罗非鱼链球菌双重荧光PCR方法的建立及临床应用  

Establishment and Clinical Application of a Duplex Fluorescent PCR Assay for Streptococcus tilapia

作　　者：丁文桂 侯文婷[3] 王自强[2] 李永福[2] 黄育浩[2] 龙海鹰 李敏 张险朋[2] Ding Wengui;Hou Wenting;Wang Ziqiang;Li Yongfu;Huang Yuhao;Long Haiying;Li Min;Zhang Xianpeng(Foshan University,Foshan,Guangdong 528231,China;Dongguan Center for Animal Disease Prevention and Control,Dongguan,Guangdong 523086,China;China Animal Health and Epidemiology Center,Qingdao,Shandong 266032,China)

机构地区：[1]佛山科学技术学院,广东佛山528231 [2]东莞市动物疫病预防控制中心,广东东莞523086 [3]中国动物卫生与流行病学中心,山东青岛266032

出　　处：《中国动物检疫》2023年第4期114-122,共9页China Animal Health Inspection

基　　金：东莞市2021年度省乡村振兴战略专项(20211800400112)。

摘　　要：为构建一种无乳链球菌(Streptococcus agalactiae)和海豚链球菌(Streptococcus iniae)的双重荧光PCR检测方法,根据筛选的海豚链球菌LysR基因和无乳链球菌Sip基因设计特异性引物和探针,优化反应浓度和退火温度,建立标准曲线,分析方法的敏感性、特异性和重复性,最后进行方法的初步应用。结果显示:当无乳链球菌引物和探针浓度分别为0.20和0.16μmol/L,海豚链球菌引物和探针浓度分别为0.20和0.32μmol/L,且退火温度为53℃时,建立的双重荧光PCR方法荧光曲线标准,线性关系线良好,有较高的扩增反应效率;无乳链球菌和海豚链球菌最低检测限分别为29.6和10.7 CFU/mL,与猪链球菌2型(Streptococcus suis serotype 2)和其他8种常见的水生动物疫病病原体无交叉反应,重复变异系数均小于2%;临床样品及人工污染样品检测结果与细菌分离鉴定方法符合率为100%。结果表明,本研究建立的罗非鱼链球菌双重荧光PCR检测方法灵敏度高、特异性强、重复性好,可同时对无乳链球菌和海豚链球菌进行检测,为罗非鱼链球菌病的临床诊断和研究提供了技术支撑。In order to establish a duplex fluorescent PCR assay for Streptococcus agalactiae and Streptococcus iniae,specific primers and probes were designed according to the screened LysR gene of Streptococcus iniae and Sip gene of Streptococcus agalactiae,the reaction concentration and annealing temperature were optimized,and a standard curve was established to analyze the sensitivity,specificity and repeatability of the method that was finally applied.The results showed that standard fluorescence curves,good linear relation lines and higher amplification reaction efficiency were observed in the established method when the concentrations of primers and probes of Streptococcus agalactiae were 0.20 and 0.16µmol/L,respectively,those of Streptococcus iniae were 0.20 and 0.32µmol/L,respectively,and the annealing temperature was 53℃;the minimum detection limits of Streptococcus agalactiae and Streptococcus iniae were 29.6 and 10.7 CFU/mL,respectively,and the method failed to crossly react with Streptococcus suis serotype 2 and other 8 common aquatic animal disease pathogens,and all the coefficients of repeated variation were less than 2%;the coincidence rate between the test results of clinical samples and artificially contaminated samples and those by bacterial isolation and identification was 100%.In conclusion,the established PCR assay was with high sensitivity,strong specificity and good reproducibility,which could be used to detect Streptococcus agalactiae and Streptococcus iniae at the same time,providing a technical support for future clinical diagnosis and researches of streptococcicosis disease in tilapia.

关 键 词：罗非鱼 无乳链球菌 海豚链球菌 双重荧光PCR 临床应用 

分 类 号：S852.61[农业科学—基础兽医学]