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

Abstract

BackgroundMost genetically modified (GM) plants contain a promoter, P35S, from the plant virus, Cauliflower mosaic virus (CaMV), and many have a terminator, TNOS, derived from the bacterium, Agrobacterium tumefaciens. Assays designed to detect GM plants often target the P35S and/or TNOS DNA sequences. However, because the P35S promoter is derived from CaMV, these detection assays can yield false-positives from non-GM plants infected by this naturally-occurring virus.ResultsHere we report the development of an assay designed to distinguish CaMV-infected plants from GM plants in a single multiplexed quantitative PCR (qPCR) reaction. Following initial testing and optimization via PCR and singleplex-to-multiplex qPCR on both plasmid and plant DNA, TaqMan qPCR probes with different fluorescence wavelengths were designed to target actin (a positive-control plant gene), P35S, P3 (a CaMV-specific gene), and TNOS. We tested the specificity of our quadruplex qPCR assay using different DNA extracts from organic watercress and both organic and GM canola, all with and without CaMV infection, and by using commercial and industrial samples. The limit of detection (LOD) of each target was determined to be 1% for actin, 0.001% for P35S, and 0.01% for both P3 and TNOS.ConclusionsThis assay was able to distinguish CaMV-infected plants from GM plants in a single multiplexed qPCR reaction for all samples tested in this study, suggesting that this protocol is broadly applicable and readily transferrable to any interested parties with a qPCR platform.

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

“…To distinguish among markers in the same reaction, a high level of multiplexing can be achieved using qPCR with different fluorophores for each target; depending on the instrument, up to five distinguishable targets have been successfully amplified in a single multiplexed qPCR reaction (Reller et al, 2013;Datukishvili et al, 2015). For example, we recently developed a multiplex qPCR assay that can distinguish CaMV infection from GM plants containing P35S in a single reaction, based on detection of four different targets: P35S, CaMV gene III, TNOS, and actin (a universal plant gene used as a positive control for the assay) (Bak and Emerson, 2019).…”

A multiplex PCR marker distinguishes between a series of four LanFTc1 alleles regulating flowering time in narrow‐leafed lupin (Lupinus angustifolius)

“…Multiplex PCR was first conceived as an efficient method for diagnosing a common sex-linked genetic disorder known as Duchenne muscular dystrophy (DMD) (Chamberlain et al, 1988). More than 30 years later, it remains a fundamental tool for rapidly and efficiently diagnos- the benefits derived from this approach has seen multiplex PCR become widely adopted within the agricultural industry for highthroughput genotyping in a variety of applications, such as germplasm characterization and MAS (Cockram et al, 2009;Yap et al, 2016), identification of genetically modified organisms (Bak & Emerson, 2019) and pathology testing (Otti et al, 2016;Viswanathan et al, 2010). The new multiplex PCR marker overcomes these critical limitations through the use of multiple, carefully positioned primers targeting individual alleles (Figure 1).…”

Detection and identification of authorized and unauthorized GMOs using high-throughput sequencing with the support of a sequence-based GMO database

“…The described qPCR-based system has a number of flaws, especially concerning the unauthorized GMOs. Even when an unexplained positive signal is observed during the qPCR screening, it does not unambiguously indicate the presence of an unauthorized event because this signal can also be caused by the presence of the naturally occurring organism in the sample ( Bak and Emerson, 2019 , Broeders et al, 2012 , Holst-Jensen et al, 2012 ). Furthermore, the presence of an unauthorized event can be concealed by the presence of authorized events harboring the same transgenic elements in the mixture.…”