Simultaneous and sensitive detection of CVB, CChMVd and CSVd mixed infections in chrysanthemum using multiplex nested RT-PCR

Abstract

Chrysanthemum plants are susceptible to infect by viral and viroid diseases. Early detection of these pathogens helps to prevent disease spread. Multiplex RT-PCR can simultaneously detect many virus and viroid in a single reaction, and applying multiplex nested PCR can improve the sensitivity of the detection. Chrysanthemum leaves were collected from cultivation areas in Northern Thailand. Multiplex nested RT-PCR using specific outer and inner primer pairs for detection of Chrysanthemum virus B (CVB), Chrysanthemum chlorotic mottle viroid (CChMVd), and Chrysanthemum stunt viroid (CSVd) were used for simultaneous detection. Among 15 randomly selected samples, one sample showed co-infections of CVB and CChMVd, and two samples showed multiple infections of all pathogens. Furthermore, multiplex nested RT-PCR detected amplicons of CChMVd and CSVd that were not detected by the first-round PCR. Also, sequence analysis was used to confirm the correction of amplified fragments and revealed that all amplicons were more than 93% identical compared to corresponding sequences deposited in the GenBank. Therefore, multiplex nested RT-PCR can be used for routine detection and diagnosis of virus and viroid diseases. This is the first detection of mixed infections (disease complex) both virus and viroid in the chrysanthemum of Thailand.