仙台病毒颗粒数实时荧光定量PCR检测方法的建立及验证

Development and application of real-time qPCR assay for detecting Sendai virus gene copy number

目的 建立并验证仙台病毒颗粒数的real-time qPCR检测方法。方法 提取仙台病毒核酸和扩增仙台病毒N基因并构建重组质粒作为参考品,建立TaqMan探针法real-time qPCR方法,并进行线性、重复性、灵敏度和精密度验证。结果 该方法可以检测到仙台病毒的基因组拷贝数,在1.024 × 103~5 × 1010拷贝数/μl范围内,线性良好,R2>99%;检测样品的加标回收率为84.56% ~119.48%。结论 与传统的检测颗粒数血凝试验相比,此方法的灵敏度更高,重复性更好,定量准确,可用于以仙台病毒为载体的疫苗颗粒数的检测,进而检测疫苗产品的比滴度。

Objective To develop a real-time quantitative PCR(qPCR)assay for detecting Sendai virus gene copy number.Methods The recombinant plasmid was constructed and a reference used to establish the real-time qPCR method with the TaqMan probe and verified for reproducibility and sensitivity.Results The linear range of the developed real-time qPCR assay was 1.024×103~5×1010 copies/μl,with an R2 value of more than 0.99.The standard recovery of the tested samples was 84.56%~119.48%.Conclusions Compared with traditional hernagglutination assay for particle number detection,this method has higher sensitivity,better repeatability and high quantitative accuracy.It can be used for the detection of particle number of vaccine with sendai virus as the carrier,so as to detect the specific activity of vaccine products.