A Simple and Simultaneous Detection Method for Two Different Viroids Infecting Chrysanthemum by Multiplex Direct RT-PCR

Abstract

The economically important Chrysanthemum stunt viroid (CSVd) and Chrysanthemum chlorotic mottle viroid (CChMVd) were detected from infected chrysanthemum (Dendranthema grandiflorum) plants by a multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay developed in this investigation. The antisense hexamer AAAGGA (5′-3′) was designed. The antisense hexamer 5′AAAGGA 3′ annealing at 5′TCCTTT3′ is located at nucleotide positions 186–191 and 245–250 of CSVd (gb: AB006737), and 231–236 of CChMVd in their sequences. Using CSVd- and CChMVd-cDNA templates which were transcribed simultaneously by the hexamer, the following multiplex PCR could detect both viroids from doubly-infected plants without nonspecific amplification. When the hexamer was used for the RT reaction, the sensitivity of detection of CSVd or CChMVd by multiplex RT-PCR was similar to that of standard RT-PCR when each viroid was detected separately. Furthermore, multiplex RT-PCR successfully detected both CSVd and CChMVd in direct templates obtained by inserting a syringe needle into the stem, leaf, and shoot tips of infected chrysanthemum plants. The direct multiplex RT-PCR method developed in this study may reduce the cost, time, and labor required for the production of viroid-free chrysanthemum plants.