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

作　　者：连海燕[1] 李建光[2] 赵晓丽[2] 吕玉峰[2] 孙宁[3] 邓丛良[2] 许志春[1] 

机构地区：[1]北京林业大学,北京100083 [2]北京出入境检验检疫局,北京101312 [3]东南大学生物科学与医学工程学院,南京210096

出　　处：《植物病理学报》2015年第3期288-296,共9页Acta Phytopathologica Sinica

基　　金：公益性行业(质检)科研专项(201110035);国家973课题(2011CB932800);质检总局课题(2013IK267)

摘　　要：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.

关 键 词：类病毒 提取方法 实时荧光定量RT-PCR 

分 类 号：S432[农业科学—植物病理学]