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.

A Current Overview of Two Viroids That Infect Chrysanthemums: Chrysanthemum stunt viroid and Chrysanthemum chlorotic mottle viroid

“…Also, a one-tube/one-step RT-PCR method was developed to detect seven viroids, including CSVd, belonging to four genera [73]. In addition, a multiplex, direct RT-PCR method was developed to detect CSVd and CChMVd in a small amount of plant tissues [74,75]. Furthermore, a previous study developed real-time PCR (TaqMan ® ) assays for detecting six viroids, including CSVd, infecting solanaceous hosts [44].…”

Development of multiplex PCR markers to distinguish evergreen and deciduous azaleas

“…This method is often used in applications requiring simple analyses, such as genotypic identification of the genes related to flower pigments in petunia (Matsubara et al. 2006) and detection of infecting viroids (Hosokawa et al. 2007).…”

“…Multiplex PCR is a rapid and cost-effective PCR-based marker analysis because sequence variation at more than one locus can be determined with one PCR reaction. This method is often used in applications requiring simple analyses, such as genotypic identification of the genes related to flower pigments in petunia (Matsubara et al 2006) and detection of infecting viroids (Hosokawa et al 2007). In this study, we compared nrDNA and cpDNA sequences between evergreen and deciduous azaleas.…”

Simultaneous Detection of Three Viroid Species Infecting Hops in China by Multiplex RT‐PCR

“…Thus, reliable protocols that can be used for quick identification of diseased plants from hop yards or cultured materials need to be established. Although multiplex RT-PCR, a method for detecting simultaneous infections by more than one viroid species in the same host, has been reported for apple (Di Serio et al 2002), citrus (Ito et al 2002;Wang et al 2009), chrysanthemum (Hosokawa et al 2007), tomato (Matsushita et al 2010) and grapevine (Hajizadeh et al 2012), no protocols have been developed for detecting concurrent viroid infections in hop. Therefore, we developed a multiplex RT-PCR for the simultaneous detection of the three viroid species known to naturally infect hops in China.…”