啤酒花矮化类病毒实时荧光定量RT-PCR检测方法的建立与应用

Development and application of RT-qPCR assay for detection of HSVd

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。

Two pairs of primers and a specific probe were designed based on the full-length sequence of HSVd to establish a rapid, sensitive and specific RT-qPCR assay for detection and quantification of Hop stunt viroid （HSVd）. The RNA standard of HSVd was prepared with transcription in vitro and the stand- ard curve was plotted. The specificity, sensitivity and reproducibility of the developed method for HSVd detection were determined. The established standard curves showed the good linear relationship between the Ct value and the logarithmic copy number of template. The correlation coefficient was 0.9988 and the amplification efficiency was 95%. The specificity of the developed method was high and there was no crossing reaction with Hop latent viroid, Grapevine yellow speckle viroid 1, Grapevine yellow speckle viroid 2 and Peach latent mosaic viroicl. The sensitivity of the method was 1.0×10^2 copy/μL, 10-fold more sensi- tive than that of routine RT-PCR assay. Both coefficients of variation were less than 3%. The results showed that the developed method was suitable for detecting HSVd rapidly and quantitatively in practical samples.