K亚群禽白血病病毒实时荧光RT-PCR检测方法的建立及初步应用  

Establishment and Application of a Real-time Fluorescent RT-PCR Detection Method for Subgroup K of Avian Leukosis Virus

作　　者：陈轩 赵海燕 杨素[1] 曹伟胜 黄海超[1] 邵建宏 赵福振 沙才华 廖秀云 CHEN Xuan;ZHAO Haiyan;YANG Su;CAO Weisheng;HUANG Haichao;SHAO Jianhong;ZHAO Fuzhen;SHA Caihua;LIAO Xiuyun;(Zhuhai Entry-Exit Inspection and Quarantine Bureau;College of Veterinary Medicine,South China Agricultural University;Key Laboratory of Zoonoses Control and Prevention of Guangdong)

机构地区：[1]珠海出入境检验检疫局检验检疫技术中心,广东珠海519000 [2]华南农业大学兽医学院,广州510642 [3]广东省动物源性人兽共患病预防与控制重点实验室,广州510642

出　　处：《病毒学报》2018年第2期224-231,共8页Chinese Journal of Virology

基　　金：国家自然科学基金(31672552);题目:禽白血病病毒新亚群受体的解析及其介导的分子感染机制;广东省自然科学基金(2016A030313403);题目:新出现K亚群禽白血病病毒的病原学研究;国家肉鸡产业技术体系岗位科学家(CARS-41-G16);岗位名称:生物安全与综合防控岗;广东省现代农业产业技术体系创新团队项目(2016LM1114);团队名称:广东省家禽产业技术体系创新团队;珠海出入境检验检疫局科技项目(ZH2016-6);题目:禽白血病K亚群病毒快速检测方法研究~~

摘　　要：根据GenBank数据库K亚群禽白血病病毒（Subgroup K avian leukosis virus,ALV-K）特异性抗原gp85的基因序列,设计一组ALV-K特异性的引物和探针,并构建阳性重组质粒。在对反应体系进行优化的基础上,建立了ALV-K的实时荧光RT-PCR检测方法并进行了特异性试验、敏感性试验和重复性试验。结果显示,本方法能对ALV-K进行特异性扩增,而对A亚群、B亚群、J亚群、E亚群禽白血病毒和新城疫病毒（NDV）、鸡传染性支气管炎病毒（IBV）、禽网状内皮组织增生病病毒（REV）、禽流感病毒（AIV）等禽病病原未见扩增。该方法最低能够检测到3.4×101拷贝数/μL的阳性质粒,灵敏度是RT-PCR的100倍。应用本方法对人工攻毒ALV-K的SPF鸡各脏器进行病毒定量分析,结果显示,在攻毒鸡体内心、肝、脾、肺、肾等脏器中均能检测到ALV-K,且肝、肾和脾中的病毒含量显著高于心和肺（P〈0.01）。According to the specific gp85 sequences of subgroup K of avian leukosis virus（ALV-K）in the GenBank database,specific primers and probe were designed,and recombinant plasmids were constructed to be used as the positive control.A real-time fluorescent reverse transcription（RT-PCR）assay specific for ALV-K was optimized and established.This assay had very good sensitivity,specificity,and repeatability.The method could detect ALV-K specifically without cross-reactions with subgroups A,B,J,or E of ALV,Newcastle disease virus,infectious bronchitis virus,reticuloendotheliosis virus or avian influenza virus.The minimum detectable concentration of positive plasmids was 3.4×101 copies/μL,which was100-fold more sensitive than RT-PCR.The assay was applied to quantitative analyses of viral content in different viscera of chickens challenged by ALV-K.We found that ALV-K could be detected in the heart,liver,spleen,lungs and kidneys of all challenged chickens.The viral contents in liver,kidneys and spleen were significantly higher than those in heart and lungs（P0.01）.

关 键 词：禽白血病病毒(ALV-K) K亚群 实时荧光RT-PCR 检测方法 应用 

分 类 号：S852.65[农业科学—基础兽医学]