甘薯病毒多重RT-PCR检测方法的建立

Establishment of Multiplex RT-PCR Detection Method of Sweet Potato Virus

为了快速进行甘薯病毒田间检测和脱毒苗诊断,该研究建立了一个能够同时检测SPCSV、SPLV和SPFMV这3种病毒的多重RT-PCR检测方法。根据GenBank公示的甘薯病毒基因序列来设计3种甘薯病毒的引物,分别对模板浓度、退火温度、Mg2+浓度和循环次数进行优化,同时对模板浓度进行10倍梯度稀释,检测多重RT-PCR灵敏度。结果显示,模板浓度为19 ng/μL、退火温度为52℃、Mg2+浓度为0 mmol/L、循环次数为40次时,可以同时扩增出大小为276 bp、570 bp、425 bp三种病毒的基因片段,多重RT-PCR检测灵敏度为10-4,单一RT-PCR检测灵敏度为10-3,多重RT-PCR检测灵敏度是单一RT-PCR的10倍。该研究使用优化的多重RT-PCR检测技术可获得稳定、灵敏、准确、高效地和同时地检测田间复合侵染的3种甘薯病毒。

In order to rapidly detect sweet potato virus and diagnose the virus-free seedlings in the field,this research established a multi-RT-PCR detection method which could simultaneously detect the three viruses including SPCSV,SPLV and SPFMV.Three pairs of primers for three sweet potato virues were designed according to the gene sequences in GenBank database.The template concentration,annealing temperature,Mg2+concentration and cycle number of PCR conditions were optimized,respectively,as well as,the sensitivity of multiple RT-PCR was measured by 10 times gradient dilution of the template concentration.The results showed that the PCR conditions with 19 ng/μL of template concentration,52℃of annealing temperature,0 mmol/L of Mg2+concentration,and 40 of PCR cycles could be amplificated the virus genes with the lengths of 276 bp,570 bp and 425 bp at the same time;Multiple RT-PCR detection sensitivity is 10-4,whereas the single RT-PCR detection sensitivity is 10-3,and the detection sensitivity of multiple RT-PCR is 10 times more than that of single RT-PCR.In this study,the multivariate RT-PCR detection technique could be used to detect the three kinds of sweet potato virus simultaneously at the stability,sensitivity,accuracy and high efficiency level.