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

Abstract

<jats:p>The DNA extraction is a critical step in Genetically Modified Organisms analysis based on real-time PCR. In this study, the CTAB and DNeasy methods provided good quality and quantity of DNA from the texturized soy protein, infant formula, and soy milk samples. Concerning the Certified Reference Material consisting of 5% Roundup Ready® soybean, neither method yielded DNA of good quality. However, the dilution test applied in the CTAB extracts showed no interference of inhibitory substances. The PCR efficiencies of lectin target amplification were not statistically different, and the coefficients of correlation (R²) demonstrated high degree of correlation between the copy numbers and the threshold cycle (Ct) values. ANOVA showed suitable adjustment of the regression and absence of significant linear deviations. The efficiencies of the p35S amplification were not statistically different, and all R² values using DNeasy extracts were above 0.98 with no significant linear deviations. Two out of three R² values using CTAB extracts were lower than 0.98, corresponding to lower degree of correlation, and the lack-of-fit test showed significant linear deviation in one run. The comparative analysis of the Ct values for the p35S and lectin targets demonstrated no statistical significant differences between the analytical curves of each target.</jats:p>

Molecular methods (digital PCR and real‐time PCR) for the quantification of low copy DNA of Phytophthora nicotianae in environmental samples

“…The efficiency of both extraction methods was evaluated by the amount and quality of DNA extracted from samples using spectrophotometric measurements, namely ratios A 260 / A 280 and A 260 / A 230 . Values between 1.8 and 2.0 for A 260 / A 280 indicate low levels of contamination by protein and aromatic substances, and values above 2 for A 260 / A 230 indicate the absence of PCR inhibitors such as polysaccharides, salts, lipids and phenolic compounds . The A 260 / A 280 values show that both methods provided good DNA quality for all samples.…”

“…According to published reports, CTAB‐extracted DNA needs further purification to be used for real‐time PCR . However, in previous studies it has been suggested that, in spite of the fact that DNA extracted using the CTAB method may possess some substances inhibitory for qPCR, dilutions were enough to remove all the inhibition …”

Detection of genetically modified organisms in soy products sold in Turkish market

“…Recently, several methods have been developed for GMO detection and quantitation, mostly based on DNA techniques, since protein-based methods are not reliable for highly processed food analysis (Branquinho et al, 2012;Randhawa et al, 2014;Fraiture et al, 2014;Song et al, 2014). However, PCR (Mullis & Faloona, 1987) have become essential for the control of the presence of genetically modified DNA.…”

Application of magnetic polymethacrylate-based microspheres for the isolation of DNA from raw vegetables and processed foods of plant origin

“…In literature, the DCt model was used not only for the analysis of gene expression (Livak & Schmittgen, 2001) but also for the comparison of real-time methods at quantification of molds in foods (Rodríguez, C ordoba, Gordillo, C ordoba, & Rodríguez, 2012) and evaluation of DNA extraction methods (Branquinho, Ferreira, & Cardarelli-Leite, 2012). In this work, we used the DCt values to monitorthe changes in DNA amplicability in the course of pickling process.…”