Toward Metrological Traceability for DNA Fragment Ratios in GM Quantification. 1. Effect of DNA Extraction Methods on the Quantitative Determination of Bt176 Corn by Real-Time PCR

Abstract

An international CCQM-P60 pilot study involving eight national metrological institutes was organized to investigate if the quantification of genetically modified (GM) corn powder by real-time PCR was affected by the DNA extraction method applied. Four commonly used extraction methods were compared for the extraction of DNA from a GM Bt176 corn powder. The CTAB-based method yielded the highest DNA template quantity and quality. A difference in the 260 nm/230 nm absorbance ratio was observed among the different extraction methods. Real-time amplification of sequences specific for endogenous genes zein and hmg as well as transgenic sequences within the cryIA(b) gene and a fragment covering the junction between the transformed DNA and the plant genome were used to determine the GM percentage. The detection of the transgenic gene was affected by the quantity and quality of template used for the PCR reaction. The Bt176 percentages measured on diluted or purified templates were statistically different depending on the extraction method applied.

Use of real-time PCR to evaluate two DNA extraction methods from food

“…Moreover, the absence of ethanol wash step, important to remove CTAB residues, may have contributed to the low DNA quality. Corbisier et al (2007) compared four extractions methods, including CTAB and DNeasy kit, in the extraction of Bt176 2% CRM. The CTAB-based method yielded the highest DNA template quantity and quality.…”

“…Many studies have been published to determine the more appropriate extraction method for each food matrix in order to minimize the influence of the extraction in the variability of DNA amplification, mainly in the quantification of low copy number target using real time PCR (CANKAR et al, 2006;CORBISIER et al, 2007;FERRARI et al, 2007;PEANO et al, 2004;SMITH;MAXWELL;DE BOER, 2005;SMITH;MAXWELL, 2007;ZIMMERMANN;LÜTHY;PAULI, 1998).…”

Testing the Robustness of Validated Methods for Quantitative Detection of GMOs Across qPCR Instruments

“…It entails adequate knowledge and sufficient control of several factors, such as the quality of the nucleic acids, appropriate choice of reference materials, comparable amplification efficiencies between calibrants and samples, stability of the event-and reference systems, and the specificity levels of the assays (Shokere et al 2009;Berdal et al 2008;Corbisier et al 2007;Cankar et al 2006;Broothaerts et al 2008 International bodies (ISO 5725 International Organization for Standardization 1994; IUPAC 1988) provide comprehensive standards describing procedures to assess analytical method performance. The Codex Alimentarius Commission recently adopted into the international food code the 'Guidelines on performance criteria and validation of methods for detection, identification and quantification of specific DNA sequences and specific proteins in foods' (Codex Alimentarius Commission 2010).…”

Extraction of Trypanosoma cruzi DNA from food: a contribution to the elucidation of acute Chagas disease outbreaks

“…The A260/A230 reflects contamination by substances such as carbohydrates, peptides, phenols, and aromatic compounds. In the case of pure samples, the ratio should be above 2 (15) .…”