Detection of Avocado Sunblotch and Other Viroids Using RNA Filter Paper Capture and RT-PCR. 2022

Deborah M Mathews, and Sohrab Bodaghi, and James A Heick, and J Allan Dodds
Department of Microbiology and Plant Pathology, University of California, Riverside, CA, USA. dmathews@ucr.edu.

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.

UI MeSH Term Description Entries
D010936 Plant Extracts Concentrated pharmaceutical preparations of plants obtained by removing active constituents with a suitable solvent, which is evaporated away, and adjusting the residue to a prescribed standard. Herbal Medicines,Plant Extract,Extract, Plant,Extracts, Plant,Medicines, Herbal
D010942 Plant Viruses Viruses parasitic on plants. Phytophagineae,Plant Virus,Virus, Plant,Viruses, Plant
D012367 RNA, Viral Ribonucleic acid that makes up the genetic material of viruses. Viral RNA
D014772 Viroids A group of pathogens comprising the smallest known agents of infectious disease. They are unencapsulated and are capable of replicating autonomously in susceptible cells. Positively identified viroids composed of single-stranded RNA have been isolated from higher plants, but the existence of DNA viroids pathogenic to animals is suspected. Viroid
D020133 Reverse Transcriptase Polymerase Chain Reaction A variation of the PCR technique in which cDNA is made from RNA via reverse transcription. The resultant cDNA is then amplified using standard PCR protocols. Polymerase Chain Reaction, Reverse Transcriptase,Reverse Transcriptase PCR,PCR, Reverse Transcriptase,Transcriptase PCR, Reverse
D027421 Persea A plant genus in the LAURACEAE family. The tree, Persea americana Mill., is known for the Avocado fruit, the food of commerce. Avocado,Persea americana,Avocados,Persea americanas,Perseas,americana, Persea

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