牛阿卡斑病RT-PCR检测方法的建立  

Establishment of a RT-PCR Detection Method for Bovine Akabane Disease

作　　者：尚佳富 李明科 徐婷婷 杨晓伟[1,3] 刘霞 张立武[3] 曹礼静 倪兴维 赵光伟 SHANG Jiafu;LI Mingke;XU Tingting;YANG Xiaowei;LIU Xia;ZHANG Liwu;CAO Lijing;NI Xingwei;ZHAO Guangwei(College of Veterinary Medicine,Southwest University,Chongqing 402460;Guizhou Provincial Center for Animal Disease Control and Prevention,Guiyang,Guizhou 550008;Chongqing Sanjiezhongxin Bioengineering Co.,Ltd.,Chongqing 402460;Chongqing Rongchang Vocational Education Center,Chongqing 402460,China)

机构地区：[1]西南大学动物医学院,重庆402460 [2]贵州省动物疫病预防控制中心,贵州贵阳550008 [3]重庆三杰众鑫生物工程有限公司,重庆402460 [4]重庆市荣昌区职业教育中心,重庆402460

出　　处：《贵州农业科学》2025年第1期55-61,共7页Guizhou Agricultural Sciences

基　　金：贵州省科技支撑计划项目(黔科合支撑〔2023〕一般022)。

摘　　要：【目的】建立牛阿卡斑病(Akabane disease,AKAD)RT-PCR实验室快速检测方法,为该病的临床防控提供技术支撑。【方法】参考前期获得的阿卡斑病毒(Akabane virus,AKAV)S基因序列(OR791104.1),针对其保守区域设计特异性扩增引物,以构建的pMD-AKAV重组质粒为模板,建立RT-PCR检测方法,并对其扩增条件进行优化,进而对该方法的特异性、敏感性和重复性进行评估,最后将所建方法对实验室保存的19份阳性和279份阴性牛血清临床样本进行符合性检测,验证该方法的准确性。【结果】所建方法的最佳反应条件为95℃预变性3 min;95℃变性15 s,55℃退火15 s,72℃延伸15 s,共37个循环;72℃延伸5 min。对牛蓝舌病病毒、多杀性巴氏杆菌、牛传染性鼻气管炎病毒和牛支原体等病原均为阴性,特异性良好;对阳性质粒检测最低限为2.5×10^(3) copies/μL,灵敏性高;重复性试验显示不同批次样本检测结果均一致,可重复性强;对实验室保存的298份(19份阳性、279份阴性)临床样本进行检测,其结果均与预期一致,符合率100%。【结论】建立的牛阿卡斑病RT-PCR检测方法特异性良好、灵敏性高、可重复性强。【Objective】A rapid RT-PCR method for detecting bovine Akabane disease(AKAD)was constructed to provide technical support for clinical prevention and control of AKAD.【Method】Firstly,based on the sequence of Akabane virus(AKAV)S gene(OR791104.1)which obtained in the previous stage,the specific amplification primers for its conserved region were designed.Secondly,taking the constructed pMD-AKAV recombinant plasmid as a standard,the RT-PCR detection method was constructed and its amplification conditions were optimized.Then,the specificity,sensitivity and repeatability of the method were evaluated.Lastly,to verify the accuracy of the method,19 positive and 279 negative clinical samples of bovine serum which stored in the laboratory were conducted the conformance checking through the detection method.【Result】The optimal reaction conditions for the established method were as follows:predenaturation at 95℃for 3 min,denaturation at 95℃for 15 s,annealing at 55℃for 15 s,extension at 72℃for 15 s,with a total of 37 cycles and then extension at 72℃for 5 min.It was negative for pathogens such as bovine bluetongue virus,Pasteurella multocida,infectious bovine rhinotracheitis and bovine Mycoplasma bovis,which illustrated a good specificity.The lowest detection limit for positive plasmid was 2.5×10^(3) copies/μL with high sensitivity.The repeatability test showed that the test results of different batches of samples were consistent and reproducible.The 298 clinic samples(19 positive samples,279 negative samples)stored in laboratory were detected and the results were all consistent with expectations,with a 100%coincidence rate.【Conclusion】The constructed RT-PCR detection method for bovine Akabane disease has good specificity,high sensitivity and strong repeatability.

关 键 词：牛 赤羽病 阿卡斑病毒 RT-PCR 检测 防控 

分 类 号：S858.23[农业科学—临床兽医学]