European Journal of Plant Pathology

, Volume 133, Issue 3, pp 765–772 | Cite as

One-step multiplex RT-PCR for simultaneous detection of four pome tree viroids



Apple scar skin viroid (ASSVd), Apple dimple fruit viroid (ADFVd), Apple fruit crinkle viroid (AFCVd), and Pear blister canker viroid (PBCVd) infect pome fruit trees. These viroids 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 mRT-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, where numerous samples must be tested for all four viroids.


Apscaviroid Apple dimple fruit viroid Apple fruit crinkle viroid Apple scar skin viroid Multiplex RT-PCR Pear blister canker viroid 



The authors would like to thank Mr. Sam Grinstead for maintenance of trees in the screenhouse and Dr. Ramesh Pokharel (Colorado State University) for pome samples from Colorado orchards.


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Copyright information

© KNPV 2012

Authors and Affiliations

  1. 1.National Germplasm Resources LaboratoryUSDA-ARSBeltsvilleUSA
  2. 2.Institute of Plant VirologyFujian Agriculture and Forestry UniversityFujianChina

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