Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits

Biotechnology-derived varieties of canola, cotton, corn and soybean are being grown in the USA, Canada and other predominantly grain exporting countries. Although the amount of farmland devoted to production of biotechnology-derived crops continues to increase, lingering concerns that unintended consequences may occur provide the EU and most grain-importing countries with justification to regulate these crops. Legislation in the EU requires traceability of grains/oilseeds, food and feed products, and labelling, when a threshold level of 0.9% w/w of genetically engineered trait is demonstrated to be present in an analytical sample. The GE content is routinely determined by quantitative PCR (qPCR) and plant genomic DNA provides the template for the initial steps in this process. A plethora of DNA extraction methods exist for qPCR applications. Implementing standardized methods for detection of genetically engineered traits is necessary to facilitate grain marketing. The International Organization for Standardization draft standard 21571 identifies detergent-based methods and commercially available kits that are widely used for DNA extraction, but also indicates that adaptations may be necessary depending upon the sample matrix. This review assesses advantages and disadvantages of various commercially available DNA extraction kits, as well as modifications to published cetyltrimethylammonium bromide methods. Inhibitors are a major obstacle for efficient amplification in qPCR. The types of PCR inhibitors and techniques to minimize inhibition are discussed. Finally, accurate quantification of DNA for applications in qPCR is not trivial. Many confounders contribute to differences in analytical measurements when a particular DNA quantification method is applied and different methods do not always provide concordant results on the same DNA sample. How these differences impact measurement uncertainty in qPCR is considered.     

Ultrasensitive quantification of mature microRNAs by real-time PCR based on ligation of a ribonucleotide-modified DNA probe

It was first demonstrated that the DNA probe modified with ribonucleotides can be efficiently ligated by using miRNA as the template. With PCR amplification of the ligated DNA probe, as low as 0.2 fM target miRNAs can be detected with high specificity.     

Cloned plasmid DNA fragments as calibrators for controlling GMOs: different real-time duplex quantitative PCR methods

Analytical real-time PCR technology is a powerful tool for implementation of the GMO labeling regulations enforced in the EU. The quality of analytical measurement data obtained by quantitative real-time PCR depends on the correct use of calibrator and reference materials (RMs). For GMO methods of analysis, the choice of appropriate RMs is currently under debate. So far, genomic DNA solutions from certified reference materials (CRMs) are most often used as calibrators for GMO quantification by means of real-time PCR. However, due to some intrinsic features of these CRMs, errors may be expected in the estimations of DNA sequence quantities. In this paper, two new real-time PCR methods are presented for Roundup Ready soybean, in which two types of plasmid DNA fragments are used as calibrators. Single-target plasmids (STPs) diluted in a background of genomic DNA were used in the first method. Multiple-target plasmids (MTPs) containing both sequences in one molecule were used as calibrators for the second method. Both methods simultaneously detect a promoter 35S sequence as GMO-specific target and a lectin gene sequence as endogenous reference target in a duplex PCR. For the estimation of relative GMO percentages both delta C_T and standard curve approaches are tested. Delta C_T methods are based on direct comparison of measured C_T values of both the GMO-specific target and the endogenous target. Standard curve methods measure absolute amounts of target copies or haploid genome equivalents. A duplex delta C_T method with STP calibrators performed at least as well as a similar method with genomic DNA calibrators from commercial CRMs. Besides this, high quality results were obtained with a standard curve method using MTP calibrators. This paper demonstrates that plasmid DNA molecules containing either one or multiple target sequences form perfect alternative calibrators for GMO quantification and are especially suitable for duplex PCR reactions.Electronic Supplementary Material Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.     

Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection

Several techniques have been developed for detection and quantification of genetically modified organisms, but quantitative real-time PCR is by far the most popular approach. Among the most commonly used real-time PCR chemistries are TaqMan probes and SYBR green, but many other detection chemistries have also been developed. Because their performance has never been compared systematically, here we present an extensive evaluation of some promising chemistries: sequence-unspecific DNA labeling dyes (SYBR green), primer-based technologies (AmpliFluor, Plexor, Lux primers), and techniques involving double-labeled probes, comprising hybridization (molecular beacon) and hydrolysis (TaqMan, CPT, LNA, and MGB) probes, based on recently published experimental data. For each of the detection chemistries assays were included targeting selected loci. Real-time PCR chemistries were subsequently compared for their efficiency in PCR amplification and limits of detection and quantification. The overall applicability of the chemistries was evaluated, adding practicability and cost issues to the performance characteristics. None of the chemistries seemed to be significantly better than any other, but certain features favor LNA and MGB technology as good alternatives to TaqMan in quantification assays. SYBR green and molecular beacon assays can perform equally well but may need more optimization prior to use.     

Microchip-based one step DNA extraction and real-time PCR in one chamber for rapid pathogen identification

Optimal detection of a pathogen present in biological samples depends on the ability to extract DNA molecules rapidly and efficiently. In this paper, we report a novel method for efficient DNA extraction and subsequent real-time detection in a single microchip by combining laser irradiation and magnetic beads. By using a 808 nm laser and carboxyl-terminated magnetic beads, we demonstrate that a single pulse of 40 seconds lysed pathogens including E. coli and Gram-positive bacterial cells as well as the hepatitis B virus mixed with human serum. We further demonstrate that the real-time pathogen detection was performed with pre-mixed PCR reagents in a real-time PCR machine using the same microchip, after laser irradiation in a hand-held device equipped with a small laser diode. These results suggest that the new sample preparation method is well suited to be integrated into lab-on-a-chip application of the pathogen detection system.     

Gold nanoparticles for one step DNA extraction and real-time PCR of pathogens in a single chamber

The optothermal properties of nanoparticles are of interest for biosensors and highly sensitive biochip applications. In this respect, the longitudinal resonance of Au nanorods was used to transform near infrared energy into thermal energy in a microfluidic chip. The resulting heat generated effectively caused pathogen lysis. Consequently the DNA was extracted out of the cell body and transferred to a PCR system. This resulted in the successful demonstration of a one step real-time PCR system for pathogen detection without removal or changing of reagents.     

Evaluation of different machines used to quantify genetic modification by real-time PCR

Quantification of genetic modification (GM) is often undertaken to test for compliance with the European Union GM labeling threshold in food. Different control laboratories will often use common validated methods, but with different models of real-time PCR machines. We performed two separate ring trials to evaluate the relative precision and accuracy of different types of real-time PCR machines used to quantify the concentration of GM maize. Both trials used dual-labeled fluorogenic probes for quantification. The first ring trial used separate GM and reference assays (a single fluorescence channel), and the second used a combined duplex assay (two simultaneous fluorescence channels). Five manufacturers and seven models--including a 96-well microtiter-plate, rotary, and portable machines--were examined. In one trial, the machine used had a significant effect on precision, but in the other it did not. Overall, the degree of variation due to the machine model was lower than other factors. No significant repeatable difference in accuracy was observed between machine models. It was not possible to use sufficient replication of machine type in each laboratory to examine all sources of variation in this study, but the results strongly indicate that factors other than machine type or manufacturer (e.g., method or laboratory) contribute more to variation in a GM quantification result.     

New FRET primers for quantitative real-time PCR

FRET primer real-time PCR chemistry depends on internally labeled primers with FRET dyes linked to their 3′ end. The best distance between the FRET dyes for obtaining the largest signal and the lowest background is six nucleotides. In this study the forward primer was labeled with FAM and the reverse primer with Texas red; the labeled primers meet in cycle two of PCR. At the end of the elongation step FAM is excited to emit fluorescence which will excite Texas red to emit new fluorescence that correlates directly with the quantity of PCR product accumulated. FRET primer techniques amplify short amplicons with unique thermal cycling steps, 0 s at 85 °C for denaturation, 7 s for annealing, and 2 s for elongation. The FRET primer technique was very efficient (92.6, 97.2, and 100%), correlation coefficients were high (1.0, 0.999, and 0.999), and total run time was very short (20, 45, and 40 min per 40 cycles with LightCycler, iCycler, and RotorGene 3000, respectively). When FRET-labeled primers were compared with similar but unlabeled primers it was observed that the FRET primer technique had a lower Ct value and was more efficient than use of unlabeled primers detected by use of SYBR Green I. Figure Schematic diagram of FRET prime real-time PCR Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Protein quantification, sandwich ELISA, and real-time PCR used to monitor industrial cleaning procedures for contamination with peanut and celery allergens

In the United States, peanut is one of the main sources of food allergens. Similarly, celery is a common allergenic food in Western Europe. Severe allergic reactions to both foods are common. Unexpected allergic reactions can occur after the consumption of celery- and peanut-free foods as a result of inadvertent cross-contaminations during manufacturing. Therefore, in cooperation with a flavor manufacturer, we monitored the cleaning process of slurry preparation equipment with regard to contaminations of follow-up products with celery and peanut compounds. Washing water samples taken after different cleaning steps and follow-up products were analyzed for the presence of celery and peanut traces with a celery-specific real-time polymerase chain reaction (PCR) and a peanut-specific sandwich enzyme-linked immunosorbent assay (ELISA). PCR and ELISA were compared with a nonspecific protein assay to evaluate whether the detection of protein traces can be a fast and cost-effective method for monitoring the effectiveness of wet cleaning procedures. Additionally, the allergenic potential of the celery and peanut mush, which were used as source material, were measured by a mediator release assay using a rat basophilic leukemia (RBL) cell line. In conclusion, the quantification of total protein in washing water was suitable for monitoring the cleaning process. Our study also revealed evidence that, in cases where wet cleaning is applicable, allergenic traces can be removed with high efficiency.     

Site-specific quantification of 5-carboxylcytosine in DNA by chemical conversion coupled with ligation-based PCR

5-Methylcytosine (5mC) is the most important epigenetic modification in mammals. The active DNA demethylation could be achieved through the ten-eleven translocation (TET) protein-mediated oxidization of 5mC with the generation of 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). It has been known that 5mC, 5hmC and 5fC play critical roles in modulating gene expression. However, unlike the 5mC, 5hmC, and 5fC, the functions of 5caC are still underexplored. Investigation of the functions of 5caC relies on the accurate quantification and localization analysis of 5caC in DNA. In the current study, we developed a method by chemical conversion in conjugation with ligation-based real-time quantitative PCR (qPCR) for the site-specific quantification of 5caC in DNA. This method depends on the selective conversion of 5caC to form dihydrouracil (DHU) by pyridine borane treatment. DHU behaves like thymine and pairs with adenine (DHU-A). Thus, the chemical conversion by pyridine borane leads to the transformation of base paring from 5caC-G to DHU-A, which is utilized to achieve the site-specific detection and quantification of 5caC in DNA. As a proof-of-concept, the developed method was successfully applied in the site-specific quantification of 5caC in synthesized DNA spiked in complex biological samples. The method is rapid, straightforward and cost-effective, and shows promising in promoting the investigation of the functional roles of 5caC in future study.     

Reducing nonspecific adhesion on cross-linked hydrogel platforms for real-time immunoassay in serum

Biointerfaces that limit nonspecific adhesion of serum proteins have been developed by relying solely on cross-linked hydrogels. In addition to being characterized for adhesion of serum proteins, immunoassay sensitivity was also investigated through a sandwich assay for rhIL-1ra. Among the compositions developed, the optimal surface is comprised of pre-cross-linked carboxymethylcellulose (CMC) and polyethyleneimine (PEI) overlaid on a cross-linked layer of poly(ethylene glycol) (PEG) and PEI and employs an anti-IgG Fc specific ligand for oriented antibody immobilization; viscoelastic modeling provides a thickness estimate of 5 nm for the hydrogel alone, rising to 33 nm after the deposition of antibodies. Alternate compositions employing a Protein A ligand and PEG at the exposed surface of the biointerface were disfavored due to an 8-fold increase in serum adhesion and retarded immobilization kinetics, respectively. Through the rapid deposition provided by hydrogels, construction of the entire biointerface, including receptor immobilization, can be completed in 1 h. Based on QCM-D measurements, estimated nonspecific serum adsorption using these compositions is as low as 1.1 ng/mm2. The immunoassay as developed requires 10 min, providing a detection limit of 500 ng/mL rhIL-1ra in 25% human serum using only 5 microg of the secondary antibody.