检测猪圆环病毒2型TaqMan荧光定量PCR方法的建立  

Establishment of TaqMan Fluorescent Quantitative PCR for Detection of Porcine Circovirus Type 2

作　　者：刘俊平[1] 庄金山[2] 孙志[2] 张建武[2] 陶琦[1] 袁世山[2] 

机构地区：[1]上海出入境检验检疫局,上海200135 [2]中国农业科学院上海兽医研究所,上海200232

出　　处：《上海交通大学学报（农业科学版）》2007年第5期483-488,共6页Journal of Shanghai Jiaotong University(Agricultural Science)

基　　金：上海市科委基金资助项目(05DZ0511)

摘　　要：针对猪圆环病毒2型(PCV2)ORF2的序列设计两对特异性引物及TaqMan探针,以感PCV2的PK-15细胞培养上清的DNA提取物为模板,分别扩增499 bp和131 bp的片段,建立了检测PCV2的TaqMan荧光PCR方法。结果表明:TaqMan荧光PCR检测PCV2的最佳探针浓度为0.5 pmol·μL-1;TaqMan荧光PCR两步法操作快捷,扩增131 bp的引物PCF1101/PCR1232检测敏感性高(3.17×102拷贝·μL-1),重复性好。在诸多检测样品中,除检测到PCV2检测指标出现阳性外,猪呼吸与繁殖障碍综合征病毒(PRRSV),日本乙型脑炎病毒(JEV),猪伪狂犬病毒(PRV),猪瘟病毒(CSFV)及PK-15细胞等检测指标均为阴性,表明特异性强;与普通PCR的检测结果(2/10)比较,本法的敏感性和对临床样品的阳性检出率(3/10)更高。上述研究表明,本方法快速、敏感、特异,具有很高的重复性,且可实时监测,能有效检测PCV2。Based on the nueleotide sequence of porcine eircovirus type 2（PCV2）, two pairs of primers and a TaqMan probe were designed and synthesized. Genomic DNA of PCV2 was obtained from the culture supematant of infected PK15 cells, two corresponding PCR fragments, 499 bp and 131 bp in-length, respectively, were amplified by the two pairs of primers. TaqMan fluorescent quantitative PCR for detection of PCV2 was established successfully with the optimal probe concentration （0.5 pmol·μL^-1）. The results showed that the two-step TaqMan fluorescent quantitative PCR was fast, the detection of the primers PCF1101/ PCR1232 amplifying 131 bp was more sensitive, with a detection limit as low as 3.17×102 copies·μL^-1. The assay specifically detected porcine cireovirus type 2 , while the detecting results of porcine reproductive and respiratory syndrome virus （PRRSV）, Japanese encephalitis virus （JEV）, porcine pseudorabies virus （PRV）, classical swine fever virus （CSFV） and PK15 cell were negative. Sensitivity and positive rate （3/10） for clinical sample of TaqMan fluorescent quantitative PCR were higher than conventional PCR. Taken together, A fast, sensitive, specific and reliable real-time PCR was established that can be used for quick quarantine of importing and exporting pigs for PCV2 active infection.

关 键 词：猪圆环病毒2型 TAQMAN 荧光PCR 检测 

分 类 号：S851.347.101[农业科学—预防兽医学]