Development of Taxon-Specific Sequences of Common Wheat for the Detection of Genetically Modified Wheat

Abstract

Qualitative and quantitative Polymerase Chain Reaction (PCR) systems aimed at the specific detection and quantification of common wheat DNA are described. Many countries have issued regulations to label foods that include genetically modified organisms (GMOs). PCR technology is widely recognized as a reliable and useful technique for the qualitative and quantitative detection of GMOs. Detection methods are needed to amplify a target GM gene, and the amplified results should be compared with those of the corresponding taxon-specific reference gene to obtain reliable results. This paper describes the development of a specific DNA sequence in the waxy-D1 gene for common wheat (Triticum aestivum L.) and the design of a specific primer pair and TaqMan probe on the waxy-D1 gene for PCR analysis. The primers amplified a product (Wx012) of 102 bp. It is indicated that the Wx012 DNA sequence is specific to common wheat, showing homogeneity in qualitative PCR results and very similar quantification accuracy along 19 distantly related common wheat varieties. In Southern blot and real-time PCR analyses, this sequence showed either a single or a low number of copy genes. In addition, by qualitative and quantitative PCR using wx012 primers and a wx012-T probe, the limits of detection of the common wheat genome were found to be about 15 copies, and the reproducibility was reliable. In consequence, the PCR system using wx012 primers and wx012-T probe is considered to be suitable for use as a common wheat-specific taxon-specific reference gene in DNA analyses, including GMO tests.

Development of Methods to Distinguish between Durum/Common Wheat and Common Wheat in Blended Flour Using PCR

“…Although detection methods of wheat 3,5) in food by PCR have been developed, it has not been possible to distinguish different wheat species. In this study, qualitative PCR methods to distinguish wheat species with two primer pairs were developed.…”

An Endogenous Reference Gene of Common and Durum Wheat for Detection of Genetically Modified Wheat

“…A DNA sequence with a single or stable low copy number should yield similar Ct values among varieties. We previously concluded that common wheat genomic DNAs have either single or double copies of the Wx012 region 21 . To estimate the copy number of the PRP region in the common wheat genome, we performed quantitative analyses of the PRP and Wx012 regions.…”

“…We calculated and compared the copy number of the Wx012 region and that of the PRP region. In this experiment, durum wheat was not included because the Wx012 system does not work on durum wheat 21 .…”

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

“…This strategy was then described in a guidance document published on the EURL-GMFF website (European Union Reference Laboratory for GM Food and Feed 2013a), together with a comprehensive review and related bioinformatics analysis on all available relevant literature on taxon-specific real-time PCR systems for the identification of T. aestivum (European Union Reference Laboratory for GM Food and Feed 2013b). The results of the study suggested that the method described by Matsuoka et al (2012), targeting ssII-D gene coding for starch synthase, and the one described by Iida et al (2005), targeting waxy-D1 gene, coding for granule-bound starch synthase, may represent good candidates to specifically identify common wheat even in complex food samples. With regard to the latter method, what was reported in the above-mentioned EURL-GMFF guidelines had to be carefully considered, i.e.…”

“…With regard to the latter method, what was reported in the above-mentioned EURL-GMFF guidelines had to be carefully considered, i.e. Baccording to in silico analyses conducted by the EURL GMFF, the primer sequences described in Iida et al 2005 are not correct. The primers (Wx012F/Wx012R) and probe (Wx-Taq 1) sequences reported by the same authors in the following article: BImai et al (2012), Food Hyg.…”