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为建立一种快速、灵敏、特异的定量检测啤酒花矮化类病毒(Hop stunt viroid,HSVd)的实时荧光定量RT-PCR(RT-qPCR)方法,设计了2对引物及特异探针,体外转录制备了RNA标准品,绘制标准曲线,并对该方法的特异性、灵敏度和重复性进行评估。建立的定量标准曲线C t值与模板拷贝数对数之间呈良好的线性关系,相关系数R2为0.9988,扩增效率为95%;该方法的特异性好,与啤酒花潜隐类病毒(HLVd)、葡萄黄斑类病毒1(GYSVd-1)、葡萄黄斑类病毒2(GYSVd-2)和桃潜隐花叶类病毒(PLMVd)均无交叉反应;灵敏度为1.0×102拷贝/μL,比普通RT-PCR高10倍;试验内及试验间重复性试验的变异系数均小于3%。研究表明该方法适用于实际样品中HSVd的快速定量检测。
In order to establish a rapid, sensitive and specific real-time fluorescence quantitative RT-PCR (RT-qPCR) method for the quantitative determination of hop stunt viroid (HSVd), two pairs of primers and specific probes were designed and in vitro transcribed RNA standards were prepared, standard curves were drawn, and the specificity, sensitivity and reproducibility of the method were evaluated. The established quantitative standard curve C t has a good linear relationship with the template copy number logarithm, the correlation coefficient R2 is 0.9988, and the amplification efficiency is 95%. The specificity of the method is in good agreement with that of the hop latent virus (HLVd ), GYSVd-1, GYSVd-2 and PLMVd. The sensitivity was 1.0 × 102 copies / μL, RT-PCR is 10 times higher; the coefficient of variation (CV) of repeatability test in the experiment and in the experiment is less than 3%. Studies have shown that this method is suitable for the rapid quantitative detection of HSVd in real samples.