猪圆环病毒2型TaqMan荧光定量PCR检测方法的建立  

Development of TaqMan fluorescent quantitative PCR for detection of porcine circovirus type 2

作　　者：张丽丽[1,2] 张志[2] 任夫波[1,2] 杨若松[2,3] 张燕霞[2] 李晓成[2] 单虎[1] 

机构地区：[1]青岛农业大学动物科技学院,山东青岛266109 [2]中国动物卫生与流行病学中心,山东青岛266032 [3]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《中国兽医科学》2010年第10期1048-1051,共4页Chinese Veterinary Science

摘　　要：根据猪圆环病毒2型(PCV2)ORF1基因的保守序列,设计合成了1对特异引物和1条探针,以PCV2全基因阳性质粒作为标准品,经反应条件优化,建立了一种检测PCV2的荧光定量PCR方法。对该方法进行特异性、敏感性和重复性试验,结果显示,该方法可特异地检测PCV2,而与PCV1等猪病病毒不发生交叉反应;该方法的灵敏度可达1×102copies/μL,比常规PCR高100倍;3次重复检测的变异系数均小于5%。用建立的荧光定量PCR和常规PCR分别对94份临床样品进行检测,荧光定量PCR的阳性检出率为51.06%,而常规PCR的阳性检出率仅为38.30%。证实,建立的方法具有快速、特异、灵敏、重复性好、定量、安全等优点,可用于PCV2的临床检测。A pair of primers and a probe were designed according to the conserved sequence of ORF1 gene of porcine circovirus type 2（PCV2）and the recombinant plasmid containing the complete genome sequence was constructed as a standard control,then a fluorescent quantitative real-time PCR（FqRT-PCR） method was developed by optimizing reaction conditions and tests of specificity,sensitivity and reproducibility were carried out.In results,the developed method could specifically detect PCV2,and had no cross- reaction with porcine circovirus type 1and other viruses.The sensitivity of this method was proved to be 1×102 copies/μL,which was 100fold better than the traditional PCR,and the coefficient of variation value was less than 5%.Among 94samples,the positive rate was 51.06%,detected by FqRT-PCR,38.30%by the traditonal PCR.The results showed that the developed method had the advantages of rapidity,specificity,reproducibility,quantitativity and sensitivity,and it was able to be applied to clinical diagnosis.

关 键 词：猪圆环病毒2型 荧光定量聚合酶链反应 TAQMAN探针 

分 类 号：S852.659.2[农业科学—基础兽医学]