One-step multiplex RT-PCR for simultaneous detection of four viroids from hop (Humulus lupulus L.)
Hop (Humulus lupulus L.) plants are hosts to several viroids; some of them can be highly aggressive, and their infection can manifest as complete plant dieback. Since molecular detection of multiple viroids can be time consuming and cost inefficient, a reliable one-step multiplex RT-PCR (mRT-PCR) was developed to detect simultaneously all four viroids infecting hops: Hop latent viroid (HLVd), Hop stunt viroid (HSVd), Apple fruit crinkle viroid (AFCVd) and Citrus bark cracking viroid (CBCVd). Several primer pairs were tested on different viroid variants from hops, citruses and grapevines, and from among them, specific primer pairs for detection of hop viroids were selected and confirmed in a single-tube assay. To improve mRT-PCR reliability and validate its effectiveness, nad5 and DRH1 genes were included as an internal control. The specificities of single and mRT-PCR assays for all four viroids were comparable. The sensitivity of mRT-PCR was compared with that of dot-blot hybridization and single RT-PCR assay on biolistically infected hop plants. The results show mRT-PCR to be more sensitive than the dot-blot and slightly less sensitive than the single RT-PCR assay. Furthermore, mRT-PCR was validated using field samples and a group of 135 hop plants, which are used in the certification scheme for planting material propagation, and the method proved to be robust, rapid and simple. Additionally, this approach can be applicable to similar methods of systematic surveys of emerging diseases and epidemiological studies.
KeywordsHop Humulus lupulus RT-PCR Viroids
The authors acknowledge the financial support of the Slovenian Research Agency (36371, P4-0077), the Administration of the Republic of Slovenia for Food Safety, Veterinary and Plant Protection, the Czech Ministry of Education (LH14255), and the European Commission (FP7-REGPOT-2012-2013-1 MODBIOLIN No. 316304). The authors would like to express their gratitude to Dr. Luitgardis Seigner, Bavarian State Research Center for Agriculture, Freising, Germany for her careful and critical reading of the manuscript, and to Prof. Dr. Teruo Sano, Faculty of Agriculture and Life Science, Gene Research Center, Hirosaki University, for providing RNA sample of AFCVd.
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Conflict of interest
All authors declare that they have no potential sources of conflict of interest.
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