番茄褪绿病毒SYBR Green I实时荧光定量PCR方法

 根据番茄褪绿病毒(Tomato chlorosis virus, ToCV)热激蛋白70(Hsp70)的基因序列,设计ToCV实时荧光定量PCR特异引物。利用重组质粒ToCV-1为标准品建立SYBR Green I实时荧光定量方法。针对引物浓度、退火温度、特异性、灵敏度、重复性和稳定性进行系列优化。结果表明,最适退火温度为63℃,最适引物浓度为0.3 μmol·L^-1。熔解曲线为特异性单峰,表明其特异性良好。建立的SYBR Green I实时荧光定量PCR较常规PCR灵敏100倍,且具有良好的重复性和稳定性。基于SYBR Green I实时荧光定量PCR技术建立的ToCV检测方法,速度快、特异性强、灵敏度高、重复性好,可以用于ToCV的定量检测。

SYBR Green I quantitative real-time PCR (qPCR) for Tomato chlorosis virus

According to the heat shock protein 70 (Hsp70) gene sequence of Tomato chlorosis virus (ToCV), a specific primer pair for ToCV real-time fluorescent quantitative PCR was designed. The recombinant plasmid ToCV-1 is used as a standard to establish a SYBR Green I real-time fluorescence quantitative method and a series of optimization include primers concentration, annealing temperature, specificity, sensitivity, reproducibility and stability were performed. The results showed that, the optimized annealing temperature is 63 ℃, and the primer concentration is 0.3 μmol·L^-1. The melting curve for specific peak proved that this method has good specificity. By comparing with the sensitivity of conventional PCR, we found that the SYBR Green I real-time fluorescence quantitative PCR was 100 times more sensitive. The method has good repeatability and stability. The rapid detection method based on SYBR Green I real-time quantitative PCR has high speed, strong specificity, high sensiti-vity and good repeatability. It can be used for the quantitative detection of ToCV.