Laboratorio de Virología de Hongos, Departamento de Biología, Facultad de Química y Biología, Universidad de Santiago de Chile, Avenida Libertador Bernardo O'Higgins 3363, Estación Central, Santiago, Chile.
Virol J. 2011 Jan 25;8:38. doi: 10.1186/1743-422X-8-38.
In most of the infected fungi, the mycoviruses are latent or cryptic, the infected fungus does not show disease symptoms, and it is phenotypically identical to a non-infected strain of the same species. Because of these properties, the initial stage in the search for fungi infected with mycoviruses is the detection of their viral genome, which in most of the described cases corresponds to double-stranded RNA (dsRNA). So to analyze a large number of fungal isolates it is necessary to have a simple and rapid method to detect dsRNA.
A rapid method to isolate dsRNA from a virus-infected filamentous fungus, Botrytis cinerea, and from a killer strain of Saccharomyces cerevisiae using commercial minicolumns packed with CF11 cellulose was developed. In addition to being a rapid method, it allows to use small quantities of yeasts or mycelium as starting material, being obtained sufficient dsRNA quantity that can later be analyzed by agarose gel electrophoresis, treated with enzymes for its partial characterization, amplified by RT-PCR and cloned in appropriate vectors for further sequencing.
The method yields high quality dsRNA, free from DNA and ssRNA. The use of nucleases to degrade the DNA or the ssRNA is not required, and it can be used to isolate dsRNA from any type of fungi or any biological sample that contains dsRNA.
在大多数感染真菌的病毒中，真菌病毒是潜伏或隐匿的，感染真菌不会表现出疾病症状，并且与同一物种的未感染菌株表型完全相同。由于这些特性，在寻找感染真菌病毒的初期阶段是检测其病毒基因组，在大多数描述的情况下，该基因组对应于双链 RNA（dsRNA）。因此，为了分析大量的真菌分离物，有必要有一种简单而快速的方法来检测 dsRNA。
开发了一种从感染丝状真菌 Botrytis cinerea 和酿酒酵母杀伤菌株的病毒中分离 dsRNA 的快速方法，使用了装有 CF11 纤维素的商业微型柱。除了是一种快速方法外，它还允许使用少量酵母或菌丝体作为起始材料，获得足够数量的 dsRNA，然后可以通过琼脂糖凝胶电泳进行分析，用酶进行部分特性分析，通过 RT-PCR 扩增并克隆到适当的载体中进行进一步测序。
该方法产生高质量的 dsRNA，无 DNA 和 ssRNA。不需要使用核酸酶降解 DNA 或 ssRNA，并且可以用于从任何类型的真菌或任何含有 dsRNA 的生物样品中分离 dsRNA。
