【摘要】 为建立检测肉食兽类细小病毒荧光定量PCR方法和探索细小病毒基因组拷贝数与病毒含量之间的相关性,在肉食兽类细小病毒VP2基因区分别设计了1对保守引物和1条TaqMan-MGB标记的水解探针,通过对貉细小病毒(RDPV)LN10-1株VP2基因上的长度为92bp的保守片段进行PCR扩增,构建标准重组质粒,由已知浓度的重组质粒通过荧光定量PCR体系建立标准曲线,建立了检测肉食兽类细小病毒基因组拷贝数的荧光定量PCR方法。结果显示,该检测方法在3.50×102～3.50×107 copies/μL范围内与Cp值呈良好的线性关系(R2可达0.999),最低检测浓度为35.0copies/μL。通过标准曲线得出RDPVLN10-1株样品的目标片段拷贝数,将拷贝数值与TCID50值进行相关性分析;分析结果显示,基因组拷贝数值与病毒TCID50值之间存在极显著的正相关关系(P<0.01),血凝效价与病毒TCID50值存在显著的正相关关系(P<0.05)。结果表明,与用血凝效价测定病毒含量的方法相比,用本试验建立的荧光定量PCR技术测得的病毒拷贝数值更能准确地代表活病毒的含量。更多还原

【Abstract】 To explore the correlation between the carnivore parvovirus genome copy number and the virus content,conservative primers and a TaqMan-MGB labeled hydrolysis probe were designed against the VP2 gene in this study.The standard recombinant plasmid was built through amplifying a 92 bp conservative fragment in the VP2 gene fragment from a RDPV LN10-1 strain,with the known concentrations of recombinant plasmid established the standard curve by quantitative PCR system.A method was established to detect carnivore parvovirus genome copy numbers quantitatively by using of fluorescent quantitative PCR.The amplification system was sensitive to detect 35.0 copies/μL.The standard curve had a good linearity when the quantity for the gene was between 3.50×102and 3.50×107 copies/μL(R2= 0.999).The gene copy number of the target gene was firstly calculated from the standard curve of RDPV LN10-1. Correlation analysis of the virus genome copy numbers and TCID50 showed that there were very significant positive correlation(P<0.01) between the genome copy numbers and TCID50. There were significant positive correlation(P<0.05) between HA titers and TCID50. The result showed that compared to the HA titers,the gene copy number in the present study was more representative of the live virus content.更多还原