Multiplex reverse transcription loop-mediated isothermal amplification for the simultaneous detection of CVB and CSVd in chrysanthemum.

A multiplex reverse transcription loop-mediated isothermal amplification (mRT-LAMP) assay was developed for the simultaneous detection of Chrysanthemum Virus B (CVB) and Chrysanthemum stunt viroid (CSVd), which are the major viral pathogens of chrysanthemum worldwide. Two sets of mRT-LAMP primers were designed for the coat protein gene of CVB and the complete nucleotide sequence of CSVd, and a restriction enzyme cleavage site was inserted into two pairs of species-specific primers. The mRT-LAMP assay was designed by combining these two sets for a total of eight primers. The mRT-LAMP method distinguished between CVB and CSVd due to the subsequent restriction enzyme analysis. The sensitivity of the mRT-LAMP method was 10(3) times higher than classical PCR regarding the detection limits for CVB and CSVd. No positive results were observed when RNA from other chrysanthemum pathogens were used as mRT-LAMP templates. The method was verified by testing chrysanthemum samples collected from Beijing and Henan Province and showed high reliability and sensitivity. The developed mRT-LAMP assay also offers an efficient, convenient, and rapid tool for screening chrysanthemum virus and viroid, especially CVB and CSVd, and can be diagnosed in a single reaction. These results suggest that the new mRT-LAMP method may be used routinely for virus and viroid surveys.

[Effect of medium components on callus induction and contents of polysaccharides in calli from Achyranthes bidentata].

OBJECTIVE: To study the effect of medium components on the callus induction and the contents of polysaccharides in calli from Achyranthes bidentata. METHOD: Leaves and stems were selected as explants. The effects of six kinds of factors including basal culture medium, carbon source, 2,4-D, 6-BA, TDZ, CH on the callus induction and the contents of ABPS in calli on the high growth point were studied by orthogonal design method. The data were analyzed with range analysis and variance analysis. RESULT: To leaf, the optimal medium of callus induction was B5 with 2 mg x L(-1) 2,4-D, 0.5 mg x L(-1) 6-BA, 30 g x L(-1) glucose and 1 g x L(-1) CH; to stem, the optimal one was B5 with2 mg x L(-1) 2,4-D, 1 mg x L(-1) 6-BA, 30 g x L(-1) glucose and 0.5 g x L(-1) CH. In order to obtain higher contents ABPS, to leaf calli, the optimal medium was LS with 1 mg x L(-1) 6-BA, 1 mg x L(-1) TDZ and 30 g x L(-1) sucrose; to stem calli, the optimal one was LS with 1 mg L(-1) 2,4-D, 0.5 mg x L(-1) 6-BA, 1 mg x L(-1) TDZ and 30 g x L(-1) glucose. CONCLUSION: The optimal media of callus induction were established with stems and leaves of A. bidentata as explants and with a view to an industrial production of polysaccharides by tissue and cell culture.