Evaluation of Double-stranded (ds) RNA from Select Isolates of Rhizoctonia solani by Real-Time PCR

Hannah Cornman, Lauren Comunale, Mary Chey, Shan Min Chin, Chin Hong Siew

Abstract


The primary purpose of this research is to develop a fungal model system at the RNA level that will accurately and rapidly identify definitive signature patterns of viruses infecting the plant pathogenic fungi Rhizoctonia solani. Three different isolates of R. solani were selected for the study that represented a single phenotype that is targeted by three different viruses. The three isolates used for current analytic purposes were strains; 357, 303, and 386. All the three isolates of R. solani had M-size dsRNA fragments that were viral in origin and exhibited considerable sequence heterogeneity. The M-size dsRNA fragments were gel-purified and cloned into pGEM®-T Vector Systems or pJET 1.2 /blunt vector. White colonies were picked and a positive clone was sent for sequencing by Retrogen, Inc. The sequence was BLAST searched in the NCBI database. The final sequence was used to design the primer and probe through Primer 3 Input version 0.4.0 (http://frodo.wi.mit.edu/). The picked forward, reverse and probe primers sequences were sent to Biosearch Technologies (http://www.biosearchtech.com/) to synthesize both the forward, reverse primers and TaqMan Probe. The 5’ end of the TaqMan probe was labeled with fluorophores dye FAM and the 3’ end with quenched dye BHQ-1. Results from such an analysis using Real-Time PCR will be presented in various combinations of the dsRNA from the three isolates and the cloned fragments from each one of them. In order to optimize the assay, the working concentrations of primers with the concentration of probe will be tested. In addition to the primes and probe concentration, the annealing temperature and number of amplification cycles will be optimized to validate sensitivity of detection.

Keywords


Rhizoctonia solani, dsRNA, primers

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