猪瘟兔化弱毒疫苗中病毒含量实时荧光定量RT-PCR检测方法的建立及初步应用

为建立敏感、特异的评价猪瘟兔化弱毒(HCLV)疫苗中病毒含量的实时荧光定量RT-PCR方法,参照中国猪瘟石门株兔化弱毒全长序列,在猪瘟兔化弱毒活疫苗基因组5'非编码区设计1对标准品引物、1对特异性引物和1条探针,建立检测猪瘟兔化弱毒活疫苗病毒含量的实时荧光定量RT-PCR方法.该方法检测的敏感度达1.20×10⁵拷贝/mL;对猪繁殖与呼吸综合征、猪乙型脑炎、仔猪副伤寒和猪伪狂犬病4种活疫苗基因组扩增结果均为阴性;重复性试验结果显示,批内变异系数为0.29%～0.39%,批间变异系数为0.32%～0.61%.应用此方法对6个不同厂家生产的7种猪瘟兔化弱毒活疫苗中病毒含量进行了检测,发现不同厂家生产的疫苗中病毒含量存在较大差异.结果表明,建立的猪瘟兔化弱毒疫苗实时荧光定量RT-PCR方法能特异地检测疫苗病毒含量,可用于初步评价猪瘟兔化弱毒疫苗抗原含量.

Development and Preliminary Application of Real-time RT-PCR Assay for Detecting Quantitation of Hog Cholera Lapinized Virus in Vaccine

To establish a rapid, sensitive and specific method for evaluation of viral load of hog cholera lapinized virus (HCLV) vaccine, a Real-time RT-PCR assay was established using two pairs of primers and an internal TaqMan probe from the 5' non-structural region of HCLV genome. The sensitivity of the assay was 1.20×10⁵ copies/mL. The assay was specific and showed no application reaction with porcine reproductive and respiratory syndrome live vaccines, porcine epidemic encephalitis B live vaccines, swine paratyphoid and pseudorabies live vaccines. The coefficient of variation (CV) of 3 different vaccine samples of HCLV was 0.29% to 0.39% in intra-assay and 0.32% to 0.61% in inter-assay, respectively. Using this method to detect 7 different vaccines of HCLV from six different plants, the results showed that there were significant differences in viral loads among the selected HCLV vaccines. In conclusion, this Real-time RT-PCR was a highly sensitive, specific and stable method which could be applied to preliminarily evaluate the viral loads of HCLV vaccines.