Effect of RNA purification systems on the detection limit of pathogenic viruses in woody fruit crops

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Viruses are major pathogens of woody fruit crops. Control of viruses infecting woody fruit crops is achieved through certification of propagating materials free from certain viruses. The official methods of virus testing in these certification programs have been based on biological indicator indexing and serology-based assays. However, these assays lack sensitivity, specificity and are time and labor-consuming. Development and implementation of highly sensitive and reliable diagnostics are crucial for the control and management of viruses and viral diseases. Nucleic acid-based diagnostic methods are advantageous over traditional biological indexing and serology-based methods for their speed, sensitivity, and specificity and versatility. Isolation of highly pure viral RNAs is pre-requisite for molecular diagnosis. However, such a technology has not been developed for woody fruit crops. The main objective of this research is to develop highly effective methodologies for isolating RNA from grapes and peaches based on commercially available RNA purification methods and through further improvement of these technologies. In a partnership with Norgen BioTeck, a biotech company located in the centre of fruit crop production in Ontario, we intend to compare RNA purification systems from five commercial sources and make further refinement for RNA purification. Quality and performance of the purified RNAs will be tested through reverse transcriptase polymerase chain reaction (RT-PCR), real time RT-PCR, and deep sequencing. Outcomes of the proposed research will be beneficial to the sustainability and betterment of the agriculture industries in Ontario and other provinces of Canada. Moreover, it would help strengthen Norgen BioTek, a young, Canada-based biotech company, through unbiased and independent validation of their products and emergence of possible new products for use in research and diagnostics in Canada and the internationally. This is a new partnership between the applicant research program and Norgen, which we hope will lead to long-term and productive collaborations.