猪PRRSV和PCV2双重TaqMan荧光定量PCR检测方法的建立及应用

Establishment of a duplex TaqMan real-time PCR assay for detection of porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 virus

为快速及定量的检测猪繁殖与呼吸综合征病毒(PRRSV)和猪圆环病毒Ⅱ型(PCV2),本研究根据GenBank中登录的PRRSV和PCV2基因组确定了其基因保守序列,分别设计其特异性探针和引物,建立了PRRSV-PCV2双重TaqMan荧光定量PCR检测方法。特异性试验显示该方法对猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪伪狂犬病毒及猪瘟病毒的检测结果均为阴性。对PRRSV和PCV2的检测灵敏度均达10拷贝/μl。重复性试验结果显示批内、批间变异系数均小于4%。应用该方法对60份已知临床病料进行检测,结果检出PRRSV和PCV2阳性感染率分别为30.67%和35%,混合感染率为11.67%,与已知结果的符合率为100%。该方法具有敏感、快速、特异、定量、重复性、稳定性好等优点,在用于PRRSV和PCV2感染的快速鉴别诊断的同时,在细胞培养特性以及疫苗生产检测中也具有广阔的应用前景。

For quantitative analysis of porcine reproductive and respiratory syndrome virus （PRRSV） and porcine circovirus type2 virus （PCV2）, a duplex TaqMan real-time PCR assay for detection of PCV2 and PRRSV was established with the specific primers and TaqMan probes of PRRSV and PCV2. The results showed that the method was specific to detect PRRSV and PCV2 without any cross reaction with PEDV, TGEV, PRV and CSFV. The sensitivity of the method was 10 copies/~L for both of PRRSV and PCV2, which was 100 times more sensitive than the normal PCR methods. The inter- and intra-assay indicated that the variations were less than 4%. Tested on 60 samples by this method, the positive rates of PRRSV and PCV2 were 30.67% and 35%, respectively, and the mixed infection rate was 11.67%, and the coincidence rate with the known results was 100%. These results demonstrate that this assay is rapid, special and sensitive for PRRSV and PCV2 detections.