Development of a qualitative, multiplex real-time PCR kit for screening of genetically modified organisms (GMOs)

Abstract

The number of commercially available genetically modified organisms (GMOs) and therefore the diversity of possible target sequences for molecular detection techniques are constantly increasing. As a result, GMO laboratories and the food production industry currently are forced to apply many different methods to reliably test raw material and complex processed food products. Screening methods have become more and more relevant to minimize the analytical effort and to make a preselection for further analysis (e.g., specific identification or quantification of the GMO). A multiplex real-time PCR kit was developed to detect the 35S promoter of the cauliflower mosaic virus, the terminator of the nopaline synthase gene of Agrobacterium tumefaciens, the 35S promoter from the figwort mosaic virus, and the bar gene of the soil bacterium Streptomyces hygroscopicus as the most widely used sequences in GMOs. The kit contains a second assay for the detection of plant-derived DNA to control the quality of the often processed and refined sample material. Additionally, the plant-specific assay comprises a homologous internal amplification control for inhibition control. The determined limits of detection for the five assays were 10 target copies/reaction. No amplification products were observed with DNAs of 26 bacterial species, 25 yeasts, 13 molds, and 41 not genetically modified plants. The specificity of the assays was further demonstrated to be 100% by the specific amplification of DNA derived from reference material from 22 genetically modified crops. The applicability of the kit in routine laboratory use was verified by testing of 50 spiked and unspiked food products. The herein described kit represents a simple and sensitive GMO screening method for the reliable detection of multiple GMO-specific target sequences in a multiplex real-time PCR reaction.

Four new SYBR®Green qPCR screening methods for the detection of Roundup Ready®, LibertyLink®, and CryIAb traits in genetically modified products

“…A number of novel approaches have been developed applying the ''Polymerase Chain Reaction'' (PCR) technology [7]. Several multiplex qPCR formats are available reducing the number of analyses and facilitating high throughput but requiring multi-channel detection devices and often including costly detection probes for at least some of the targets [8][9][10]. In other cases, the applied chemistries are quite complex or are to be combined with other technologies that are less appropriate for routine applications [11][12][13][14][15][16].…”

Multiplex PCR assays for qualitative detection and identification of the GT73, Ms8, Rf3 and T45 varieties of genetically modified oilseed rape

“…The first category comprises screening tests targeting for DNA sequences widely used in the construction of various GM crops. The target sequence includes promoters (e.g., 35S promoter of the cauliflower mosaic virus -CaMV 35S promoter) or terminators (e.g., terminator of the cauliflower mosaic virus -CaMV T-35S and terminator of the nopaline synthase gene -T-NOS), which allow proper expression of the introduced transgene as well as selection markers commonly present in vectors used for genetic modification (nptII -neo-mycin phosphotransferase II; bla -β-lactamase) (Marmiroli et al, 2008;Dörries et al, 2010). The positive amplification of one of these elements does not always indicate the presence of GMO, because they naturally occur in some viruses, bacteria and plants; therefore, this method is associated with a particular risk of false positive results (Holst-Jensen et al, 2012).…”

“…Finally, to overcome this problem, event-specific assays have been developed, which are the most specific reactions for identification of GMOs. They target a unique site comprising a junction between the transgenic insert and the host genome (Holst-Jensen et al, 2003;Yang et al, 2006;Marmiroli et al, 2008;Dörries et al, 2010). Different varieties of polymerase chain reactions are commonly used for monitoring of GMOs in foods and animal feeds (Shin et al, 2013;Kim et al, 2014;Meriç et al, 2014;Datukishvili et al, 2015;Turkec et al, 2016).…”

“…Different varieties of polymerase chain reactions are commonly used for monitoring of GMOs in foods and animal feeds (Shin et al, 2013;Kim et al, 2014;Meriç et al, 2014;Datukishvili et al, 2015;Turkec et al, 2016). Nowadays real-time PCR (qPCR) has become a gold standard in the routine analysis of GMOs, allowing very sensitive detection and especially quantification of GMOs in test samples (Dörries et al, 2010;Elsanhoty et al, 2013;Özgen Arun et al, 2013, Zdjelar et al, 2013Fernandes et al, 2014;Kim et al, 2014;SantaMaria et al, 2014;Turkec et al, 2016). However, to quantify GMO content using qPCR, it is necessary to plot standard curves for both the reference gene and transgen, using serial dilutions of DNA extracted from the reference material, which is time-consuming.…”

Screening for six genetically modified soybean lines by an event-specific multiplex PCR method: Collaborative trial validation of a novel approach for GMO detection

“…Validated duplex, triplex and pentaplex assays combining element-and constructspecific real-time PCR methods are available and allow time-and cost-reduced GMO screening (Waiblinger et al 2008;Bahrdt et al 2010;Dorries et al 2010;Huber et al 2013). These multiplex TaqMan PCR assays are based on probes labelled by up to five different fluorescent dyes for simultaneous detection of the different target sequences.…”