基于recA基因的qPCR与RAA-LFD检测鳗败血假单胞菌方法的建立与应用  

Establishment and application of qPCR and RAA-LFD based on recA gene for detection of Pseudomonas anguilliseptica

作　　者：王一霖 艾明齐 蒋奇滨 彭焜 周柯宇 樊威 欧阳萍[1,2] 陈德芳 黄小丽 耿毅[1,2] WANG Yilin;AI Mingqi;JIANG Qibin;PENG Kun;ZHOU Keyu;FAN Wei;OUYANG Ping;CHEN Defang;HUANG Xiaoli;GENG Yi(Institute of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,China;Sichuan Aquatic Animal Disease Control and Prevent Center,Chengdu 611130,China;Neijiang Academy of Agricultural Sciences,Neijiang 641000,China;Institute of Animal Science and Technology,Sichuan Agricultural University,Chengdu 611130,China)

机构地区：[1]四川农业大学动物医学院,四川成都611130 [2]四川省水生动物疫病监控中心,四川成都611130 [3]四川省内江市农业科学院,四川内江641000 [4]四川农业大学动物科技学院,四川成都611130

出　　处：《南方水产科学》2025年第2期138-148,共11页South China Fisheries Science

基　　金：四川省重点研发项目(2022YFN0012);四川省淡水鱼产业技术体系创新团队建设项目(SCCXTD-25-15);四川省自然科学基金项目(24NSFSC0381);中央引导地方发展科技项目(2024ZYD0317)。

摘　　要：为实现鳗败血假单胞菌(Pseudomonas anguilliseptica,PA)早期感染的快速诊断,基于recA基因建立了2种检测方法:SYBR Green I实时荧光定量PCR(SYBR Green I real-time quantitative PCR)和重组酶介导等温扩增结合侧流层析试纸条(Recombinase-mediated isothermal amplification combined with lateral flow dipstick,RAA-LFD)。以PA的管家基因recA为靶标,设计筛选出1对qPCR特异性引物、1对RAA特异性引物和RAA探针,并通过同源重组构建标准品质粒pUC18-recA,以建立2种检测方法。将所建立的方法应用于PA感染的大口黑鲈(Micropterus salmoides)组织样本检测,并测定PA载量。结果表明,建立的qPCR方法最低DNA检测浓度为2.816×10^(2)拷贝·μL^(-1),模板量与Ct值在构建的标准曲线中呈现良好的线性关系(r^(2)=0.9992),且具有较强的特异性和较高的稳定性;RAA-LFD方法的最低DNA检测浓度为2.816×10^(4)拷贝·μL^(-1),检测时间最快可达15 min,显色较为稳定且特异性强。应用结果显示,qPCR和RAALFD方法的阳性样本检出率分别为87.50%和85.00%,较普通PCR方法明显提高;其中,qPCR方法可准确测定PA感染宿主组织中的菌体载量,肾中的载量最高,达3.533×10^(7)拷贝·ng^(-1)。建立的2种方法特异性均较好,其中qPCR方法灵敏性更高,RAA-LFD方法则时效性更强,均可用于PA早期感染的检测,且qPCR方法还可对感染宿主体内的菌体载量进行定量分析。For rapid diagnosis of early infection with Pseudomonas anguilliseptica(PA),we established SYBR Green I real-time quantitative PCR and recombinase-mediated isothermal amplification combined with lateral flow dipstick(RAA-LFD)based on recA gene for rapid and specific detection.A pair of qPCR specific primers,a pair of RAA specific primers and a RAA probe were designed and screened based on the recA gene of PA.The standard quality plasmid pUC18-recA was constructed by homolo-gous recombination to establish the two detection methods.The established methods were applied to detect PA-infected large-mouth bass(Micropterus salmoides)tissue samples and PA load was determined.The minimum DNA detection con-centration of the qPCR method was 2.816×10^(2) copies·μL^(-1).There was a good linear relationship between the template amount and Ct value in the standard curve(r^(2)=0.9992),and the method had strong specificity and stability.The minimum DNA detec-tion concentration of the RAA-LFD method was 2.816×10^(4) copies·μL^(-1).The detection time of the RAA-LFD method was 15 min,and the color development was relatively stable and the specificity was strong.The application results show that the positive de-tection rates of qPCR and RAA-LFD were 87.50%and 85.00%,respectively,which were significantly higher than that of the common PCR method.The established qPCR method could accurately measure the bacterial load in the tissues of PA infected hosts.The highest bacterial load was found in the kidney(3.533×10^(7) copies·ng-1).Both methods can be used for rapid detection of early PA infection.The established qPCR can also be used to quantitatively analyze the bacterial load in infected hosts.

关 键 词：鳗败血假单胞菌 大口黑鲈 SYBR Green I real-time quantitative PCR RAA-LFD 菌体载量 

分 类 号：S941.42[农业科学—水产养殖]