樱桃病毒A实时荧光定量PCR检测技术的建立与应用

Establishment and application of real-time fluorescence quantitative PCR detection technology for cherry virus A

在樱桃病毒A(CVA)mp基因保守区域设计了3对检测引物,经特异性筛选后,获得可用于病毒定量研究的引物。制备质粒标准品,建立标准曲线,同时验证该方法的灵敏度和特异性,并应用于田间果树样品CVA定量检测。最终成功筛选出1对检测效率高、特异性强的引物(CVA-dF2、CVA-dR2),基于SYBR Green I荧光染料建立反转录实时荧光定量PCR检测CVA的方法。该方法重复性好、灵敏度高,无需借助内参基因即可准确检测目的病毒载量,绝对定量标准曲线斜率为-3.5746,决定系数R2为0.9986,扩增效率为0.9044,比常规RT-PCR检测灵敏度高10倍。该方法的建立为CVA定量研究提供了有力工具,可用于果树中CVA批量检测或低丰度病毒样品检测。

Three pairs of detection primers were designed in the conserved region of cherry virus A(CVA)mp gene.After specific screening,primers were obtained that could be used for virus quantitative research.Preparation of plasmid standards,and establishment of standard curves were conducted the sensitivity and specificity of this method,were verified and it is applied to the quantitative detection of CVA in field fruit tree samples.One pair of primers with high detection efficiency and strong specificity(CVA-dF2,CVA-dR2)was successfully screened,and a real-time reverse transcription fluorescence quantitative PCR method for detecting CVA was established based on SYBR Green I fluorescent dye.This method had good repeatability and high sensitivity.It could accurately detect the target viral load without the help of internal reference genes.The slope of the absolute quantitative standard curve was-3.5746,the coefficient of determination R2 was 0.9986,and the amplification efficiency was 0.9044,which was 10 times higher than the sensitivity of conventional RT-PCR detection.The establishment of this method provided a powerful tool for quantitative research on CVA,which could be used for batch detection of CVA in fruit trees or detection of low abundance virus samples.