猪瘟、猪圆环病毒病和猪繁殖与呼吸综合征寡核苷酸芯片诊断方法的建立与初步应用  

Establishment and application of oligonucleotide microarray diagnostic technique for detection of porcine reproductive and respiratory syndrome virus,classical swine fever virus and porcine circovirus type 2 virus

作　　者：叶芬[1] 蔡家利[2] 王昱 李应国 

机构地区：[1]西南大学动物科技学院,重庆400715 [2]重庆理工大学药学与生物工程学院,重庆400054 [3]重庆市出入境检验检疫局,重庆400020

出　　处：《中国兽医学报》2011年第10期1395-1399,共5页Chinese Journal of Veterinary Science

基　　金：重庆市科技攻关资助项目(2009CC05)

摘　　要：猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)和猪圆环病毒Ⅱ型(PCV-2)是引发猪繁殖障碍的主要病原,建立其基因芯片快速检测体系,对开发猪繁殖障碍快速检测试剂盒具有重要意义。根据GenBank中已发表的PRRSV、CSFV和PCV-2的病毒基因组序列,设计合成特异性引物和特异性较强的60mer的寡核苷酸探针,并将探针按所设计阵列固定于表面经氨基化修饰的玻片上,制备出寡核苷酸芯片。通过引物标记及特异性验证,建立了带标记引物的多重PCR检测体系,从而产生大量可与寡核苷酸探针特异性互补的带标记的DNA片段。将标有荧光染料的扩增产物与芯片上寡核苷酸探针杂交,扫描、分析芯片上荧光信号。结果表明,芯片上各样本对应探针位点呈现阳性荧光信号,而阴性对照和空白对照则基本不能检测到荧光信号。分别用基因芯片检测方法和PCR/RT-PCR检测60份临床病料,两者符合率高达92%,表明该检测技术能够用于临床病料的检测及快速诊断PRRSV、CSFV和PCV-2。Porcine reproductive and respiratory syndrome virus（PRRSV）,classical swine fever virus（CSFV） and porcine circovirus type 2（PCV-2） are main pathogens of porcine reproduction barrier diseases.The establishment of the oligonucleotide microarray diagnostic technique is very important for developing porcine reproductive problems detection kit.Based on the similarity of the viral sequences deposited in the GenBank database,species-specific 60-mer oligonucleotide probes（oligoprobes） as the capture probes were designed and immobilized on the surface of amino modified slides.Primers were designed and labeled with Cy3.Multiplex PCR was used to prepare abundant fluorescently-tagged target DNA.Following purification,the labeled PCR products were hybridized to the microarray.The presence of the virulence genes was determined by a fluorescent signal above the background noise.When the spots of capturing probes was corresponded with samples a positive result was displayed,while in the negative and blank controls showed no signal.Then the 60 clinical samples were identified with oligonucleotide microarray diagnostic technique and PCR/RT-PCR,respectively.Compared with the sensitivities of two methods,the their compliance rate was 92%.The results indicated that the oligonucleotide microarray diagnostic technique could be applied to clinical samples for diagnosis of PRRSV,CSFV and PCV-2.

关 键 词：多重PCR 基因芯片 PCV-2 CSFV PRRSV 

分 类 号：S852.65[农业科学—基础兽医学]