POSTERS: Pathogen detection, quantification and diagnosis
Towards the development of a multiplexed qPCR assay to distinguish Cauliflower mosaic virus (CaMV) infection from GM plants
Joanne Emerson - University of California, Davis. Aurelie Bak- University of California, Davis
To facilitate transgene expression, many genetically modified (GM) plants contain a promoter from the plant virus, Cauliflower mosaic virus (CaMV). The relative ubiquity of this CaMV 35S promoter (P35S) in GM constructs means that assays designed to detect plant GMOs often target the P35S DNA sequence. However, because this promoter is derived from CaMV, these detection assays can yield false-positives from non-GM plants infected by these naturally-occurring viruses or their relatives within the Caulimoviridae. Here we report the development of an assay designed to distinguish CaMV-infected plants from GM plants in a single multiplexed qPCR reaction. With literature-derived PCR primers targeting P35S, TNOS (a terminator often used in GM constructs), and P3 (a CaMV-specific gene), we have successfully amplified target sequences in plasmids cloned to contain these genes, both in PCR and qPCR reactions. We have also successfully tested these PCR primers on plant DNA extracts from organic watercress and both organic and GM canola, all with and without CaMV infection. Four qPCR probes with different fluorescence wavelengths have been designed to target P35S, TNOS, P3, and actin (a positive-control plant gene). Results from multiplexing these probes with the aforementioned primers in single qPCR reactions containing template DNA from organic and GM plants with and without CaMV infection will be presented as a demonstration of the utility of this new assay.