A novel multiplex quantitative DNA array based PCR (MQDA-PCR) for quantification of transgenic maize in food and feed

Abstract

We have developed a novel multiplex quantitative DNA array based PCR method (MQDA-PCR). The MQDA-PCR is general and may be used in all areas of biological science where simultaneous quantification of multiple gene targets is desired. We used quantification of transgenic maize in food and feed as a model system to show the applicability of the method. The method is based on a two-step PCR. In the first few cycles bipartite primers containing a universal 5' 'HEAD' region and a 3' region specific to each genetically modified (GM) construct are employed. The unused primers are then degraded with a single-strand DNA-specific exonuclease. The second step of the PCR is run containing only primers consisting of the universal HEAD region. The removal of the primers is essential to create a competitive, and thus quantitative PCR. Oligo nucleotides hybridising to internal segments of the PCR products are then sequence specifically labelled in a cyclic linear signal amplification reaction. This is done both to increase the sensitivity and the specificity of the assay. Hybridisation of the labelled oligonucleotides to their complementary sequences in a DNA array enables multiplex detection. Quantitative information was obtained in the range 0.1-2% for the different GM constructs tested. Seventeen different food and feed samples were screened using a twelve-plex system for simultaneous detection of seven different GM maize events (Bt176, Bt11, Mon810, T25, GA21, CBH351 and DBT418). Ten samples were GM positive containing mainly mixtures of Mon810, Bt11 and Bt176 DNA. One sample contained appreciable amounts of GA21. An eight-plex MQDA-PCR system for detection of Mon810, Bt11 and Bt176 was evaluated by comparison with simplex 5' nuclease PCRs. There were no significant differences in the quantifications using the two approaches. The samples could, by both methods, be quantified as containing >2%, between 1 and 2%, between 0.1 and 1%, or <0.1% in 43 out of 47 determinations. The described method is modular, and thus suited for future needs in GM detection.

Transgenic Residues in Soybean-based Foods

“…For the detection of transgenic soybean, multiplex PCR was developed for accurate and simultaneous detection of epsps and nos fragments, both corresponding to the introduced gene in Roundup Ready soybean, using two primer pairs, one for the 35S-epsps gene and the other for nos. Rudi et al (2003) designed a multiplex PCR-based assay for quantification of DNA. This method is based on two stages of PCR; in the first few cycles, bipartite primers containing the termination 5 'universal sequence and the 3' region of a specific sequence were used for each of the events that were analyzed genetically.…”

Adventitious presence of GMOs: Scientific overview for Canadian grains

“…Their EasyRead GMO Chip uses an enzyme-linked system to generate a dye that can be seen with a naked eye, and this may have applicability in the future (Fagan 2004). A quantitative system using PCR and microarray technology (multiplex quantitative DNA array based PCR; MQDA-PCR) has also been reported (Rudi et al 2003). However, the procedure is time consuming, as it requires two PCR steps, labeling and hybridization with probes.…”

Highly selective single nucleotide polymorphism recogniton by a chiral (5S) PNA beacon

“…[3][4][5] Heterogeneous formats for performing such assays have become increasingly available on account of the advancement of gene chip and DNA microarray technologies, [6][7][8] which can be used, in combination with multiplex PCRs, for the specific detection of DNA tracts also in food analysis. [9][10][11][12] An important class of probes is represented by molecular beacon (MB) oligonucleotides, which are modified with a fluorophore and a quencher (or a quenching surface) at each end, held together by a dsDNA stem made of complementary sequences; this structure allows to produce a fluorescence ''switch-on'' upon interaction with the target DNA. [13][14][15] PNA beacons [16][17][18] and the related ''light up probes'' [19][20][21] have also been recently described, displaying the advantages of higher selectivity and simpler design.…”