奶牛流产三种病原多重PCR检测方法的建立与初步应用  

Establishment and application of a multiplex PCR assay for simultaneously detecting three pathogens inducing abortions in dairy cows

作　　者：李彬 王辉 刁梓洋 翟云逸 陈家露 周栋 刘伟[1] 靳亚平[1] 王爱华[1] LI Bin;WANG Hui;DIAO Ziyang;ZHAI Yunyi;CHEN Jialu;ZHOU Dong;LIU Wei;JIN Yaping;WANG Aihua(Key Laboratory of Animal Biotechnology,Ministry of Agriculture and Rural Affairs,College of Veterinary Medicine,Northwest A&F University,Yangling 712100,Shaanxi,China;Jiagedaqi District Animal Husbandry and Aquatic Products Service Center,Jiagedaqi 165000,Heilongjiang,China;Guangdong Haid Animal Husbandry and Veterinary Research Institute,Guangzhou 511400,Guangdong,China)

机构地区：[1]西北农林科技大学动物医学院农业农村部动物生物技术重点实验室,陕西杨凌712100 [2]加格达奇区畜牧水产服务中心,黑龙江加格达奇165000 [3]广东海大畜牧兽医研究院有限公司,广东广州511400

出　　处：《微生物学通报》2025年第2期613-622,共10页Microbiology China

基　　金：国家自然科学基金(32373016)。

摘　　要：【背景】布鲁氏菌(Brucella)、牛传染性鼻气管炎病毒(infectious bovine rhinotracheitis virus,IBRV)和犬新孢子虫(Neospora caninum)是引起奶牛流产的重要病原,目前针对上述病原的血清学检测方法,存在特异性低或操作复杂等问题,PCR或RT-PCR方法仅能对单一病原进行检测;多重PCR可同时对多种病原进行检测,可极大地提高病原的检测效率。【目的】建立布鲁氏菌、牛传染性鼻气管炎病毒和犬新孢子虫的多重PCR检测方法,对临床样品进行快速检测。【方法】针对布鲁氏菌omp25基因、牛传染性鼻气管炎病毒gB基因和犬新孢子虫SRS2基因设计特异性引物,优化多重PCR的退火温度、引物浓度、延伸时间和循环次数;以沙门氏菌(Salmonella)、大肠杆菌(Escherichia coli)、隐孢子虫(Cryptosporidium)、弓形虫(Toxoplasma gondii)和圆环病毒标准核酸验证多重PCR反应的特异性,以含靶基因的重组质粒验证多重PCR的敏感性;对55份流产奶牛阴道拭子进行检测。【结果】多重PCR的退火温度为59℃,延伸时间为40 s,循环次数为30,各引物浓度为1μmol/L;对omp25基因和SRS2基因的检测下限为4×10^(1) copies,对gB基因的检测下限为4×10^(2 )copies;与其他病原无交叉反应;55份样品中检出布鲁氏菌阳性9份,牛传染性鼻气管炎病毒阳性3份,犬新孢子虫阳性7份,其中布鲁氏菌和犬新孢子虫混合感染1份。【结论】成功构建了3种病原的多重PCR检测方法,能够对临床样品进行快速、准确的检测。[Background]Brucella,infectious bovine rhinotracheitis virus(IBRV),and Neospora caninum are major pathogens that cause abortions in dairy cows.Current serological methods for detecting the above pathogens are characterized by low specificity and complex operation,while PCR and RT-PCR methods are only capable of detecting single pathogens.Multiplex PCR enabling the detection of multiple pathogens at the same time greatly increases the detection efficiency.[Objective]To establish a multiplex PCR assay for rapidly detecting Brucella,IBRV,and N.caninum in clinical samples.[Methods]Specific primers were designed for Brucella omp25,IBRV gB,and N.caninum SRS2,and the annealing temperature,primer concentration,extension time,and number of cycles of the multiplex PCR assay were optimized.The specificity of the multiplex PCR assay was examined with the standard nucleic acids of Salmonella,Escherichia coli,Cryptosporidium,Toxoplasma gondii,and circovirus.The sensitivity of the multiplex PCR assay was tested with the recombinant plasmids carrying the target genes.Finally,the established assay was employed to test 55 vaginal swabs from cows suffering from abortions.[Results]The optimal conditions of the multiplex PCR assay were annealing temperature of 59℃,the extension time of 40 s,30 cycles,and the primer concentration of 1μmol/L.The lower limit of detection was 4×10^(1) copies for omp25 and SRS2 and 4×10^(2) copies for gB.The established assay showed no cross-reactivity with other pathogens.Nine out of 55 samples were detected positive for Brucella,3 positive for IBRV,and 7 positive for N.caninum.Particularly,one sample was subjected to mixed infection by Brucella and N.caninum.[Conclusion]A multiplex PCR assay for three pathogens was successfully constructed,enabling rapid and accurate detection of clinical samples.

关 键 词：奶牛 流产 多重PCR 布鲁氏菌 牛传染性鼻气管炎病毒 犬新孢子虫 

分 类 号：S858.23[农业科学—临床兽医学]