Critical points of DNA quantification by real-time PCR – effects of DNA extraction method and sample matrix on quantification of genetically modified organisms

BACKGROUND: Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. RESULTS: Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was chosen as the primary criterion by which to evaluate the quality and performance on different matrixes and extraction techniques. The effect of PCR efficiency on the resulting GMO content is demonstrated. CONCLUSION: The crucial influence of extraction technique and sample matrix properties on the results of GMO quantification is demonstrated. Appropriate extraction techniques for each matrix need to be determined to achieve accurate DNA quantification. Nevertheless, as it is shown that in the area of food and feed testing matrix with certain specificities is impossible to define strict quality controls need to be introduced to monitor PCR. The results of our study are also applicable to other fields of quantitative testing by real-time PCR

Quantitative Analysis of Food and Feed Samples with Droplet Digital PCR

<div><p>In this study, the applicability of droplet digital PCR (ddPCR) for routine analysis in food and feed samples was demonstrated with the quantification of genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (qPCR) is currently used for quantitative molecular analysis of the presence of GMOs in products. However, its use is limited for detecting and quantifying very small numbers of DNA targets, as in some complex food and feed matrices. Using ddPCR duplex assay, we have measured the absolute numbers of MON810 transgene and <i>hmg</i> maize reference gene copies in DNA samples. Key performance parameters of the assay were determined. The ddPCR system is shown to offer precise absolute and relative quantification of targets, without the need for calibration curves. The sensitivity (five target DNA copies) of the ddPCR assay compares well with those of individual qPCR assays and of the chamber digital PCR (cdPCR) approach. It offers a dynamic range over four orders of magnitude, greater than that of cdPCR. Moreover, when compared to qPCR, the ddPCR assay showed better repeatability at low target concentrations and a greater tolerance to inhibitors. Finally, ddPCR throughput and cost are advantageous relative to those of qPCR for routine GMO quantification. It is thus concluded that ddPCR technology can be applied for routine quantification of GMOs, or any other domain where quantitative analysis of food and feed samples is needed.</p> </div

Precision of the duplex ddPCR assay as a function of the target concentration.

<p>MON810 content measured by ddPCR in five series of seven target concentrations. The target MON810 content (3.85%) is indicated by a dotted line. Acceptance criterion for precision is ±25% of the target content (from 2.89% to 4.81%) represented by the dashed lines. Error bars represent the standard deviation of the measured MON810% by ddPCR at each target concentration (five replicates per target concentration).</p

Dynamic range of the ddPCR duplex assay.

<p>Five replicates for each data point. Error bars represent the standard deviation between the five replicates at each target concentration.</p

Summary table of qPCR, ddPCR and cdPCR performance for MON810 detection and quantification.

<p>qPCR: data produced in this study, or obtained from the literature, when indicated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062583#pone.0062583-Burns1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062583#pone.0062583-EuropeanUnionReferenceLaboratoryforGM1" target="_blank">[22]</a></p><p>ddPCR: data produced in this study.</p><p>cdPCR: data produced on a BioMark System (Fluidigm, South San Francisco) using the 12.765 digital arrays (Fluidigm) and obtained from the literature <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062583#pone.0062583-Corbisier1" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062583#pone.0062583-Burns1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062583#pone.0062583-Bhat1" target="_blank">[19]</a>.</p><p>Repeatability through the dynamic range: assessed through the coefficient of variation (Cv) of the target copy numbers or the MON810 content between repeats.</p><p>Trueness: assessed through the calculation of the bias between the MON810 content measured and the target MON810 content. * For our study, trueness is indicated only when qPCR and ddPCR results could be compared to a third, independent value (obtained from the CRM provider or proficiency test organizer).</p><p>Time for results/96 well plate: Total time needed from DNA pipetting to the analysis of the results; reaction mixes are already prepared.</p><p>Price/sample if 96-well plate: Price based on material and reagent costs available at NIB, including labor cost.</p><p>N.A.: not evaluated.</p