猪圆环病毒2型TaqMan荧光定量PCR检测方法的建立与初步应用

Establishment and preliminary application of TaqMan fluorescence quantitative polymerase chain reaction method for detection of porcine circovirus type 2

参照GenBank收录的猪圆环病毒2型(PCV2)基因组全序列选择保守区域设计合成了引物及其探针,并对其进行了筛选;对荧光定量PCR的反应条件进行了优化,建立了TaqM an荧光定量PCR检测方法.用建立的检测方法对临床采集的组织病料进行了检测,并与常规PCR检测方法作比较.结果显示,所建立的方法灵敏度可达3×101拷贝.μL-1,比常规PCR检测方法灵敏度高10倍,而与猪圆环病毒1型(PCV1)、猪伪狂犬病毒(PRV)、猪瘟病毒(CSFV)、猪繁殖和呼吸综合征病毒(PRRSV)没有交叉反应,具有灵敏度高、特异性强、操作简便等优点,适合于PCV2的流行病学调查和临床诊断.

The probes and primers were designed and synthesized according to the conserved gene porcine eireovirus type 2 （PCV2） available in GenBank, and then reaction parameters were optimized to develop a real-time TaqMan fluorescence quantitative PCR assay. The tissue samples from pigs were detected by using the established quantitative PCR assay, and the method was compared with that of routine PCR. The results indicated that the developed quantitative PCR assay could detect 3 × 10^1 copys · μL^-1 of plasmid DNA and its sensitivity was 10 times higher than that of the routine PCR, and had no cross reaction with porcine cireovirus type 1 （PCV1） , classical swine fever virus （CSFV） , porcine reproductive and respiratory syndrome virus （PRRSV） and pseudorabies vi- rus （PRV）. The real-time TaqMan fluorescence quantitative PCR assay which is specific, sensitive and accurate can be used for the diagnosis of PCV2 infection.