迟缓爱德华氏菌荧光PCR方法的建立及初步应用  

Establishment and Application of a Fluorescent PCR for Edwardsiella tarda

作　　者：叶毅飞 梁国华 吴志鹏[1] 殷进方[1] 龙海鹰 周柱辉 莫钻兰 邓海燕 谢翠美 林其明[1] 李敏 张险朋[1,2] Ye Yifei;Liang Guohua;Wu Zhipeng;Yin Jinfang;Long Haiying;Zhou Zhuhui;Mo Zuanlan;Deng Haiyan;Xie Cuimei;Lin Qiming;Li Min;Zhang Xianpeng(Dongguan Center for Animal Disease Prevention and Control,Dongguan 523086,Guangdong,China;Dongguan Key Laboratory of Zoonosis,Dongguan 523086,Guangdong,China;Hengli Township Center for Agricultural Technology Service,Dongguan 523460,Guangdong,China)

机构地区：[1]东莞市动物疫病预防控制中心,广东东莞523086 [2]东莞市人兽共患病重点实验室,广东东莞523086 [3]东莞市横沥镇农业技术服务中心,广东东莞523460

出　　处：《中国动物检疫》2024年第9期89-96,共8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：为构建一种灵敏、特异、准确的迟缓爱德华氏菌(Edwardsiellatarda)检测方法,以gyr B为靶基因设计特异性引物和探针,经过优化反应体系和退火温度,建立了迟缓爱德华氏菌荧光PCR方法。随后对该方法的敏感性、特异性和重复性进行了评估,并应用该方法进行临床样品检测。结果显示:当引物和探针浓度分别为250和200 nmol/L,退火温度为55℃时,建立的迟缓爱德华氏菌荧光PCR方法荧光曲线标准,线性关系良好,最低检测限可达12.4 CFU/m L,且1 CFU/m L的细菌经35℃培养1 h后即可被检出;与猪链球菌2型以及无乳链球菌等14种常见水生动物病原体无交叉反应;重复性试验变异系数均小于0.95%;在100份临床样品中,检出阳性22份,与细菌分离鉴定结果一致。结果表明,本研究建立的迟缓爱德华氏菌荧光PCR检测方法敏感性高、特异性强、重复性好,为迟缓爱德华氏菌病的临床诊断和研究提供了技术支撑。In order to construct a rapid,specific and accurate method for detection of Edwardsiella tarda,specific primers and probe were designed targeting at gyr B gene,a fluorescent PCR was established after optimizing the reaction system and annealing temperature,followed by evaluation on its sensitivity,specificity and reproducibility,and then detection of clinical samples.The results revealed that the established method had a standard fluorescence curve with a good linearity when the concentrations of primers and probe were 250 and 200 nmol/L,respectively,and the annealing temperature was at 55℃.The lowest detection limit was 12.4 CFU/m L,and bacteria with the initial concentration of 1 CFU/m L could be detected by the method after being cultivated for 1 hour under 35℃;no cross reaction with 14 common aquatic animal pathogens such as Streptococcus suis type 2 and Streptococcus agalactiae was observed;the coefficients of variation of repeatability tests were all less than 0.95%;and 22 out of 100 clinical samples were detected positive,which was consistent with bacterial isolation and identification results.In conclusion,clinical diagnosis and research of Edwardsiella tarda disease were technically supported by the established method that was with high sensitivity,strong specificity and good reproducibility.

关 键 词：迟缓爱德华氏菌 荧光PCR 线性关系 特异性 变异系数 临床应用 

分 类 号：S851[农业科学—预防兽医学]