Real-time polymerase chain reaction detection of cauliflower mosaic virus to complement the 35S screening assay for genetically modified organisms.
Labeling of genetically modified organisms (GMOs) is now in place in many countries, including the European Union, in order to guarantee the consumer's choice between GM and non-GM products. Screening of samples is performed by polymerase chain reaction (PCR) amplification of regulatory sequences frequently introduced into genetically modified plants. Primers for the 35S promoter from Cauliflower mosaic virus (CaMV) are those most frequently used. In virus-infected plants or in samples contaminated with plant material carrying the virus, false-positive results can consequently occur. A system for real-time PCR using a TaqMan minor groove binder probe was designed that allows recognition of virus coat protein in the sample, thus allowing differentiation between transgenic and virus-infected samples. We measured the efficiency of PCR amplification, limits of detection and quantification, range of linearity, and repeatability of the assay in order to assess the applicability of the assay for routine analysis. The specificity of the detection system was tested on various virus isolates and plant species. All 8 CaMV isolates were successfully amplified using the designed system. No cross-reactivity was detected with DNA from 3 isolates of the closely related Carnation etched ring virus. Primers do not amplify plant DNA from available genetically modified maize and soybean lines or from different species of Brassicaceae or Solanaceae that are natural hosts for CaMV. We evaluated the assay for different food matrixes by spiking CaMV DNA into DNA from food samples and have successfully amplified CaMV from all samples. The assay was tested on rapeseed samples from routine GMO testing that were positive in the 35S screening assay, and the presence of the virus was confirmed.
National Institute of Biology, Department of Plant Physiology and Biotechnology, Vecna pot 111, 1000 Ljubljana, Slovenia. katja.cankar@nib.si
Katarina
K
Cankar
Maja
M
Ravnikar
Jana
J
Zel
Kristina
K
Gruden
Natasa
N
Toplak
D001483:Base Sequence; D017796:Caulimovirus / Q000235:genetics*; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004279:DNA, Viral; D005504:Food Analysis; D030841:Food, Genetically Modified; D016679:Genome, Viral; D008854:Microscopy, Electron; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D030781:Organisms, Genetically Modified / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013025:Glycine max / Q000378:metabolism; D013045:Species Specificity; D019076:Transgenes; D003313:Zea mays / Q000378:metabolism
D017931:DNA Primers; D018744:DNA, Plant; D004279:DNA, Viral; D004247:DNA