贵州农业科学2025，Vol.53Issue(1)：55-61,7.DOI:10.3969/j.issn.1001-3601.2025.01.007
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
摘要
Abstract
[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:pre-denaturation 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 2.5×103 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Key words
cattle/Akabane disease/Akabane virus/RT-PCR/detection/prevention and control分类
农业科技引用本文复制引用
尚佳富,李明科,徐婷婷,杨晓伟,刘霞,张立武,曹礼静,倪兴维,赵光伟..牛阿卡斑病RT-PCR检测方法的建立[J].贵州农业科学,2025,53(1):55-61,7.基金项目
贵州省科技支撑计划项目(黔科合支撑[2023]一般022) （黔科合支撑[2023]一般022）
