Bar-Joseph M, Rosner A, Moscovitz M, Hull R
J Virol Methods. 1983 Jan;6(1):1-8. doi: 10.1016/0166-0934(83)90062-9.
A simple procedure for the isolation of double-stranded (ds) RNA from virus-infected plants is described. The method is based on grinding plant tissue in 4% p-aminosalicylic acid and recovery of ds RNA by phenol extraction and precipitation with 30% ethanol. The presence of both negative and positive virus RNA strands in RNA fractionated in agarose gels was verified by Northern blot hybridization with polynucleotide kinase labelled genomic RNA or complementary DNA (cDNA) probes. The procedure enabled detection of three major ds RNA species (MWs 4.2, 1.05 and 0.48 X 10(6)) and at least 4 minor bands with estimated MWs of 3.5, 2.5, 2.2 and 2.0 X 10(6) in Nicotiana tabacum plants systemically infected with tobacco mosaic virus (TMV). Cucumber mosaic virus (CMV)-infected Pachystachys coccinea plants contained 2 minor bands of MWs 0.49 and 0.35 X 10(6) in addition to the previously described 4 major ds RNAs and ds CARNA 5 (MW 0.22 X 10(6)). The patterns of ds RNA are useful for diagnosing natural infections of CMV and TMV in N. glauca plants and of citrus tristeza virus in Citrus spp.
本文描述了一种从病毒感染植物中分离双链（ds）RNA的简单方法。该方法基于在4%对氨基水杨酸中研磨植物组织，并通过苯酚提取和30%乙醇沉淀回收dsRNA。通过用多核苷酸激酶标记的基因组RNA或互补DNA（cDNA）探针进行Northern印迹杂交，验证了在琼脂糖凝胶中分级分离的RNA中病毒正负链RNA的存在。该方法能够检测到在系统感染烟草花叶病毒（TMV）的烟草植株中存在三种主要的dsRNA种类（分子量分别为4.2、1.05和0.48×10⁶）以及至少4条分子量估计为3.5、2.5、2.2和2.0×10⁶的次要条带。除了先前描述的4种主要dsRNA和ds CARNA 5（分子量0.22×10⁶）外，感染黄瓜花叶病毒（CMV）的朱缨花植株还含有分子量为0.49和0.35×10⁶的2条次要条带。dsRNA模式可用于诊断格劳卡烟草植株中CMV和TMV的自然感染以及柑橘属植物中柑橘衰退病毒的感染。
