猪瘟兔化弱毒疫苗株TaqMan荧光定量RT-PCR检测方法的建立及初步应用

Development and preliminary application of fluorogenic TaqMan quantitative RT-PCR for detection of hog cholera lapinized virus

为建立一种快速鉴定猪瘟兔化弱毒疫苗株(HCLV)的方法,本研究根据GenBank中登录的HCLV株基因组序列,在Erns基因序列区内设计一对引物和一条TaqMan探针,建立了检测HCLV的荧光定量RT-PCR(FQRT-PCR)方法。该方法检测的灵敏度可达3.84拷贝/μL;而对猪细小病毒、猪伪狂犬病毒、猪繁殖与呼吸障碍综合征病毒、牛病毒性腹泻病毒基因组扩增结果均为阴性。实验组批内变异系数为1.05%～1.84%,批间变异系数为3.70%～5.43%。通过对32批HCLV半成品抗原和9批成品疫苗,分别用经典的兔检法测定兔体感染量(RID)和新建立的FQRT-PCR方法进行检测比较,两者有较好的相关性。结果表明,该方法具有快速、敏感、特异性强、稳定性好等优点,对HCLV生产配制及成品检验具有指导作用。

A fluorogenic quantitative RT-PCR （FQ RT-PCR） assay of the hog cholera lapinized virus （HCLV） was established using a pair of primers and an internal TaqMan fluorogenic probe derived from the Ems region of HCLV genome. The assay was specific and showed no cross reaction with porcine parvovirus virus （PPV）, pseudorabies virus （PRV）, porcine reproductive and respiratory syndrome virus（PRRSV）, bovine viral diarrhea virus （BVDV）. The sensitivity of the assay was 3.84 copies/uL. The coefficient of variation （CV） of 3 different samples of HCLV was 1.05 % to 1.84 % in intra-assay and 3.70 % to 5.43 % in inter-assay respectively. Tests on 32 cell cultures and 9 vaccines of HCLV showed that the FQ RT-PCR produced similar but more accurate results compared to the conventional rabbit fever reaction. In conclusion, the method has the advantages of high sensibility, specificity and stability, which could be applied to evaluating the viral loads and vaccination of HCLV.