实时定量PCR对猪圆环病毒2型感染猪体内病毒分布规律的研究  

Detection of porcine circovirus type 2 distributions in the infected pigs by real-time quantitative PCR

作　　者：危艳武[1] 刘长明[1] 袁婧[1] 黄立平[1] 张朝霞[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/猪传染病研究室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2008年第12期924-928,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家"948"计划(2006-Z6);国家科技支撑计划项目(2006BAD06A07)

摘　　要：根据发表的猪圆环病毒2型(PCV2)ORF2基因序列设计了一对特异引物,并以克隆重组质粒作为阳性标准品,采用SYBR-Green I嵌合荧光染料建立了一种用于检测PCV2的实时定量PCR方法。该方法在107～101范围内具有良好的线性关系,相关系数为R2=0.997,扩增效率为E=0.920;对质粒标准品检测下限值为8.2拷贝/μL,检测变异系数低于2.0%。该方法与常规PCR及过氧化物酶单层细胞试验(IPMA)比较,其敏感性提高103倍以上。采用该法对PCV2人工感染猪的心、肝、脾、肺、肾、腹股沟淋巴结等组织中病毒核酸载量检测结果表明,在多种脏器中均可检测到病毒,其中腹股沟淋巴结、扁桃体和脾脏中病毒含量较高(为3.4×109拷贝/g～1.7×1010拷贝/g),这表明病毒主要在免疫器官增殖,导致淋巴细胞耗损。对来自国内不同地区的28份临床发病猪病料进行病毒DNA定量检测,有半数以上病料的病毒载量达到109-10拷贝/g。实验表明,实时定量PCR可用于PCV2核酸定量检测,为该病毒体内外定量检测提供了一种技术手段。A real-time quantitative PCR was developed for detection of porcine circovirus type 2 （PCV2） using primers derived from the ORF2 sequence. The assay had a dependent coefficient R2=0.997, amplified efficiency E=0.92, and detection limit of 8.2 plasmid DNA copies/μL, with variation coefficient less than 2.0 %. The sensitivity of the assay was 103 times higher than that of normal PCR and immunoperoxidase monolayer assay （IPMA）. Test on PCV2 DNA samples extracted from tissues of infected pig showed that the viral DNA loads in the inguinal lymph nodes, tonsil and spleen were higher than those of other tissues, indicating that PCV2 mainly propagated in the immune organs, which led to lymphocyte depletion. A total of 28 cases of postweaning multisystemic wasting syndrome （PMWS） were detected by the assay and about half cases had a virus load of 10^9-10 copies/g. These results demonstrated that the real-time quantitative PCR was a sensitive and reliable tool for detection of PCV2 DNA in the cell culture or pig＇s tissues.

关 键 词：猪圆环病毒2型 实时定量PCR 病毒体内分布规律 

分 类 号：S858.285.3[农业科学—临床兽医学]