摘要
为建立一种新型、稳定、可普遍应用的诊断布病的方法,将重组酶聚合酶扩增(recombinase polymerase amplification,RPA)与胶体金侧向流试纸条技术(lateral flow dipstick,LFD)相结合。根据布鲁氏菌bcsp31基因序列保守区设计1套标有生物素Biotin的特异性引物和1条标记dSpacer、C3 Spacer的特异性探针,通过对反应条件、体系的优化,以及对敏感性、特异性以及模拟样本的分析,最后进行临床应用,建立一种可以检测布鲁氏菌的RPA-LFD方法。结果显示,成功建立了布鲁氏菌RPA-LFD检测方法,最佳反应条件为闭合手掌中孵育12 min;引物与探针的最佳比为3.5∶1;RPA-LFD方法的最低检测限为5.89 CFU/m L,能够特异地检测出布鲁氏菌,检测大肠杆菌等其他细菌结果均为阴性;用RPA-LFD方法检测模拟样本和临床样品的结果与ELISA方法一致。上述结果表明,本研究建立的布鲁氏菌RPA-LFD检测方法特异性强、灵敏性高、操作简单,可广泛应用于临床检测。
In order to establish a new,stable and universally applicable method for the diagnosis of Brucella.Recombinase polymerase amplification(RPA)-Lateral flow dipstick(LFD) technique was used.According to the conserved region with relatively high homology in the bcsp31 gene sequence of Brucella,a set of specific primers labeled with Biotin homology in the bcsp31 gene sequence of Brucella,a set of specific primers labeled with Biotin was designed,and a specific probe for labeling d Spacer and C3 Spacer was combined to establish RPA-LFD detection method.The results showed that the Brucella RPA-LFD method was successfully established.The optimal reaction conditions were incubation in closed palm for 12 min.The optimal ratio of primers to probes was 3.5 ∶ 1.The established RPA-LFD detection method has good specificity and sensitivity.The detection limit was 5.89 CFU/m L.The specificity test showed that RPA-LFD was only positive for Brucella and negative for other bacteria.These results indicated that the established detection method for Brucella based on RPA-LFD was specific,sensitive,simple operation and could be widely used.
作者
张珊珊
李楠
郝镯
孟轲音
刘军
ZHANG Shan-shan;LI Nan;HAO Zhuo;MENG Ke-yin;LIU Jun(Laboratory of Military Veterinary Medicine,Institute of Military Medical Sciences,Academy of Military Sciences/Jilin Province Key Laboratory of Zoonosis Prevention and Control,Changchun 130122 yChina)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2021年第10期1215-1220,共6页
Chinese Veterinary Science
基金
内蒙古自治区科技重大专项(2019ZD006)。
