K亚群禽白血病病毒实时荧光定量PCR检测方法的建立及应用  

Establishment and application of a real-time PCR method for the detection of subgroup K avian leukosis virus

作　　者：王瑞明 都玉印 相英杰 郭龙宗 李玉燕 李阳 李建亮[1,2,3] 赵鹏 王一新[1,2,3] 崔言顺 WANG Rui-ming;DU Yu-yin;XIANG Ying-jie;GUO Long-zong;LI Yu-yan;LI Yang;LI Jian-liang;ZHAO Peng;WANG Yi-xin;CUI Yan-shun(College of Animal Science and Veterinary Medicine,Shandong Agricultural University,Tai’an,Shandong 271018,China;Shandong Province Key Laboratory of Animal Biotechnology and Disease Control and Prevention,Shandong Agricultural University,Tai’an,Shandong 271018,China;Shandong Province Engineering Technology Research Center of Animal Disease Control and Prevention,Shandong Agricultural University,Tai’an,Shandong 271018,China;Shandong Yisheng Livestock&Poultry Breeding Co.,Ltd.,Yantai,Shandong 264001,China;China Animal Health and Epidemiology Center,Qingdao,Shandong 266114,China)

机构地区：[1]山东农业大学动物科技学院,山东泰安271018 [2]山东省动物生物工程与疾病防治重点实验室,山东泰安271018 [3]山东省畜禽疫病防制工程技术研究中心,山东泰安271018 [4]山东益生种畜禽股份有限公司,山东烟台264001 [5]中国动物卫生与流行病学中心,山东青岛266114

出　　处：《中国兽医学报》2020年第3期524-529,共6页Chinese Journal of Veterinary Science

基　　金：国家“十三五”重点研发计划资助项目(2018YFD0501404);山东省自然科学基金博士基金资助项目(ZR2018BC047);山东省“双一流”奖补资金项目(2017003)。

摘　　要：根据K亚群禽白血病病毒(ALV-K)gp85基因序列设计1对引物,对反应条件和反应体系进行优化,建立了快速检测ALV-K的SYBR GreenⅠ实时荧光定量PCR方法。该方法在1~10^7 copies/μL范围内具有良好的线性关系,灵敏度达到1copies/μL,是常规PCR方法的100倍。该方法与经典亚群ALV及其他常见禽免疫抑制病病毒无交叉反应,批间与批内重复性试验变异系数均小于2%。将ALV-K原型株JS11C1通过卵黄囊接种途径感染SPF鸡,运用本试验建立的实时荧光定量PCR方法,对感染鸡体内各器官的病毒分布及病毒载量进行了检测。结果显示,在感染鸡的心脏、脾脏、肺脏、肾脏等器官中均可检测到病毒,其载量无明显差异。结果表明,本试验所建立的实时荧光定量PCR方法具有敏感度高、特异性强和重复性好的优点,可用于ALV-K的快速、定量检测,该方法同时为ALV-K致病机制的研究提供了良好的技术手段。To establish a SYBR Green I real-time PCR method for the rapid detection of subgroup K avian leukosis virus(ALV-K),apair of primer specific for ALV-Kgp85gene was synthesized,and the reaction condition and system were optimized.This real-time PCR method had a perfect linearity ranged from 1to 10^7 copies/μL,and the sensitivity of the method was 1copies/μL,which was 100times than the conventional PCR assay.No cross reactions were observed with ALV of classical subgroups and other common avian immunosuppressive viruses,and both of intra-assay and inter-assay coefficient of variation were less than 2%.SPF chickens were infected with JS11C1,the prototype strain of ALV-K,by yolk sac inoculation,and the distribution and viral load within different tissues in infected chickens were detected by the method established in this study.The result showed that ALV-K could be detected in heart,spleen,lung,kidney and some other organs,while there was no obvious difference of viral loads in those tissues.In brief,the real-time PCR method established in this study was specific and sensitive with repeatability,which could be used for the rapid and quantitative detection of ALV-K.This method also provided a good technical means for the research of the pathogenic mechanism of ALV-K.

关 键 词：K亚群禽白血病病毒(ALV-K) 实时荧光定量PCR 病毒载量 

分 类 号：S852.65[农业科学—基础兽医学]