Sensitive and simultaneous analysis of five transgenic maizes using multiplex polymerase chain reaction, capillary gel electrophoresis, and laser‐induced fluorescence

Abstract

The benefits of using multiplex polymerase chain reaction (PCR) followed by capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) for the simultaneous detection of five transgenic maizes (Bt11, T25, MON810, GA21, and Bt176) are demonstrated. The method uses a hexaplex PCR protocol to amplify the five mentioned transgenic amplicons plus the zein gene used as reference, followed by a CGE-LIF method to analyze the six DNA fragments. CGE-LIF was demonstrated very useful and informative for optimizing multiplex PCR parameters such as time extension, PCR buffer concentration and primers concentration. The method developed is highly sensitive and allows the simultaneous detection in a single run of percentages of transgenic maize as low as 0.054% of Bt11, 0.057% of T25, 0.036% of MON810, 0.064% of GA21, and 0.018% of Bt176 in flour obtaining signals still far from the detection limit (namely, the signal/noise ratios for the corresponding DNA peaks were 41, 124, 98, 250, 252, and 473, respectively). These percentages are well below the minimum threshold marked by the European Regulation for transgenic food labeling (i.e., 0.5-0.9%). A study on the reproducibility of the multiplex PCR-CGE-LIF procedure was also performed. Thus, values of RSD lower than 0.67 and 6.80% were obtained for migration times and corrected peak areas, respectively, for the same sample and three different days (n = 12). On the other hand, the reproducibility of the whole procedure, including four different multiplex PCR amplifications, was determined to be better than 0.66 and 23.3% for migration times and corrected peak areas, respectively. Agarose gel electrophoresis (AGE) and CGE-LIF were compared in terms of resolution and sensitivity for detecting PCR products, demonstrating that CGE-LIF can solve false positives induced by artifacts from the multiplex PCR reaction that could not be addressed by AGE. Moreover, CGE-LIF provides better resolution and sensitivity. To our knowledge, these results demonstrate for the first time that multiplex PCR-CGE-LIF is a solid alternative to determine multiple genetically modified organisms in maize flours in a single run.

Recent advances in the application of capillary electromigration methods for food analysis

“…The combined use of molecular techniques together with CE has, therefore, brought about an impressive analytical procedure that combines the selectivity and the sensitivity increases provided by any molecular technique with the speed of analysis, resolving power and low sample requirements of CE techniques. Thus, PCR-based techniques combined with CE separation have demonstrated to be a powerful analytical method for: (i) detection of genetically modified organisms, also called transgenic foods [95,96]; (ii) detection of food-borne pathogens and food-spoilage bacteria [97][98][99][100][101]; and (iii) species identification [102,103], when e.g., fraudulent substitution, addition or contamination are suspected in foodstuffs. These applications include analysis of transgenic maize [95,96], detection of lactic acid bacteria [97], detection of spoilage bacterium Alicyclobacillus acidoterrrestris [98], detection of toxigenic fungi from dry cured meat (MEKC was also used to study secondary metabolites produced by fungal strains) [99], detection of food-borne pathogens, mainly Salmonella [100,101], identification of porcine, caprine, and bovine meats [102], and identification of molecular markers of Glu-1 genes in wheat allowing selection of wheat genotypes of higher breadmaking quality [103].…”

“…Recently, a new PCR-CGE-LIF approach to quantitatively determine the content of genetically modified Bt maize in foods was developed [95]. Also, a new multiplex-PCR-CGE-LIF procedure was shown to be useful for the sensitive and simultaneous analysis of five transgenic maizes in a singe run [96]. We have also developed a new multiplex-PCR-CGE-LIF procedure for the detection and differentiation of several food-spoilage lactic acid bacteria [97].…”

Recent advances in the application of capillary electromigration methods for food analysis

“…Following the first works published on GMOs analysis by PCR-CGE-LIF [71][72][73][74][75] some new applications have come out also on this topic. Thus, a PCR-CGE-LIF method for GMO detection involved the use of fluorescent labeled primers with different dyes to perform the simultaneous PCR amplification in a single tube [62].…”

Detection of microbial food contaminants and their products by capillary electromigration techniques

“…On the other hand, with multiplex PCR more than one target sequence can be amplified in a single reaction allowing the sensitive detection of several pathogens in a single analysis. However, for a successful multiplex PCR assay it is important to optimize the reaction parameters to avoid the formation of spurious amplification products and uneven amplification of target sequences [71]. This novel concept of PCR was exploited in combination with CGE-LIF to achieve simultaneous detection of several bacteria in a single analysis [72,73].…”