啤酒花潜隐类病毒RNA提取方法及检测技术的研究

Research on RNA extraction and detection based on MNP-RT-qPCR assay of Hop latent viroid

本研究采用纳米磁珠提取法(Magnetic Nanoparticles,MNP)、改良的Li Cl沉淀法、CTAB法、TRIzol法和RNeasy Plant M ini Kit 5种方法提取感染啤酒花潜隐类病毒(Hop latent viroid,HLVd)的啤酒花叶片总RNA,结果显示Li Cl沉淀法、CTAB法和M NP法提取的RNA的质量较好,而M NP法具有提取时间短、操作简便,环境友好和批量提取的优势,适用于啤酒花潜隐类病毒RNA的快速提取。体外转录制备HLVd RNA标准品,利用实时荧光定量RT-PCR绘制标准曲线,并对其特异性、灵敏度进行评估。应用纳米磁珠提取啤酒花总RNA,结合实时荧光RT-PCR技术,建立了HLVd的快速、高效的M NP-RT-q PCR定量检测方法。

In this study,total RNA of hop leaves infected by Hop latent viroid（ HLVd） w as extracted by M agnetic Nanoparticles（ M NP）,Li Cl precipitation,CTAB,TRIzol methods and RNeasy Plant M ini Kit. The comparison result indicated that the RNA extracted by M NP,Li Cl and CTAB had high quality. And the M NP method had the advantages of short extraction time,simple operation,friendly environment and batch extraction. The M NP method w as more suitable for rapid extraction of HLVd RNA. The RNA standard of HLVd w as prepared by transcription in vitro and the standard curve w as plotted. The specificity and sensitivity of the developed method for HLVd detection w ere determined. A rapid,sensitive and specific M NP-RT-q PCR assay for detection of HLVd w as established.