Department of Microbiology and Plant Pathology, University of California, Riverside, CA, USA.
Methods Mol Biol. 2022;2316:219-233. doi: 10.1007/978-1-0716-1464-8_19.
This method originated due to the need to quickly and sensitively detect Avocado sunblotch viroid (ASBVd) in nursery and field trees in California. Optimum sampling protocols were developed for leaf collection from different sized trees based on size and branching as well as for fruit. An ethanol containing buffered extract from 1 g of ground leaf tissue was used as the source of RNA. The extract was absorbed onto small pieces (disks) of Whatman No. 1 filter paper which were then washed and dried. RNA was eluted from the filter paper using sterile water and used as a template in a standard single-tube RT-PCR reaction. The RNA adsorbed on the filter paper disks was quite stable, and the disks could be stored for over 1 year and shipped worldwide at ambient temperature with no noticeable decline in the quality or quantity of the resulting RT-PCR products. The filter paper capture method was expanded to the detection of other viroids including Potato spindle tuber viroid, Peach latent mosaic viroid, and Chrysanthemum stunt viroid and was tested with some viruses as well with minor modifications of the standard protocol.
这种方法源于在加利福尼亚州的苗圃和田间树木中快速、灵敏地检测鳄梨日斑类病毒（ASBVd）的需要。根据大小和分枝情况，为不同大小的树木采集叶片以及果实制定了最佳的采样方案。将 1 克研磨后的叶片组织的含乙醇缓冲提取物用作 RNA 的来源。将提取物吸附到沃特曼 No.1 滤纸上的小块（圆盘）上，然后进行洗涤和干燥。使用无菌水从滤纸上洗脱 RNA，并将其用作标准单管 RT-PCR 反应中的模板。吸附在滤纸上的 RNA 圆盘非常稳定，圆盘可以储存超过 1 年，并在环境温度下运往世界各地，不会导致 RT-PCR 产物的质量或数量明显下降。滤纸捕获方法已扩展到其他类病毒的检测，包括马铃薯纺锤块茎类病毒、桃潜隐花叶类病毒和菊花矮化类病毒，并对一些病毒进行了检测，对标准方案进行了一些修改。
