Development of a New Gene Diagnosis Method for Plant Viruses and Viroids

植物病毒和类病毒基因诊断新方法的开发

• 批准号: 08660046
• 负责人: SANO Teruo
• 金额: $ 1.66万
• 依托单位: Hirosaki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08660046/
• 关键词: gene diagnosis; virus; microplate hybridization; viroid

1 : To setablish nucleic acid hybridization assay protocol that is carried in mult-well microplates, we have examined several factors affecting the sensitivity. Nucleic acids (mainly RNA) were dfficiently adsorbed on polystirene multi-well microplate by denaturing in a solution containing 50% formamide, 6.125% formaldehyde, 1X SSC,for 10 min at 68C.Hybridization procedure was greatly simplified comparing to the traditional membrane hybridization by eliminating several steps that are essential for membrane hybridization, that is, a part of washing, RNase A treatment and pre-hybridization steps.Finally by using digoxigenine (DIG)-labeled cRNA and p-nitrophenylphosphate for the probe and substrate, we have established a simple and sensitive microplate hybridization (MPH) that is sensitive enough to detect picograms of virus and viroid RNAs.2 : We have further examined the sensitivity of the MPH method by comparing to the traditional serological diagnosis method, such as ELISA.In case of hop latent viruse, MPH was 40 to 100 times more sensitive that ELISA.3 : We have developed MPH probes specific for ten viroids (HSVd, CEVd, ASSVd, PBCVd, PSTVd, HLVd, CbVd1, GYSVd, CSVd and CVdIII) and eight viruses (CMV,CMV-satellite RNA,HLV,TuMV,WCMV,MNSV,PVY and HMV).

1.为了建立适用于多孔微孔板的核酸杂交检测方法，我们考察了影响其灵敏度的几个因素。在含有50%甲酰胺、6.125%甲醛、1×SSC的溶液中，68℃变性10min，使核酸(主要是核糖核酸)有效地吸附在聚苯乙烯多孔微板上。与传统的膜杂交相比，杂交过程大大简化，省去了膜杂交中必不可少的几个步骤，即洗涤、核糖核酸酶A处理和杂交前步骤。最后以地高辛标记的cRNA和对硝基苯磷酸为探针和底物，我们建立了一种简单、灵敏的微孔板杂交(MPH)方法，它足以检测病毒和类病毒的皮克图。2.通过与传统的血清学诊断方法(如ELISA)的比较，进一步检验了MPH方法的敏感性。对于HOP潜伏病毒，MPH的灵敏度是ELISA的40-100倍。3：我们开发了针对10种类病毒(HSVd、CEVd、ASSVd、PBCVd、PSTVd、HLVd、CbVd1、GYSVd、CSVd和CVdIII)和8种病毒(CMV、CMV卫星RNA、HLV、TuMV、WCMV、MNSV、PVY和HMV)的MPH探针。

项目成果

Satoshi, Sugiyama and Teruo, Sano: "The detection of cucumber mosaic virus satellite RNA from melon by microplate hybridization" Ann.Rept.Plant Prot.North.Japan. Vol.48. 99-100 (1997)

Satoshi, Sugiyama 和 Teruo, Sano：“通过微板杂交从甜瓜中检测黄瓜花叶病毒卫星 RNA”Ann.Rept.Plant Prot.North.Japan。

Teruo Sano&A.Ishiguro: "A simple and sensitive non-radioactive microplate hybridization for the detection and quantification of picograms of viroid and viral RNA" Arch.Phytopath.Pflanz.30. 303-312 (1996)

佐野辉夫

Teruo Sano: "A simple and sensitive non-radioactive microplate hybridization for the detection and quantification of picograms of viroid and viral RNA" Arch.Phytopath pflanz.30巻. 303-312 (1996)

Teruo Sano：“一种简单而灵敏的非放射性微孔板杂交，用于检测和定量皮克类病毒和病毒 RNA”Arch.Phytopath pflanz.Volume 30. 303-312 (1996)

Teruo, Sano & Akira, Ishiguro: "A simple and sensitive non-radioactive microplate hybridization for the detection and quantification of picograms of viroid and viral RNA" Arch.Phytopath pflanz.Vol.30. 303-312 (1996)

佐野辉雄

Teruo, Sano and Akira, Ishiguro: "Viability and pathogenicity of intersubgroup viroid chimera suggest possible involvement of the terminal right region in replication" Virology. Vol.240. 238-244 (1998)

Teruo, Sano 和 Akira, Ishiguro：“亚群间类病毒嵌合体的活力和致病性表明末端右侧区域可能参与复制”病毒学。