猪繁殖障碍性疫病多重PCR诊断方法的建立及其应用  

Establishment and application of a mutiplex PCR diagnosis method for reproductive failure of swine diseases

作　　者：贾怀杰[1] 陈国华[1] 何小兵[1] 王晓霞[1] 房永祥[1] 窦永喜[1] 瞿惠玲[1,2,3] 景志忠[1] 

机构地区：[1]中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,农业部兽医公共卫生重点实验室,甘肃兰州730046 [2]甘肃农业大学动物医学院,甘肃兰州730070 [3]甘肃省兽医局,甘肃兰州730030

出　　处：《甘肃农业大学学报》2010年第6期6-11,共6页Journal of Gansu Agricultural University

基　　金：国家自然基金项目(30871884);国家支撑计划(2006BAD31B03);甘肃省支撑计划(0804NKCA076)

摘　　要：根据GenBank已发表的猪瘟病毒(CSFV)、猪圆环病毒2型(PCV2)、猪繁殖与呼吸综合征病毒(PRRSV)和猪伪狂犬病病毒(PRV)的序列,经DNAstar生物学软件和BLAST序列分析,利用Oligo6.0软件分别设计并合成了4对特异性扩增引物.在初步优化单项PCR反应退火温度和反应体系的基础上,建立了同时检测CSFV、PCV2、PRRSV和PRV的多重PCR诊断方法.结果表明:该方法具有良好的特异性、敏感性和重复性;对30份临床样品进行检测,结果发现,CSFV、PRRSV、PCV2和PRV的阳性率分别为46.7%、43.3%、53.4%、10.0%,其中,PRV与PCV2、CSFV与PCV2和CSFV与PRRSV混合感染的阳性率分别为6.7%、36.7%和10.0%;PRV、PCV2和PRRSV单一感染的阳性率分别为3.3%、10.0%和33.3%.说明所建立的多重PCR诊断方法,可以应用于CSFV、PRRSV、PCV2和PRV混合感染所引起的猪繁殖障碍性疫病的临床鉴别诊断.According to the sequences published in the GenBank,the sequences of relevant genes of swine fever virus （CSFV）,porcine circovirus type 2 （PCV2）,porcine reproductive and respiratory syndrome virus （PRRSV） and porcine pseudorabies virus （PRV） were analyzed by biological softwares such as DNAStar and BLAST,and four pairs of special primers were designed by the Oligo 6.0 software.On the basis of initial optimization of individual PCR reaction annealing temperature and the reaction system,a multiple PCR diagnostic method for simultaneously detecting the four viruses of CSFV,PCV2,PRRSV and PRV was established.And then,this method was used for detection of 30 clinical samples.The results showed that positive rate of CSFV,PRRSV,PCV2,PRV were 46.7 %,43.3 %,3.4 %,10 % respectively.Among them,the positive rate of PRV-PCV2,CSFV-PCV2 and CSFV-PRRSV co-infection were 6.7 %,36.7 % and 10 % respectively,while positive rate of PRV,PCV2 and PRRSV single infection was 3.3 %,10 % and 33.3 % respectively.The results showed that the multiplex PCR had good specificity,sensitivity,reproducibility,and could be used in clinical differential diagnosis.

关 键 词：猪瘟病毒 猪圆环病毒2型 猪繁殖与呼吸综合征病毒 猪伪狂犬病毒 多重PCR 

分 类 号：S858.28[农业科学—临床兽医学] S854.43[农业科学—兽医学]