毒害艾美耳球虫疫苗ENYS株TaqMan qPCR检测方法的建立  

Methodology Development of TaqMan qPCR for E.necatrix Vaccine ENYS Strain

作　　者：靳浩展 肖文婉 廖申权 戚南山 李娟 吕敏娜 蔡海明 胡俊菁 林栩慧 宋勇乐 陈祥杰 朱易斌 尹理君 张健騑 张旭 孙铭飞 JIN Haozhan;XIAO Wenwan;LIAO Shenquan;QI Nanshan;LI Juan;LV Minna;CAI Haiming;HU Junjing;LIN Xuhui;SONG Yongle;CHEN Xiangjie;ZHU Yibin;YIN Lijun;ZHANG Jianfei;ZHANG Xu;SUN Mingfei(Foshan University,Foshan Guangdong 528231;Key Laboratory of Avian Influenza and Other Major Poultry Diseases Prevention and Control,Key Laboratory of Livestock Disease Prevention of Guangdong Province,Institute of Animal Health,Guangdong Academy of Agricultural Sciences,Guangzhou Guangdong 510640)

机构地区：[1]佛山科学技术学院,广东佛山528231 [2]广东省农业科学院动物卫生研究所/农业农村部禽流感等家禽重大疫病防控重点实验室/广东省畜禽疫病防治研究重点实验室,广东广州510640

出　　处：《广东畜牧兽医科技》2024年第4期1-8,共8页Guangdong Journal of Animal and Veterinary Science

基　　金：广东省农业科学院协同创新中心项目(XTXM202202);广东省畜禽疫病防治研究重点实验室项目(2023B1212060040);科技创新战略专项资金(高水平农科院建设)(202110TD,202122TD);广东省现代农业产业技术体系创新团队建设专项(2023KJ119)。

摘　　要：为了对E.necatrix ENYS株在生产中进行质控及对临床应用效果的监测,该研究对ENYS株和ENGD株进行全基因组重测序,结合生物信息学分析建立了一种TaqMan qPCR检测技术。结果显示,En-marker5测序结果与预期相符,最优的引物浓度和探针浓度分别为500nM和250 nM,标准曲线的斜率为-3.249,相关系数为0.999,可以通过Ct值对模板浓度定量,该方法检测下线为290个拷贝/反应和50个卵囊/反应,灵敏度是传统PCR的10倍;该方法可以特异性检测EnYS株,不能扩增其他种的疫苗株和野毒株以及肠道病原菌;重复性检测的变异系数小于0.6%,重复性良好。本研究针对E.necatrix EnYS株开发了一种灵敏度高、特异性强、稳定性好的TaqMan qPCR检测方法,为E.necatrix EnYS株的安全性评估及临床应用提供技术支持。In order to control the quality of E.necatrix ENYS in production and monitor its clinical application,this study performed whole genome resequencing on ENYS and ENGD strains,and established a TaqMan qPCR detection technology combined with bioinformatics analysis.The results showed that the En marker5 sequencing results were consistent with expectations,with the optimal primer and probe concentrations of 500 nM and 250 nM,respectively.The slope of the standard curve was-3.249,and the correlation coefficient was 0.999.The template concentration can be quantified by the Ct value.This method detected 290 copies/reactions and 50 follicles/reactions,with a sensitivity 10 times higher than traditional PCR;This method can specifically detect ENYS strains,but cannot amplify other vaccine strains,wild⁃type strains,and intestinal pathogens;The coefficient of variation for repeatability testing is less than 0.6%,indicating good repeatability.This study developed a highly sensitive,specific,and stable TaqMan qPCR detection method for the E.necatrix ENYS strain,providing technical support for the safety evaluation and clinical application of the E.necatrix ENYS strain.

关 键 词：毒害艾美耳球虫 减毒活疫苗 全基因组重测序 分子标记 TaqMan qPCR 

分 类 号：S852.7[农业科学—基础兽医学]