1.华中农业大学动物医学院，武汉 430070;2.农业微生物资源发掘与利用全国重点实验室，武汉 430070;3.湖北洪山实验室，武汉 430070;4.湖北省兽医流行病学国际科技合作基地/农业农村部兽用诊断制剂创制重点实验室/ 农业农村部反刍动物生物制品重点实验室，武汉 430070
徐恩红，E-mail： xeh0311@163.com
郭爱珍，E-mail： aizhen@mail.hzau.edu.cn
S852.61
宁夏回族自治区重点研究发计划项目（2021BEF02028）；国家现代农业产业技术体系（肉牛/牦牛）专项（CARS-37）；湖北省重点研发计划项目（2020BBA055）
- XU Enhong 1,2,3
- QI Mingpu 1,2,3
- XIANG Zhijie 1,2,3
- HU Changmin 2
- CHEN Yingyu 1,2,3
- CHEN Jianguo 2
- CHEN Xi 2
- GUO Aizhen 1,2,3,4
GUO Aizhen
College of Veterinary Medicine，Huazhong Agricultural University，Wuhan 430070，China;State Key Laboratory of Agricultural Microbiology，Wuhan 430070，China;Hubei Hongshan Laboratory，Wuhan 430070，China;Hubei International Scientific and Technological Cooperation Base of Veterinary Epidemiology/Key Laboratory of Development of Veterinary Diagnostic Products，Ministry of Agriculture and Rural Affairs/ Key Laboratory of Ruminant Biological Products，Ministry of Agriculture and Rural Affairs，Wuhan 430070，China
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1.College of Veterinary Medicine，Huazhong Agricultural University，Wuhan 430070，China;2.State Key Laboratory of Agricultural Microbiology，Wuhan 430070，China;3.Hubei Hongshan Laboratory，Wuhan 430070，China;4.Hubei International Scientific and Technological Cooperation Base of Veterinary Epidemiology/Key Laboratory of Development of Veterinary Diagnostic Products，Ministry of Agriculture and Rural Affairs/ Key Laboratory of Ruminant Biological Products，Ministry of Agriculture and Rural Affairs，Wuhan 430070，China
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为了提高牛呼吸疾病综合征多病原混合感染的临床诊断效率，以牛支原体（Mycoplasma bovis，M.b）oppD/F基因、多杀性巴氏杆菌（Pasteurella multocida，P.m）ompH基因、溶血性曼氏杆菌（Mannheimia haemolytica，M.h）gcp基因、牛传染性鼻气管炎病毒（infectious bovine rhinotracheitis virus，IBRV）gB基因、牛呼吸道合胞体病毒（bovine respiratory syncytial virus，BRSV）以及牛副流感病毒（bovine parainfluenza virus type，BPIV） 3型a和c基因型（BPIV-3a，-3c）的N基因等为检测靶标，分别设计特异性引物和Taqman探针，通过优化反应条件，采用3管7联的组合方式建立了7种病原体的多联实时荧光定量PCR检测方法。结果显示，该方法仅对本试验的7种病原有特异性反应，与其他常见病原无交叉反应。对M.b、P.m、M.h、IBRV、BRSV、BPIV-3a和BPIV-3c质粒标准品的最低检测限分别为102、102、101、102、102、102和101 拷贝/μL。组内变异系数小于2.5%，组间变异系数小于5.5%。平行应用该方法和常规PCR方法对临床采集的115份有呼吸道症状牛的鼻拭子进行检测，P.m阳性率36.65%，M.b阳性率27.83%，M.h阳性率25.22%，IBRV阳性率11.30%，BPIV-3c阳性率8.57%，BRSV阳性率0.95%；其中混合感染率为26.1%。共检测到11种混合感染模式，主要由M.b与其他病原体的混合感染，占72.7%（8/11）；M.b/P.m混合感染的检出率最高，占60%（18/30）；M.b、P.m、M.h在混合感染中出现率排前三，其占比分别为73.3%（22/30）、73.3%（22/30）和43.3%（13/30）；其次为IBRV，占26.7%（8/30）； BPIV-3c 占13.3%（4/30）。以上结果表明，该方法具有较高的分析敏感性和特异性，可用于牛呼吸疾病综合征多病原感染的联合检测。
In order to improve the clinical diagnosis efficiency of bovine respiratory disease complex （BRD） with multiple pathogen mixed infection，this study established a real-time quantitative PCR （qPCR） for multiple and rapid detection of seven common pathogens of BRD including Mycoplasma bovis （M.b），Pasteurellae multocida （P.m），Mannheimia haemolytica （M.h），bovine infectious rhinotracheitis virus （IBRV），bovine syncytial virus （BRSV），bovine parainfluenza virus type 3 genotype a （BPIV-3a） and c （BPIV-3c），the specific primers and TaqMan probes were designed and synthesized for the oppD/F gene of M.b，gcp gene of M.h，ompH gene of P.m，gB gene of IBRV，N genes of BRSV，BPIV-3a and BPIV-3c，respectively. After optimizing the reaction conditions，a multiple qPCR was established for simultaneous detection of the above seven pathogens in three tubes. This method specifically amplified these seven pathogens，rather than other major pathogens commonly found in cattle，indicating a high specificity. For M.b，P.m，M.h，IBRV，BRSV，BPIV-3a and BPIV-3c，the analytical sensitivity as limit of detection （LOD） was 102 copies /μL，102 copies /μL，101 copies /μL，102 copies /μL，102 copies /μL，102 copies /μL，102 copies /μL and 101 copies /μL，respectively，suggesting a high sensitivity. In addition，the coefficient of variation of the method was less than 2.5% within group and 5.5% between groups，indicating a good repeatability. Furthermore，115 clinical nasal swabs were parallelly detected by this method and conventional PCR，and the positivity was 27.83% for M.b，36.65% for P.m，25.22% for M.h，11.30% for IBRV，0.95% for BRSV，8.57% for BPIV-3C，with the proportion of coinfection was 26.1%. A total of 11 mixed infection patterns were detected，among them，the coinfection rate of M.b with other pathogens was the highest，accouning for 72.7% （8/11）. Within the 30 coinfection cases，the detection rate of M.b/P.m coinfection was the highest （60%，18/30）； the top three pathogens present in the coinfection were M.b，P.m，and M.h with frequency of 73.3% （22/30），73.3% （22/30） and 43.3% （13/30），respectively； followed by IBRV （26.7%，8/30） and BPIV-3c （13.3%，4/30）. Overall，this method has high sensitivity and specificity，and potential application in the clinical detection of single pathogen and multiple pathogens causing BRD.
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徐恩红,祁明普,项志杰,胡长敏,陈颖钰,陈建国,陈曦,郭爱珍.牛呼吸疾病综合征七病原联合检测多重qPCR方法的建立[J].华中农业大学学报,2023,42(2):38-47
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- 收稿日期:2022-11-01
- 在线发布日期: 2023-03-31
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