Real-Time PCR of cDNA of Tomato Mosaic Virus (ToMV) RNA

Kelsey Marie Hayden, Mary Chey

Abstract


A protocol for successful detection of ssRNA from infected leaf tissue always yielded high quality RNA.  In this project, a combination of degenerate primers was used to amplify part of the polymerase gene of the ToMV viral genome, followed by nested PCR that resulted in increased amplicon specificity and sensitivity of detection.  This project allowed overcoming problems of generic detection and identification of ToMV RNA by nested Real-Time PCR involving DI-containing primers. The Real-Time PCR of the cDNA generated by reverse transcription was done using DNA Master SYBR Green I from Roche in an Eppendorf RealPlex© Real-Time PCR Thermocycler.  The amplicon profile of the various treatments will be presented. Little or no amplification was detected in the negative controls.  The cT values were used as a measure to test the specificity of the primer design.  The non-specificity cT value was close to 27.5.  All Real-Time PCR results that had stable PCR products resulted in melting temperature ™ values of close to 84.5 0C.

Keywords


Tomato mosaic virus; epidemiology; PCR RNA

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