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

Cauliflower mosaic virus (CaMV) Biology, Management, and Relevance to GM Plant Detection for Sustainable Organic Agriculture

“…As a result of these farming trends and, in some countries, regulation of GMOs, international trade can necessitate quantification of the GMO content of a given product. Thus, numerous GMO detection methods are emerging (Kamle et al, 2017;Salisu et al, 2017;Umesha and Manukumar, 2018), but the potential for crop infection with CaMV can lead to false-positive results in the most commonly used GMO detection assays, due to the use of a CaMVderived P35S promoter in many GM constructs (Lipp et al, 1999;Becker and Ulrich, 2018;Lübeck, 2019). This potential for false-positive GMO detection is particularly relevant for organic farmers trying to meet regulatory requirements for non-GM plants in a sustainable international market.…”

“…Some CaMV strains (D4 and W260) are also able to infect Solanaceae species, such as devil's trumpets (genus Datura) and tobacco plants (genus Nicotiana) (Scholelz and Shepherd, 1988). CaMV genetic variants have been described in different host species with different symptoms, virulence, and transmission rates (Covey et al, 1990;Al-Kaff and Covey, 1994;Doumayrou et al, 2013;Yasaka et al, 2014), and recent studies have identified a high diversity of CaMV genomic sequences (Farzadfar et al, 2014;Gong and Han, 2017;Becker and Ulrich, 2018;Sukal et al, 2018).…”

“…Thus, most GMO detection methods are based on marker sequences for P35S and TNOS detected through polymerase chain reaction (PCR) or quantitative PCR (qPCR) (Holden et al, 2010;Wu et al, 2014;Fraiture et al, 2015). However, it is known that CaMV infection of non-GM plants can yield false-positive results in some GMO detection assays, due to the presence of the P35S region in both the CaMV genome and many GMO constructs (Figures 1A,B; Wolf et al, 2000;Chaouachi et al, 2008;Becker and Ulrich, 2018). Although a detailed consideration of A. tumefaciens infections is beyond the scope of this mini-review, A. tumefaciens is naturally found in the soil and can infect plants, so assays designed to detect TNOS can yield false-positive GMO results too, as described elsewhere (Wolf et al, 2000;Escobar and Dandekar, 2003;Yang et al, 2013;Nabi et al, 2016;Becker and Ulrich, 2018).…”

Multiplex quantitative PCR for single-reaction genetically modified (GM) plant detection and identification of false-positive GM plants linked to Cauliflower mosaic virus (CaMV) infection

“…Also, the efficacy of the P3 and P35S primers on divergent CaMV strains is to be determined. For example, a recent in silico test designed to evaluate the specificity of common CaMV primers to a wide diversity of CaMV strains detected 100% sequence homology for the P3 primers used here to 54% (forward) and 78% (reverse) of 96 tested CaMV strains, with the number and locations of SNPs variable among strains [10]. Although these P3 primers were expected to perform the best among the pre-existing primers in that study, primers that target a more diverse range of CaMV strains could be considered for future improvements to this assay, for example, newly designed CaMV ORFV primers with three degenerate base-pairs per primer to target all 96 of the tested strains [10].…”

“…Submitted). However, it is known that CaMV infection of non-GM plants can yield false-positive results from GM plant detection assays, due to the presence of the P35S region in both the CaMV genome and many GM crops [9, 10]. Therefore, a non-GM plant infected with CaMV could be incorrectly identified as a genetically modified organism (GMO).…”