Finding the Joker among the Maize Endogenous Reference Genes for Genetically Modified Organism (GMO) Detection

Abstract

The comparison of five real-time polymerase chain reaction (PCR) methods targeted at maize ( Zea mays ) endogenous sequences is reported. PCR targets were the alcohol dehydrogenase (adh) gene for three methods and high-mobility group (hmg) gene for the other two. The five real-time PCR methods have been checked under repeatability conditions at several dilution levels on both pooled DNA template from several genetically modified (GM) maize certified reference materials (CRMs) and single CRM DNA extracts. Slopes and R(2) coefficients of all of the curves obtained from the adopted regression model were compared within the same method and among all of the five methods, and the limit of detection and limit of quantitation were analyzed for each PCR system. Furthermore, method equivalency was evaluated on the basis of the ability to estimate the target haploid genome copy number at each concentration level. Results indicated that, among the five methods tested, one of the hmg-targeted PCR systems can be considered equivalent to the others but shows the best regression parameters and a higher repeteability along the dilution range. Thereby, it is proposed as a valid module to be coupled to different event-specific real-time PCR for maize genetically modified organism (GMO) quantitation. The resulting practicability improvement on the analytical control of GMOs is discussed.

Identification of single target taxon-specific reference assays for the most commonly genetically transformed crops using digital droplet PCR

“…These results indicate that the presence of a single target site for hmg and ZmAdh1 and of two targets for aldolase is not limited to only one reference material, but is confirmed for both CRMs used in the study. Both the assay for hmg and for ZmAdh1 could be used in GMO quantification, although one particular reaction mix recipe for hmg was reported to be more stable in terms of quantification performance according to Paternò et al (2009) . The assay for aldolase instead targets two sequences that amplify with different efficiencies due to the presence of mismatches in the reverse primer and in the probe ( Fig.…”

In-depth analysis of the endogenous reference genes used in the quantitative PCR detection systems for rice

“…A similar situation also exists for other crop species [25], such as maize, rapeseed and cotton. The maize and rapeseed-specific reference gene detection systems have been systematically compared and validated [15][16][17]. The results of these studies revealed that the comparison and validation of such reference genes is essential to standardize GM detection and to obtain comparable results between different assays.…”

“…The use of various endogenous reference genes in rice detection systems could cause confusion similar to that which has occurred with maize and rapeseed [14][15][16][17]. One study has investigated the amplicon SNPs of some rice genes [18], but the mutual commutability among these genes and the advantages and flaws of each gene for the quantitative detection of GM rice have not been evaluated.…”

In-House Validation and Comparison of Two Wheat (Triticum aestivum) Taxon-Specific Real-Time PCR Methods for GMO Quantification Supported by Droplet Digital PCR

“…With regard to the choice, as already stated in a previous study (Paternò et al 2009), from both a theoretical and technical point of view, there is no reason for changing the PCR reference system according to the GM event to be quantified (except for very peculiar situations related to food matrix and amplicon length). Therefore, the use of a single taxon-specific PCR reference system for enforcement purposes is not only advisable for practical reasons but also sensible from a scientific perspective.…”