尼罗罗非鱼无乳链球菌微滴式数字PCR检测方法的建立及临床应用

Establishment and Clinical Application of a Droplet Digital PCR Detection Method for Streptococcus agalactiae in Tilapia

通过设计筛选引物和探针、优化反应浓度和退火温度,构建一种尼罗罗非鱼(Oreochromis niloticus)无乳链球菌(Streptococcus agalactiae)的微滴式数字PCR(dd PCR)检测方法,分析该方法的敏感性、特异性及重复性,并应用于临床样品检测。结果显示:当引物、探针浓度分别为0.9μmol·L^(-1)、0.3μmol·L^(-1)且退火温度为56.9℃时,建立的罗非鱼无乳链球菌dd PCR方法阴、阳性微滴分布界限明显,平均拷贝数高,有较高扩增反应效率;线性关系线良好(R^(2)=0.997 3),最低检测限为2.56 copies·μL^(-1);与猪链球菌2型、鱼类海豚链球菌和其他5种常见的水生动物疫病病原体无交叉反应;重复变异系数为3.15%;临床样品检测结果与实时荧光PCR方法结果的符合率100%,与细菌分离鉴定方法结果符合率为94.12%。结果表明,建立的罗非鱼无乳链球菌dd PCR检测方法灵敏度高、特异性强、重复性好,可对罗非鱼无乳链球菌感染的临床样品进行定量检测,为尼罗罗非鱼无乳链球菌的研究提供有益参考。

A droplet digital PCR(ddPCR)method for detection of Streptococcus agalactis in tilapia was established by designing and screening primers and probes,optimizing reaction concentration and annealing temperature,the sensitivity,specificity and repeatability of the method were analyzed,and finally the method was applied to clinical samples for detection.The results showed that the ddPCR method established here had obvious distribution boundary between negative and positive droplets,high average copy number and high amplification efficiency at primer concentration of 0.9μmol·L^(-1),probe concentration of 0.3μmol·L^(-1) and the annealing temperature of 56.9℃.The linearity was found to be good(R^(2)=0.9973),with the minimum detection limit of 2.56 copies·μL^(-1),and without cross reaction with Streptococcus suis type 2,S.dolphinus and other five common pathogens of aquatic animal diseases.There was repeated coefficient variation of 3.15%and 100%of the coincidence rate between the results of clinical samples and real-time fluorescence PCR method.The coincidence rate between the results of bacterial isolation and identification method was shown to be 94.12%,indicating that the established ddPCR method for detection of S.agalactis in tilapia has high sensitivity,strong specificity and good reproducibility,and can be used for quantitative detection of clinical samples infected by S.agalactis in tilapia,which provides a useful reference for the study of S.agalactis in tilapia.