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Title: One-Step Multiplex RT-PCR for Simultaneous Detection of Four Pome Tree Viroids

item LIN, LIMING - Fujian Agricultural & Forestry University
item Li, Ruhui
item Mock, Raymond
item Kinard, Gary

Submitted to: European Journal of Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/25/2012
Publication Date: N/A
Citation: N/A

Interpretive Summary: Viroids are small segments of genetic code (RNA) that are not covered by a protein coat or shell. They can infect plants, including pome fruit trees such as apples and pears, and cause a wide range of diseases. If viroids are present in pome trees, it is often difficult to detect them before propagative cuttings are shipped to countries around the world thereby unknowingly spreading these disease causing agents. Here we report an improved method to test for and detect four pome tree viroids that is fast, reliable and inexpensive. A multiplex reverse transcription polymerase chain reaction (mRT-RCR) procedure was developed to detect the four viroids simultaneously. This procedure uses reagents and enzymes to amplify a portion of the genetic material of the viroids. All four viroids can be tested for in one reaction tube, which saves money on reagents, requires less time, and streamlines laborious and repetitive steps that can increase the chances for contamination. This test has the potential to be used routinely in programs that test fruit tree material for viroids, and ship plant material worldwide.

Technical Abstract: Apple scar skin viroid (ASSVd), Apple dimple fruit viroid (ADFVd), Apple fruit crinkle viroid (AFCVd), and Pear blister canker viroid (PBCVd) cause natural infections in pome (apple, pear, quince) fruit trees. These viroids are found worldwide and are important quarantine pathogens for the international movement of pome germplasm. A single-step multiplex reverse transcription polymerase chain reaction assay (mRT-PCR) was developed for the simultaneous detection of these viroids. Four pairs of primers specific for each of the four viroids were used to amplify PCR products of different sizes that can be resolved by agarose gel electrophoresis. Amplification of a plant 18S rRNA was included in the assay as an internal control. Amplicons of 371 bp (AFCVd), 270 bp (ADFVd), 186 bp (ASSVd), 120 bp (PBCVd), and 844 bp (18S rRNA) were obtained in both uniplex and multiplex RT-PCR assays. The identities of the amplification products were confirmed by sequencing. The specificities and limits of detection for all four viroids by uniplex and mRT-PCR assays were comparable. The assay was further validated using samples from pome trees inoculated with all four viroids, as well as field samples from commercial orchards in Colorado. All four viroids were detected from inoculated pear trees and up to three viroids were detected from inoculated apple trees. This is a simple, rapid and cost-effective technique to detect these four viroids in fruit trees. The procedure is especially applicable to certification and quarantine programs that test numerous samples for all four viroids.