A Real-Time Quantitative PCR Detection Method Specific to Widestrike Transgenic Cotton (Event 281-24-236/3006-210-23)

Abstract

In compliance with global regulations on transgenic crops, a real-time quantitative PCR method specific to Widestrike transgenic cotton (event 281-24-236/3006-210-23, OECD Unique Identifier DAS-24236-5/DAS-21023-5) was established on the basis of the DNA sequences in the junction between the transgene insert and cotton genome. The optimized method consists of a DNA extraction method for cotton seeds and three PCR systems corresponding to a cotton-specific endogenous reference DNA sequence SAH7 (Sinapis Arabidopsis Homolog 7) and specific detection of event 281-24-236 and event 3006-210-23. The method performance including specificity, sensitivity, accuracy, and precision was determined at a dynamic range of Widestrike DNA levels from 0.04% to 5.0%. The limits of detection (LOD) and quantification (LOQ) were < or =0.04% and < or =0.09%, respectively, at 100 ng of DNA sample per reaction. The quantification results using either the event 281-24-236 or 3006-210-23 system were consistent, and the relative deviation from the expected (true) value was in the range of +/-25%. The robustness of the method was demonstrated by a series of tests with deviations from the optimized assay parameters such as annealing temperature, extension time, PCR instrument, interlaboratory transferability, etc. All the measurements from these tests met the criteria set by EU JRC-CRL (European Commission Joint Research Centre-Community Reference Lab). This real-time quantitative PCR method is accurate and robust, and is recommended as a global benchmark method for the detection and quantification of Widestrike cotton. The method including description, protocol, and performance results is available on the JRC-CRL website (http://gmo-crl.jrc.it/statusofdoss.htm).

Review of methods for the detection and quantification of adulteration of rice: Basmati as a case study

“…As of today, real time PCR assay is regarded as the most sought after method for accurate quantification of the nucleic acids. It is a reliable as well as commonly used method to detect foreign DNA in genetically modified (GM) food samples (Baeumler et al 2006;Leimanis et al 2006). In case of GM food, detection and quantification of transgene has been relatively straight forward because transgenes are distinct "foreign" DNA elements in the host genome, which can be specifically amplified with high accuracy (detection) and sensitivity (quantification) by real time PCR (Nakamura et al 2013).…”

Fast-Tracking Determination of Homozygous Transgenic Lines and Transgene Stacking Using a Reliable Quantitative Real-Time PCR Assay

“…It is important that the amplification of the internal reference does not change, even under conditions when target gene amplification may alter dramatically [14]. Ideally, an internal reference gene should be species-specific, having single or low copy number per haploid genome, and exhibiting low heterogeneity across genotypes within a species [1,15]. To screen genes appropriate for use as an internal reference, the following eight genes, present as one or two copies in the rice genome, were chosen: starch branching enzyme (RBE4) [16], sucrose phosphate synthase (SPS), rice root-specific gene (gos9), eukaryotic elongation factor 1-alpha (eEF1α), rice actin gene (RAc1), 1-deoxy-Dxylulose-5-phosphate reductoisomerase (dxr), and trs-like genes (Os3bet3 and Os4trs20).…”

“…It has led to the development of commercial crops with improved agronomic characteristics [1]. Rapidly obtaining homozygous lines shortens the breeding duration for generating transgenes, particularly when multiple genes or transgenes are stacked once the viability of primary transformants has been obtained.…”

A novel trait-specific real-time PCR method enables quantification of genetically modified (GM) maize content in ground grain samples containing stacked GM maize

“…Therefore, many countries have mandated the labeling of foods containing a specified threshold level of GM crops (0.9 % in the European Union, 3 % in Korea and 5 % in Japan) [1]. To monitor the content of GM crops such as maize [2][3][4][5][6][7][8], soybean [2][3][4][8][9][10] and other crops [11][12][13] in foods, in general, the quantitative real-time polymerase chain reaction (qPCR) has been used. In several Abstract Stacked genetically modified (GM) maize is increasingly produced; thereby, current event-specific quantitative real-time polymerase chain reaction (qPCR) methods have led to the overestimation of GM organism (GMO) content compared with the actual weight/weight percentage of GM organism in maize samples.…”