Nsp9基因实时荧光定量PCR检测方法的建立及其在感染细胞过程中表达量的变化

试验旨在建立猪繁殖与呼吸综合征病毒（porcine reproductive and respiratory syndrome virus，PRRSV）Nsp9基因的实时荧光定量PCR检测方法，并探究其在感染细胞过程中表达量的变化。根据PPRSVNsp9基因序列设计引物，建立基于SYBR Green Ⅰ检测模式的实时荧光定量PCR。结果显示，PRRSV Nsp9基因在1×10⁴~1×10⁸拷贝/μL范围内有很好的线性关系，扩增产物的熔解曲线只有1个单特异峰，无引物二聚体。该方法与猪戊肝病毒（HEV）、猪流感病毒（SIV）、伪狂犬病病毒（PRV）基因组均无交叉反应，可重复性好，组内变异系数小，可用于临床PRRSV检测。在病毒感染细胞过程中，Nsp9基因表达量逐步升高，36 h达到最高。本研究为探究病毒复制规律与临床疫苗生产奠定了理论基础。

Establishment of the Quantitative Real-time PCR Method for PRRSV Nsp9 Gene Rapid Detection and Its Expression in PRRSV Infected Cells

The study was conducted to establish a quantitative Real-time PCR method for PRRSV Nsp9 gene rapid detection and study its expression in PRRSV infected cells.A pair of specific primers targeted to Nsp9 of PRRSV was designed and a Real-time PCR method based on SYBR Green Ⅰ fluorescent was developed for the quantization of PRRSV.The melting curve analysis using SYBR Green Ⅰ dye showed one specific peak,and no primer dimers peak was observed.No amplification was detected from HEV,SIV and PRV samples by this method.There was good reproducibility and low variation coefficient.The quantitative Real-time PCR method developed in this study would be useful for rapid laboratory diagnosis and epidemiology investigation of PRRSV.During the process of virus infecting cells,the expression level of Nsp9 increased gradually,and it got the highest at 36 h.This study laid the theoretical basis to explore the law of virus replication and clinical vaccine production.