A simple and simultaneous detection method for two different viroids infecting Chrysanthemum by multiplex direct RT-PCR

Munetaka Hosokawa, Hayato Shiba, Takashi Kawabe, Akiko Nakashima, Susumu Yazawa

Research output: Contribution to journalArticlepeer-review

14 Citations (Scopus)

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. JSHS

Original languageEnglish
Pages (from-to)60-65
Number of pages6
JournalJournal of the Japanese Society for Horticultural Science
Volume76
Issue number1
DOIs
Publication statusPublished - 2007 Jan

Keywords

  • Chrysanthemum chlorotic mottle viroid (CChMVd)
  • Chrysanthemum stunt viroid (CSVd)
  • Direct PCR
  • Hexamer
  • Multiplex RT-PCR

ASJC Scopus subject areas

  • Horticulture

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