Improved detection and quantification of cauliflower mosaic virus in food crops: assessing false positives in GMO screening based on the 35S promoter

The 35S promoter (P35S) of the cauliflower mosaic virus (CaMV) is frequently used in screening tests as a first step in detecting and quantifying genetically modified organisms (GMOs) in food and feed samples. However, PCR reactions targeting P35S do not distinguish between GMOs and naturally occurring CaMV introduced by infection or contamination, resulting in false positives. Initial attempts at specific CaMV detection were based on a limited number of CAMV sequences. To account for the broad variability in the viral genome, we designed a new real-time PCR assay based on the genomic sequences of 96 CaMV isolates collected worldwide. This system amplified a 112 bp fragment of ORFV in the double-stranded genome, with complete sequence homology to the primer and probe targets of the tested genomes. Tests on a CaMV isolate collection and GMOs revealed improved specificity compared to previously published methods as well as high sensitivity, with limits of detection (LOD) and quantification (LOQ) of three copies of CaMV.