非洲绿猴肾细胞中猪圆环病毒1型的检测

Detection of porcine circovirus type 1 in African green monkey kidney cells

目的建立轮状病毒疫苗污染事件中的外源因子猪圆环病毒1型（porcine circovirus type1，PCV1）感染非洲绿猴肾细胞（Vero细胞）的检测方法。方法培养感染PCV1的Vero细胞6d后，通过定性PCR、定量PCR、电子显微镜（电镜）检查和原位杂交检测PCV1的复制与增殖。结果从感染PCV1的Vero细胞培养物提取的DNA，其PCR扩增产物琼脂糖凝胶电泳可见特异性条带；定量PCR检测显示，感染6d后病毒总拷贝数是感染前的794．3倍；定性和定量PCR的最低检出浓度均为10^2.0拷贝／ml；电镜观察到PCV1特征性形态病毒颗粒；原位杂交检测到出现深色阳性信号的PCV1阳性细胞。结论PCV1感染Vero细胞后的复制增殖可以通过上述方法进行检测。

Objective To develop detection methods for porcine circovirus type 1 （PCVI） -- the adventitious agent in rotavirus vaccine contamination incident, in African green monkey kidney cell （Vero ceil）. Methods Six days after PCV1 infection, virus replication and proliferation in Vero ceils were tested with qualitative PCR, quantitative PCR （qPCR）, electron microscopy （EM） and in situ hybridization （ISH）. Results PCR products amplified from PCVl-infeeted Vero cell DNA extracts presented specific band in agarose gel electrophoresis. According to qPCR, PCV1 copy number increased to 794. 3 times of the original level. The detection limits of qualitative PCR and qPCR were both 102.0 copies/ml. Characteristic viral particles were observed by EM. ISH showed dark positive signal of PCV1 in infected cells. Conclusion The replication and proliferation of PCV1 in infected Vero cells can be detected by the 4 methods used.