SYBR Green Ⅰ实时PCR对猪细小病毒体外复制动态分析

根据已经发表的猪细小病毒（PPV）的VP2基因序列,设计2对特异性引物,建立了检测PPV的SYBR GreenⅠ实时PCR方法。该方法最小检出量为12个PPV拷贝。模板稀释度在108范围内呈良好的线性关系,与猪圆环病毒2型、猪繁殖与呼吸综合征病毒、乙型脑炎病毒、猪瘟病毒、伪狂犬病病毒无交叉反应。应用本方法对PPV在体外感染细胞后的复制动态进行了观察,并绘制了病毒的体外增殖曲线。数据换算为每瓶中细胞内、外病毒拷贝数,结果显示细胞外病毒含量在接毒初始的36h逐渐下降,随后开始逐步增加;接毒后84h培养液中的病毒含量（1.739×1010拷贝）逐渐超过细胞内的病毒含量（1.321×1010拷贝）;在接毒后108h培养液中病毒含量达到峰值（7.626×1010拷贝）,随即病毒含量开始快速下降。细胞内病毒粒子在接毒后24h内为对数增长期,然后为缓慢增长期,至接种后72h达到复制峰值（1.425×1010拷贝）,并维持至108h。与病毒复制动态变化相对应的细胞病变是从细胞聚集、开始形成空斑到约80%的细胞病变产生,108h之后随着细胞的大量死亡,细胞内、外病毒数量都开始急剧减少。

Dynamic analysis of porcine parvovirus in vitro replication by SYBR GreenⅠ real-time PCR assay

According to the structural protein gene（VP2） sequence of porcine parvovirus（PPV）,two pairs of specific primers were designed and a real-time polymerase chain reaction（real-time PCR） with SYBR GreenⅠ for quantification detection of PPV was developed.The detection limit of the SYBR GreenⅠ reaction was 12 PPV copies/reaction.The assay was linear at a 108 dilution range of template concentrations.Other porcine pathogens involved in reproductive disorders such as porcine circovirus type 2（PCV-2）,porcine reproductive and respiratory virus（PRRSV）,Japanese encephalitis virus（JEV）,classical swine fever virus（CSFV） and Aujeszky＇s disease virus（PRV） were not detected by this assay.Dynamic analysis of PPV replication in vitro by this assay traced a curve of PPV proliferation.The data is converted into intracellular and extracellular virus copy number in a culture flask,the results indicated that the amount of the extracellular virus copies gradually decreased from 0 h to 36 h post inoculation,and then began to increase.The copies of virus in the cultural supernatants（1.739×1010 copies） were higher than that in the cells（1.321×1010 copies） at 84 h post inoculation.The copies produced a peak at 108 h post inoculation（7.626×1010 copies） and then rapidly reduced.While intracellular virus particles showed a logarithmic growth phase within the first 24 h post inoculation followed by a slow growth until 72 h post inoculation（1.425×1010 copies）,and reached a peak of titer and maintained a high level until 108 h post inoculation.Cytopathic effect（CPE） corresponding to the process of the virus growth was characterized by the cell aggregation,and about 80% plaque formation.With the large number of cell death,virus copies both intracellular and extracellular began to sharply reduce at 108 h after inoculation.