplex PCR method could be used to detect single or mixed infection of DTMUV,RA and DPV,and the PCR product sizes were 220,308,551 bp,respectively.The optimal final primer concentration in the reaction system was 1.0μmol/L of primers DTMUV-S and DTMUV-A,0.5μmol/L of primers RA-S and RA-A,0.5μmol/L of primers DPV-S and DPV-A.The multiplex PCR method could detect three kinds of pathogen nucleic acids at the annealing temperature between 46.5℃and 63.8℃C,and the optimal annealing temperature was 57.0℃.The minimum detection amounts of nucleic acids for DTMUV,DPV and RA by multiplex PCR were 6.570,0.600,0.440μg/L,respectively.In terms of clinical sample detection results,the coincidence rate of multiplex PCR and single PCR was 98.1%.In summary,the multiplex PCR can be used for the rapid diagnosis of simple or mixed infection of DTMUV,DPV and RA,and provides technical support for the epidemiological investigation and the rapid and accurate formulation of targeted prevention and control measures.

关 键 词：多重PCR 鸭坦布苏病毒 鸭瘟病毒 鸭疫里默杆菌 诊断 

分 类 号：S852.61[农业科学—基础兽医学] S852.65[农业科学—兽医学]