兔源支气管败血波氏杆菌荧光定量PCR检测方法的建立  

Development of a fluorescence quantitative PCR assay for detection of Bordetella bronchiseptica in rabbits

作　　者：苏进博 王锦祥[2] 殷光文[1] SU Jinbo;WANG Jinxiang;YIN Guangwen(College of Future Technology,Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,China;Institute of Animal Husbandry and Veterinary Medicine,Fujian Academy of Agriculture Sciences,Fuzhou,Fujian 350013,China)

机构地区：[1]福建农林大学未来技术学院,福建福州350002 [2]福建省农业科学院畜牧兽医研究所,福建福州350013

出　　处：《福建农林大学学报（自然科学版）》2025年第1期110-116,共7页Journal of Fujian Agriculture and Forestry University:Natural Science Edition

基　　金：福建省科技计划公益类专项(2022R10260012);国家现代农业产业技术体系项目(CARS-43-G-5)。

摘　　要：【目的】建立兔源支气管败血波氏杆菌(Bb)的快速检测方法,用于兔波氏杆菌病的诊断。【方法】以Bb的fimX基因为靶基因,设计特异性引物并构建质粒标准品。在此基础上建立检测兔源Bb的SYBR GreenⅠ荧光定量PCR方法,对该方法的特异性、敏感性和重复性进行评价,并将该方法初步应用于临床样品的检测。【结果】荧光定量PCR方法仅对兔源Bb的检测结果呈阳性,对其他常见兔源病原菌的检测结果均呈阴性;对质粒标准品的最低检测限为13.8拷贝·μL^(-1),敏感性是普通PCR方法的10倍;批内重复性试验的变异系数小于2%,批间重复性试验的变异系数小于3%;对68份临床样品进行检测,阳性检出率为39.71%,比普通PCR方法的高2.95%。【结论】建立的SYBR GreenⅠ荧光定量PCR方法特异性强、敏感性高、重复性好,且对临床样品的阳性检出率比普通PCR方法高,为兔源Bb的快速检测和防控提供了技术支持。【Objective】The study aimed to develop an assay for the rapid detection of Bordetella bronchiseptica in rabbits.【Method】A SYBR GreenⅠ-based fluorescence quantitative PCR assay was developed by designing primers and constructing plasmid standard based on the fimX gene of B.bronchiseptica.After evaluating its specificity,sensitiveness and repeatability,the assay was preliminarily used for the detection of clinical samples.【Result】The assay had no cross-reaction with other rabbit-sourced bacterial pathogens,and was only positive for B.bronchiseptica in rabbits,with a detection limit of 13.8 copies·μL^(-1)of standard plasmid,which was 10-fold higher than that of conventional PCR assay.The variation coefficients for intra-test and inter-test were less than 2%and 3%,respectively.The detection rate of 68 clinical samples via the assay was 39.71%,which was 2.95%higher than that of the conventional PCR assay.【Conclusion】The fluorescence quantitative PCR assay based on SYBR GreenⅠprovided an alternative method for the rapid detection of B.bronchiseptica in rabbits with high specificity,high sensitivity and good repeatability,which is helpful for the prevention and control of B.bronchiseptica in rabbits.

关 键 词：兔 兔波氏杆菌病 支气管败血波氏杆菌 荧光定量PCR方法 检出率 

分 类 号：S858.291[农业科学—临床兽医学]