莲藕中甘薯潜隐病毒实时荧光定量PCR检测技术的建立及应用

甘薯潜隐病毒莲藕分离物(sweet potato latent virus-lotus,SPLV-lotus)在江苏省莲藕产区普遍引起发病,并且该分离物序列与已报道的SPLV甘薯分离物存在较大差异。为进一步监测SPLV-lotus在莲藕上的发生情况,针对SPLV-lotus的CP基因设计并优化特异性引物QSPLV-lotus3-F/QSPLV-lotus3-R,通过优化引物浓度和退火温度条件,构建标准曲线,建立了SPLV-lotus实时荧光定量PCR(Real-time fluorescent quantitative polymerase chain reaction,RT-qPCR)检测技术。该方法特异性强,可以快速检测出SPLV-lotus,灵敏度为普通PCR的100倍,可广泛应用于脱毒莲藕SPLV-lotus的精确检测。

Development and Evaluation of a Real-time Fluorescent Quantitative PCR Assay for Detection of Sweet Potato Latent Virus-Lotus in Lotus Plants

During the survey of lotus viruses disease，sweet potato latent virus-lotus was found to occur at high frequencies in Jiangsu province, China. And there is a significant sequence difference between SPLV-lotus and sweet potato isolates. In order to detect SPLV more accurately，a specific RT-qPCR assay based on SYBR GreenⅠfluorescent dye was established. A specific primer pair QSPLV- lotus3-F/QSPLV-lotus3-R was designed based on the CP gene of SPLV-lotus，and a standard curve was constructed under the optimized primer concentration and annealing temperature. The results showed that the RT-qPCR assay had higher specificity and was 100 times more sensitive for SPLV detection than conventional RT-PCR. This method can be widely used in batch inspection of field samples and be suitable for the detection and identification of virus-free lotus root seedlings.