实时定量PCR检测马传染性贫血病毒载量方法的建立和应用  

Quantification of equine infectious anemia virus RNA in horse plasma by a real-time PCR assay

作　　者：梁华[1] 张晓燕[1] 沈弢[1] 童骁[1] 马燕[1] 李红梅[2] 相文华[2] 沈荣显[2] 邵一鸣 

机构地区：[1]中国疾病预防控制中心性病艾滋病预防控制中心病毒与免疫研究室,北京100050 [2]中国农业科学院哈尔滨兽医研究所

出　　处：《中华微生物学和免疫学杂志》2005年第12期1035-1039,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金(30371319);人事部留学人员科技活动项目择优资助经费

摘　　要：目的建立实时定量PCR检测血浆病毒载量的方法,对马传染性贫血病毒(equine infectious anemia virus,EIAV)强毒株攻毒马和疫苗免疫攻毒马血浆中病毒载量进行了跟踪检测,探讨病毒载量和临床疾病状态的相关性.方法以EIAV强毒株LN40序列为标准,在gag保守区设计1对引物和Taqman探针,用于实时定量PCR扩增,用含扩增目的基因的体外转录RNA作标准品,获得标准曲线,对扩增样品进行准确定量.强毒株LN40直接攻毒,或者疫苗株DLV免疫马6个月后用强毒株LN40进行攻毒,跟踪检测攻毒马及免疫攻毒马血浆中EIAV载量情况.结果反应在101～109copies/ml之间具有良好的线性关系,反应的检出下限为10 copies/ml.强毒攻毒马出现发热并最终死亡,发热期间马血浆中EIAV载量与体温呈正相关,载量最高达107 copies/ml.免疫攻毒马未出现发热,其血浆EIAV载量的总体水平低于强毒攻毒马,攻毒后3个月低至10 copies/ml以下.结论成功建立了实时定量PCR检测血浆EIAV载量的方法,并证实了用实时荧光定量PCR检测EIAV病毒载量的方法来监测动物感染状态具有可行性,为EIAV致病机制研究和弱毒疫苗免疫保护机制的研究提供良好的技术平台.Objective To develop a new quantification method for equine infectous anemia virus （EIAV） RNA by real-time PCR. Methods A pair of primers and a Taqman probe were designed on the bases of conservative sequence of strain LN40 for the amplification of real-time PCR and transcripted RNA containing the amplified fragment was used as a standard to quantify the sample RNA. To detect viral load in the plasma of horses challenged by DLV and/or LN40. Results The EIAV real-time PCR assay had a sensitivity of 10 copies/nil, with a dynamic range of detection between 10^1 and 10^9 copies/ml. The horses attacked by LN40 had fever and then died. During the fever phase, the viral load was positively related to body temperature. As a whole, the viral load of horses challenged by LN40 after being vaccinated by DLV was lower than that of horses directly challenged by LN40, and showed less than 10 copies/ml after three months of being challenged by LN40. Conclusion We successfully established a method for the quantification of EIAV RNA in plasma, and the feasibility of infectious state observation by detecting EIAV viral load using real-time PCR was proved. This method has potential application for investigating the pathogenesis of the virulent EIAV and the protective mechanism of the EIAV vaccine.

关 键 词：马传染性贫血病毒 实时定量PCR 病毒载量 

分 类 号：R446.5[医药卫生—诊断学]