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.