Detection of Avocado Sunblotch and Other Viroids Using RNA Filter Paper Capture and RT-PCR

Abstract

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.

Keywords: Avocado sampling; Avocado sunblotch viroid; RNA filter paper capture; RT-PCR; Viroid detection.