牛传染性鼻气管炎病毒和牛病毒性腹泻病毒双重PCR检测方法的建立与应用
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S855.3

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东西部科技合作项目(2017BY078);宁夏奶牛育种专项(2019NYYZ05)


Establishment and Application of a Dual PCR Assay for Detection of Infectious Bovine Rhinotracheitis Virus and Bovine Viral Diarrhea Virus
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    摘要:

    为建立一种针对牛传染性鼻气管炎病毒(infectious bovine rhinortracheitis virus,IBRV)和牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)的双重PCR检测方法,根据GenBank中IBRV gB(UL27)和BVDV 5'-UTR基因序列分别合成特异性引物,优化反应条件,建立了能够同时检测IBRV和BVDV的双重PCR方法,并对其特异性、敏感性、重复性进行了测试。结果显示:该方法对IBRV、BVDV和IBRV/BVDV可分别扩增出500、198、500/198 bp的特异性目标条带,对牛呼吸道合胞体病毒、牛冠状病毒、牛细小病毒、牛纽布病毒和牛支原体检测结果均为阴性;对混合液中IBRV、BVDV的最低检测限分别为104和103 copies/μL;3次重复性试验结果一致。采用建立的方法对79份临床可疑牛血清样品进行IBRV、BVDV和IBRV+BVDV检测,阳性率分别为12.66%、17.72%和8.86%,与IBRV和BVDV单重PCR检测结果符合率分别为90.00%和93.33%,两种病原混合感染的阳性符合率为100%。结果表明,本研究建立的IBRV和BVDV双重PCR检测方法具有良好的特异性、敏感性及重复性,为兽医临床快速诊断IBRV和BVDV提供了一种方便、快捷的分子生物学检测手段。

    Abstract:

    In order to establish a dual PCR assay for detection of infectious bovine rhinotracheitis virus(IBRV)and bovine viral diarrhea virus(BVDV),specific primers were synthesized according to IBRV gB(UL27)and BVDV 5'-UTR sequences registered in GenBank,after optimization of the reaction conditions,a dual PCR assay capable of simultaneous detection of IBRV and BVDV was established,followed by the tests of its specificity,sensitivity and repeatability. The results showed that,for IBRV,BVDV and IBRV/BVDV,the specific target bands of 500,198 and 500/198 bp could be amplified,respectively,and negative results were obtained from detection of bovine respiratory syncytial virus,bovine coronavirus,bovine parvovirus,bovine newbrucellovirus and Mycoplasma bovis;the minimum detection limits for IBRV/BVDV mixture was 104 and 103 copies/μL,respectively;consistent results were obtained from three repeatability tests. 79 clinically suspected bovine serum samples were detected for IBRV,BVDV and IBRV + BVDV by the established method,with the positive rates of 12.66%,17.72% and 8.86%,respectively,which were 90.00% and 93.33% consistent with the results by single PCR assay for IBRV and BVDV,and the positive coincidence rate of mixed infection was 100%. In conclusion,the established method,with good specificity,sensitivity and repeatability,could be used as a convenient and fast molecular biological detection tool for rapid diagnosis of IBRV and BVDV in veterinary practice.

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鲍显伟,李小龙,石亚楠,梁晓珊,李昊,王雪妍,许立华.牛传染性鼻气管炎病毒和牛病毒性腹泻病毒双重PCR检测方法的建立与应用[J].《中国动物检疫》编辑部,2023,40(1):115-120.

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