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

• Main Authors: Maria Regina Branquinho, Renata Trotta Barroso Ferreira, Paola Cardarelli-Leite
• Format: Article
• Language: English
• Published: Sociedade Brasileira de Ciência e Tecnologia de Alimentos 2012-03-01
• Series: Food Science and Technology
• Subjects: extração de DNA; organismos geneticamente modificados; OGM; análise de alimentos; PCR em tempo real; linearidade
• Online Access: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0101-20612012000100017&lng=en&tlng=en

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.