Novel Reference Molecules for Quantitation of Genetically Modified Maize and Soybean

Abstract

New quantitation methods based on a real-time polymerase chain reaction (PCR) technique were developed for 5 lines of genetically modified (GM) maize, including MON810, Event176, Bt11, T25, and GA21, and a GM soy, Roundup Ready. Oligonucleotide DNA, including specific primers and fluorescent dye, labeled probes, were designed for PCRs. Two plasmids were constructed as reference molecules (RMs) for the detection of GM maize and GM soy. The molecules contain the DNA sequences of a specific region found in each GM line, universal sequences used in various GM lines, such as cauliflower mosaic virus 35S promoter and nopaline synthase terminator, and the endogenous DNA sequences of maize or soy. By using these plasmids, no GM maize and GM soy were required as reference materials for the qualitative and quantitative PCR technique. Test samples containing 0, 0.10, 0.50, 1.0, 5.0, and 10% GM maize or GM soy were quantitated. At the 5.0%level, the bias (mean–true value) ranged from 2.8 to 19.4% and the relative standard deviation was <5.2%. These results show that our method involving the use of these plasmids as RMs is reliable and practical for quantitation of GM maize and GM soy.

Quantification of Genetically Modified Soybeans Using a Combination of a Capillary-Type Real-Time PCR System and a Plasmid Reference Standard

“…[5][6][7][8][9][10][11][12][13][14][15][16][17][18] Furthermore, many real-time PCR systems based on fluorescent detection, such as TaqMan chemistry, have been developed to identify and quantify GM soybeans, GM maize, and GM varieties of other agricultural commodities. [19][20][21][22][23][24][25][26][27][28] Real-time PCR systems using TaqMan chemistry are based on the use of a fluorescent TaqMan probe that monitors the formation of the PCR product during each cycle of the reaction. In addition, most commonly, GMO quantification by quantitative real-time PCR methods is calculated from the ratio of the target transgenic specific DNA sequence copy number vs. the DNA sequence copy number of the respective target plant species (taxon gene sequence).…”

A Specific Qualitative Detection Method for Peanut (Arachis Hypogaea) in Foods Using Polymerase Chain Reaction

“…For the quantitative or qualitative detection of genetically modified crops, many analytical methods using polymerase chain reaction (PCR) have been developed because of its specificity, sensitivity and applicability (Akiyama et al . 2002a; Kuribara et al . 2002; Matsuoka et al .…”

Decision Support for the Comparative Evaluation and Selection of Analytical Methods: Detection of Genetically Modified Organisms as an Example

“…Methods, selected for evaluation and comparison (assessment) in this study, are listed in Table 1. Four of the methods represent the qPCR system currently used in routine GMO diagnostics covering different applications: simplex and multiplex screening/identification (Alary et al 2002;Kuribara et al 2002;Pla et al 2013) and simplex quantification (Holck et al 2002). Two additional qPCR applications (SIMQUANT; Berdal et al 2008) use qPCR chemistry together with the limiting dilutions principle, which is near to the idea of the ddPCR-based methods, of which two were included Dobnik et al 2015).…”