罗非鱼戴维斯黄杆菌实时定量PCR检测方法的建立及应用  

Establishment and application of real-time quantitative PCR detection method for Flavobacterium davisii in Nile tilapia(Oreochromis niloticus)

作　　者：叶小灵 许莹萱 王世锋 周永灿[1,2] 郭伟良 张冬冬[1,2] Ye Xiaoling;Xu Yingxuan;Wang Shifeng;Zhou Yongcan;Guo Weiliang;Zhang Dongdong(Hainan Provincial Key Laboratory for Tropical Hydrobiology and Biotechnology,Haikou 570228;School of Marine Biology and Fisheries,Hainan University,Haikou 570228,China)

机构地区：[1]海南省热带水生生物技术重点实验室,海南海口570228 [2]海南大学海洋生物与水产学院,海南海口570228

出　　处：《海南大学学报（自然科学版）》2024年第3期258-267,共10页Journal of Hainan University（Natural Science）

基　　金：国家自然科学基金项目(32303046)。

摘　　要：戴维斯黄杆菌(Flavobacterium davisii)是柱形病病原之一,严重影响罗非鱼养殖产业.为了实现对其快速和准确的诊断,本研究以TonB基因为靶基因构建了重组质粒,建立了戴维斯黄杆菌的实时定量PCR检测方法,并将其应用到罗非鱼感染戴维斯黄杆菌后的载菌量测定研究.结果表明:特异性引物TonB-F/R与不同基因型的黄杆菌及罗非鱼其他常见病原无交叉反应,所建立的绝对定量检测方法是常规PCR检测方法灵敏度的100倍,其重复性和稳定性良好;罗非鱼感染戴维斯黄杆菌后其鳃组织载菌量显著高于其他组织,符合该疾病的侵染特点.综上所述,该实时定量PCR检测方法具有快速、高灵敏度和强特异性等优势,可准确评估戴维斯黄杆菌的感染程度,对柱形病的预防具有重要价值.Flavobacterium davisii,a pathogen of columnaris disease,significantly impacts the tilapia aquacul⁃ture industry.In order to diagnostic columanris disease in Nile tilapia quickly and exactly,in the report,the re⁃combinant plasmid containing TonB gene(iron complex protein,TonB)was constructed,the specific PCR prim⁃ers,TonB-F/R,targeting TonB,were designed,and a reliable absolute quantitative PCR detection method was established and was used for detecting bacterial loads in tilapia infected with the pathogen.The results demon⁃strated that the specific primers,TonB-F/R,did not cross-react with different genotypes of Flavobacterium or other common pathogens in tilapia.The sensitivity of the established absolute quantification method was 100 times higher than that of conventional PCR,with excellent repeatability and stability.The bacterial loads in the gill tissues of tilapia infected with F.davisii were significantly higher than that in other tissues,which was con⁃sistent with the infection characteristics of the disease.In summary,the real-time qPCR detection method offers advantages of rapidity,high sensitivity,and strong specificity,which can perform the accurate assessment of F.davisii infection levels.The method is valuable for the prevention and control of columnaris disease.

关 键 词：罗非鱼 戴维斯黄杆菌 实时定量PCR检测 

分 类 号：S963.211[农业科学—水产养殖]