Teresa Esteve

A Specific Real-Time Quantitative PCR Detection System for Event MON810 in Maize YieldGard® Based on the 3′-Transgene Integration Sequence

The increasing presence of transgenic plant derivatives in a wide range of animal and human consumables has provoked in western Europe a strong demand for appropriate detection methods to evaluate the existence of transgenic elements. Among the different techniques currently used, the real-time quantitative PCR is a powerful technology well adapted to the mandatory labeling requirements in the European Union (EU). The use of transgene flanking genomic sequences has recently been suggested as a means to avoid ambiguous results both in qualitative and quantitative PCR-based technologies. In this study we report the identification of genomic sequences adjacent to the 3′-integration site of event MON810 in transgenic maize. This genetically modified crop contains transgene sequences leading to ectopic expression of a synthetic CryIA(b) endotoxin which confers resistance to lepidopteran insects especially against the European corn borer. The characterization of the genome–transgene junction sequences by means of TAIL-PCR has facilitated the design of a specific, sensitive and accurate quantification method based on TaqMan chemistry. Cloning of event MON810 3′-junction region has also allowed to compare the suitability of plasmid target sequences versus genomic DNA obtained from certified reference materials (CRMs), to prepare standard calibration curves for quantification.

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.

Development of real-time PCR systems based on SYBR® Green I, Amplifluor and TaqMan® technologies for specific quantitative detection of the transgenic maize event GA21

Abstract Maize GA21 line has integrated several tandemly repeated copies of the r-act 5-enol-pyruvylshikimate-3-phosphate synthase construct used for plant transformation. We were able to amplify a nucleotide sequence corresponding to the polylinker plasmid vector flanked by the r-act promoter and nopaline synthase 3′-terminator. A method for specific detection and quantification of Roundup Ready ® transgenic maize line GA21 DNA using conventional and real-time PCR and based on this transgenic sequence is described. GA21 specific primers and probe were designed targeting the vector–promoter junction region and amplifying a 72-bp DNA fragment. Quantification methods were optimized through three different real-time PCR chemistries, i.e. SYBR ® Green I, Amplifluor™ and TaqMan ® . All three methods proved to be specific, highly sensitive and reliable for both identification and quantification of GA21 DNA. Plasmid pGAivr containing single copies of the GA21 and invertase amplicons was constructed for use as external standard in calibration curves. Using pGAivr , a TaqMan ® based real-time PCR assay was optimized in duplex format targeting the maize species-specific ivr1 gene and the GA21 junction region. The detection limit of the method was 0.01% GA21, which is far below the established threshold for accidental presence of genetically modified organisms (GMO), this method therefore being suitable for use in routine GMO analysis.

A rapid and direct real time PCR-based method for identification of Salmonella spp.

The aim of this work was the validation of a rapid, real-time PCR assay based on TaqMan technology for the unequivocal identification of Salmonella spp. to be used directly on an agar-grown colony. A real-time PCR system targeting at the Salmonella spp. invA gene was optimized and validated through a four times repeated blind experiment performed in two different laboratories including 50 Salmonella spp. with representative strains from each of the 5 different Salmonella subgenera and 30 non-Salmonella strains. Both parameters DeltaR(n) (fluorescence intensity of template through a normalized reporter value) and C(T) (cycle at which the fluorescence intensity achieved a pre-established threshold) were analyzed. Overall mean DeltaR(n) and C(T) values for Salmonella strains (2.14+/-0.87 and 15.30+/-0.90, respectively) were statistically different from values for non-Salmonella strains, allowing the establishment of cut-off DeltaR(n) and C(T) values based on 95% confidence intervals that allowed the correct identification of all strains tested in each independent experiment. The accuracy of this assay in terms of inclusivity and exclusivity was 100%. Moreover, the PCR system proved to be especially convenient because the pre-mix containing all PCR reagents except for the bacterial cells could be kept at -20 degrees C for at least 1 month before its use. The optimized TaqMan real-time PCR assay is a useful, simple and rapid method for routine identification of Salmonella spp., irrespective of the particular subgenus.

Assessment of real-time PCR based methods for quantification of pollen-mediated gene flow from GM to conventional maize in a field study

Maize is one of the main crops worldwide and an increasing number of genetically modified (GM) maize varieties are cultivated and commercialized in many countries in parallel to conventional crops. Given the labeling rules established e.g. in the European Union and the necessary coexistence between GM and non-GM crops, it is important to determine the extent of pollen dissemination from transgenic maize to other cultivars under field conditions. The most widely used methods for quantitative detection of GMO are based on real-time PCR, which implies the results are expressed in genome percentages (in contrast to seed or grain percentages). Our objective was to assess the accuracy of real-time PCR based assays to accurately quantify the contents of transgenic grains in non-GM fields in comparison with the real cross-fertilization rate as determined by phenotypical analysis. We performed this study in a region where both GM and conventional maize are normally cultivated and used the predominant transgenic maize Mon810 in combination with a conventional maize variety which displays the characteristic of white grains (therefore allowing cross-pollination quantification as percentage of yellow grains). Our results indicated an excellent correlation between real-time PCR results and number of cross-fertilized grains at Mon810 levels of 0.1–10%. In contrast, Mon810 percentage estimated by weight of grains produced less accurate results. Finally, we present and discuss the pattern of pollen-mediated gene flow from GM to conventional maize in an example case under field conditions.

Real-Time and Conventional Polymerase Chain Reaction Systems Based on the Metallo-Carboxypeptidase Inhibitor Gene for Specific Detection and Quantification of Potato and Tomato in Processed Food

In this paper, a method for the specific detection and quantification of potato and tomato DNA in food samples with the use of conventional and real-time polymerase chain reaction (PCR) is described. This method is adequate for use in food quality routine assays involving highly processed samples for which very tiny amounts of DNA are expected. Detection was achieved by amplifying a region of the metallo-carboxypeptidase inhibitor gene from either the potato (PCI) or the tomato (MCPI) and by using specific primers complementary to the propeptide regions of these inhibitors, which were found to differ for the potato and tomato proproteins. Conventional and real-time PCR systems were based on the same potato- or tomato-specific primer pairs, and quantification was carried out with a TaqMan chemistry-based probe. The methods developed proved to be very specific and sensitive and highly reliable for the identification and quantification of DNA from both plant species. In addition, the construction of plasmids pPAT and pTOM, suitable for use as external calibration standards for the elaboration of comparative amplification profiles, is reported. Limits of detection and quantification with the use of these plasmid standards are given. Specificity and copy number conservation among different cultivars were analyzed, and the reliability of these systems was tested through their application to the analysis of commercial food samples including potato and/or tomato as components.

“Parallel” and “Antiparallel Tail‐Clamps” Increase the Efficiency of Triplex Formation with Structured DNA and RNA Targets

Sequence‐specific triple‐helix structures can be formed by parallel and antiparallel DNA clamps interacting with single‐stranded DNA or RNA targets. Single‐stranded nucleic acid molecules are known to adopt secondary structures that might interfere with intermolecular interactions. We demonstrate the correlation between a secondary structure involving the target—a stable stem predicted by in silico folding and experimentally confirmed by thermal stability and competition analyses—and an inhibitory effect on triplex formation. We overcame structural impediments by designing a new type of clamp: “tail‐clamps”. A combination of gel‐shift, kinetic analysis, UV thermal melting and thermodynamic techniques was used to demonstrate that tail‐clamps efficiently form triple helices with a structured target sequence. The performance of parallel and antiparallel tail‐clamps was compared: antiparallel tail‐clamps had higher binding efficiencies than parallel tail‐clamps both with structured DNA and RNA targets. In addition, the reported triplex‐stabilizing property of 8‐aminopurine residues was confirmed for tail‐clamps. Finally, we discuss the possible use of this improved triplex technology as a new tool for applications in molecular biology.

Efficient sequence-specific purification of Listeria innocua mRNA species by triplex affinity capture with parallel tail-clamps

Parallel clamps can interact in a sequence‐specific manner with homopyrimidine DNA and RNA oligonucleotides to form triplexes. For longer nucleic acids, we have previously demonstrated the inhibitory effect of DNA‐target secondary structures on triplex formation. We further designed a modification of these molecules—that is, tail‐clamps formed by addition of a tail sequence to the parallel clamp—and proved efficient binding of the molecules with structured single‐stranded DNA targets. Here we explore the possible application of the tail‐clamp strategy for triplex formation with RNA targets, which are typically found as strongly folded single‐stranded molecules. Efficient and specific binding of a tail‐clamp designed to form a parallel triplex with Listeria innocua iap mRNA sequences has been verified by UV melting curves and triplex affinity capture techniques. Furthermore, we show for the first time the formation of stable complexes of mRNA with tail‐clamps not only under acidic but also under neutral and slightly basic pH conditions. These results signify a further step towards the possible applications of triplexes with mRNA molecules; research, analytical, and therapeutic uses can be envisaged. As an example, our tail‐clamp‐based triplex affinity capture assay allowed the specific capture and recovery of iap mRNA molecules from an L. innocua total RNA solution with 45 % yield.