南方水稻黑条矮缩病毒一步双重RT-PCR检测技术及其应用  

Detection of Southern rice black-streaked dwarf virus using one-step dual RT-PCR

作　　者：王强[1] 周国辉[1] 张曙光[1] 

机构地区：[1]华南农业大学资源环境学院,广州510642

出　　处：《植物病理学报》2012年第1期84-87,共4页Acta Phytopathologica Sinica

基　　金：广东省科技攻关项目(2008B020100004);公益性行业(农业)科研专项(201003031)

摘　　要：南方水稻黑条矮缩病毒（Southern rice blackstreaked dwarf virus，SRBSDV）是呼肠孤病毒科（Reoviridae）斐济病毒属（Fijivirus）的一个建议新种，自然条件下。An one-step dual RT-PCR method was developed for Southern rice black-streaked dwarf virus （SRBSDV） detection from host plants and insect vector white-backed planthopper （Sogatella furcifera Horvath, Hemiptera： Delphacidae）, from which two cDNA fragments of the viral genome S5 and S10 were amplified simultaneously. Two primer pairs, S5-F1/S5-R2 （ 5＇-ttacaactggagaagcattaacacg-3＇/5＇-atgaggtattgcgtaactgagcc -3＇ ） and S10-oF/S10-oR （ 5＇-cgcgtcatctcaaactacag -3 ＇/5 ＇-tttgtcagcatctaaagcgc -3＇ ）, were selected from 40 primer pairs based on SRBSDV genome sequences, and amplified viral $5 ORF1 fragment （819 bp） and cp gene fragment （ 682 bp）, respectively. Using total RNA extracts from infected plant leaf tissue or in- dividual planthopper adult as templates, one step RT-PCR reaction in the conditions of annealing temperature of 53℃ with the final concentrations of 240 nmol/L S5-FI/S5-R2 and 120 nmol/L S10-oF/S10-oR generated a similar yield of two amplicons in argrose electrophoresis analysis. Sequence analysis confirmed the correct result of the amplification. Additionally, several commercal RNA extraction kits was proven to be fit for the template preparation, and the protocol for total RNA extraction from plant leaf tissue and individual planthopper was simplified by sample grinding direct in eppendorf tube. This method can be used for SRBSDV detection of dried or 70% alcochol soaked planthopper samples stored over one month in room temprature.

关 键 词：水稻黑条矮缩病毒 检测技术 南方 PCR 应用 RT 呼肠孤病毒科 VIRUS 

分 类 号：S435.111.4[农业科学—农业昆虫与害虫防治]