Development of real-time polymerase chain reaction (qPCR) technique for quantitative detection of chrysanthemum chlorotic mottle viroid (CChMVd) and chrysanthemum stunt viroid (CSVd) in chrysanthemum
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Abstract
Chrysanthemum is a famous ornamental plant in the world, especially in Thailand. The production and propagation of chrysanthemum are achieved by vegetative propagation, which is a fast, easy, convenient method and many plantlets are obtained. However, this method is risk for viroid contamination. To disinfect viroid contamination, the meristem tip culture has been used for generating viroid-free plantlets. The real-time polymerase chain reaction (qPCR) which was developed to detect chrysanthemum chlorotic mottle viroid (CChMVd) and chrysanthemum stunt viroid (CSVd) from chrysanthemum plantlets, obtained from meristem tip culture for verification of viroid-free plantlets production, using new sets of primers showed that annealing at 50°C was suited for both CChMVd and CSVd detection. The standard curve analysis was illustrated and found that the lowest quantification cycle (Cq) values for detecting CChMVd and CSVd were 5.42 and 18.81, respectively. Moreover, the lowest copies number of CChMVd and CSVd which could be detected were 1 × 108 and 1 × 109 copies/µl, respectively. The qPCR techniques were applied to detect both viroids from chrysanthemum plantlets and revealed that all plantlets were free from viroid-contamination. Therefore, the qPCR with high sensitivity and specificity can be used as a main technique for detecting CChMVd and CSVd in chrysanthemum.
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