空肠弯曲菌RPA-LFD快速检测方法的建立及评价  

Establishment and evaluation of RPA-LFD rapid detection method for Campylobacter jejuni

作　　者：叶景芬 武绍碧 陈世雄 龙宥茨 廖怡雯 罗雪 杨琦[1,2] YE Jingfen;WU Shaobi;CHEN Shixiong;LONG Youci;LIAO Yiwen;LUO Xue;YANG Qi(College of Animal Science,Guizhou University,Guiyang 550025,China;Institute of AnimalDisease Research of Guizhou,Guiyang 550025,China)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]贵州省动物疫病研究所,贵州贵阳550025

出　　处：《中国兽医学报》2024年第12期2579-2584,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(32260884)。

摘　　要：为建立特异、快速、便捷的空肠弯曲菌(Campylobacter jejuni,C.jejuni)检测方法,以空肠弯曲菌的hip0基因为靶标,设计了1组特异性引物及1条探针,下游引物和探针的5′端分别标记生物素、荧光素,通过对反应时间及温度的优化,建立了C.jejuni-RPA-LFD检测方法,并对其进行了灵敏度评估及模拟样本检测。结果显示,C.jejuni-RPA-LFD与肺炎克雷伯菌、大肠杆菌、假单胞菌、蜡样芽胞杆菌、巴氏杆菌、奇异变形杆菌、鼠伤寒沙门菌无交叉反应,最佳反应体系为37℃、25min,其灵敏度可达3.93×10^(0)拷贝/μL,模拟检测中,可检测出1×10^(2) CFU/mL的空肠弯曲菌污染粪便样本。结果表明,建立的C.jejuni-RPA-LFD具有特异性好、简便、快速、灵敏度高等优点,为畜禽养殖业基层现场、快速诊断空肠弯曲菌及控制其传播提供有效途径。In order to establish a specific,rapid and convenient method for the detection of Campylobacter jejuni(C.jejuni).A set of specific primers and a probe that do not cause false positives were designed with the hipO gene of C.jejuni as the target,and the 5′ends of the downstream primers and probes were labeled with biotin and fluorescein,respectively.C.jejuni-RPA-LFD had no cross-reactivity with Klebsiella pneumoniae,Escherichia coli,Pseudomonas,Bacillus cereus,Pasteurella,Proteus mirabilis,and Salmonella typhimurium,and the optimal reaction system was 37℃,25min,and its sensitivity could reach 3.93×10^(0)copies/μL,and 1×10^(2) CFU/mL of C.jejuni contaminated stool samples could be detected in the simulated detection.The C.jejuni-RPA-LFD established in this study has the advantages of good specificity,simplicity,rapidity and high sensitivity,which provides an effective way for the rapid diagnosis of C.jejuni and the control of the spread of C.jejuni at the grassroots level of livestock and poultry farming.

关 键 词：空肠弯曲菌 RPA-LFD hipO基因 

分 类 号：S852.61[农业科学—基础兽医学] R535[农业科学—兽医学]