Marta I. Hernández

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.

A microarray-based detection system for genetically modified (GM) food ingredients

A multiplex DNA microarray chip was developed for simultaneous identification of nine genetically modified organisms (GMOs), five plant species and three GMO screening elements, i.e. the 35S promoter, the nos terminator and the nptII gene. The chips also include several controls, such as that for the possible presence of CaMV. The on-chip detection was performed directly with PCR amplified products. Particular emphasis was placed on the reduction of the number of PCR reactions required and on the number of primers present per amplification tube. The targets were biotin labelled and the arrays were detected using a colorimetric methodology. Specificity was provided by specific capture probes designed for each GMO and for the common screening elements. The sensitivity of the assay was tested by experiments carried out in five different laboratories. The limit of detection was lower than 0.3% GMO for all tests and in general around 0.1% for most GMOs. The chip detection system complies with the requirements of current EU regulations and other countries where thresholds are established for the labelling of GMO.

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.

Long‐range interaction for dimers of atmospheric interest: dispersion, induction and electrostatic contributions for O2O2, N2N2 and O2N2

Electric multipole moments, static dipole polarizabilities, and dynamic dipole, quadrupole, and mixed dipole‐octupole polarizabilities of molecular oxygen and nitrogen in their ground electronic states have been obtained by means of high level multiconfigurational ab initio calculations. From these properties, we have obtained electrostatic, dispersion, and induction coefficients for the long‐range interactions of the O2uf8ffO2, N2uf8ffN2, and O2uf8ffN2 dimers. Our data is a comprehensive and consistent set that for N2uf8ffN2 shows a very good agreement with previous accurate calculations, whereas for quantities involving open‐shell O2 represents a considerable improvement over previous estimations. Moreover, the long‐range interaction is analyzed and compared for the different interacting partners. It is found that the C8 dispersion interaction plays a nonnegligible role and that the induction component is only important for a detailed description of the highest order anisotropy terms in the spherical harmonics expansion of the long‐range potential. It is also found that the total long‐range interaction is quite similar in O2uf8ffO2 and O2uf8ffN2, and that differences with N2uf8ffN2 are mainly because of the important role of the electrostatic interaction in that dimer. Comparison with high level supermolecular calculations indicates that the present long‐range potentials are accurate for intermolecular distances larger than about 15 bohr.

Wave packet study of the Ne2I2 fragmentation dynamics: a four degrees of freedom model

Abstract We present a four degrees of freedom model that accounts for all possible fragmentation mechanisms in the vibrational predissociation of the Ne 2 I 2 van der Waals cluster. Calculations are performed by wave packet propagation and results are interpreted in terms of an analytical sequential model leading to a good agreement with experimental data.

Time-dependent Hartree study of lifetimes for the Ne2I2 van der Waals cluster

Abstract The vibrational predissociation of the Ne2I2 van der Waals cluster is studied by a time-dependent Hartree type approach. Lifetimes for different initial vibrational states of I2 are computed and compared with the experimental results. The validity of this approach for studying vibrational predissociation is explored as well as the limitations and advantages.

A time-dependent wave packet approach for reaction and dissociation in H2 + H2

Abstract A time-dependent wave packet method to study four center (4C) reactions in competition with collision-induced dissociation is presented and applied to the H 2 +H 2 system, using the potential of Aguado et al. [J. Chem. Phys. 101 (1994) 4004] and a reduced dimensionality model. The calculated probabilities are better converged than previous time-independent quantum calculations and, in addition, compare quite satisfactorily with quasiclassical trajectory calculations. Tunneling effects near reaction thresholds are, however, important. For the 4C reaction in H 2 ( v 1 =10)+H 2 ( v 2 =0), it is estimated that such effects are dominant for temperatures below 1200 K, with decreasing values for more excited initial states or for the dissociation process.

Wave packet dynamics of H2(v1=8-14)+H2(v2=0-2): the role of the potential energy surface on different reactive and dissociative processes.

A time-dependent wave packet method has been used to study different competing products of H(2)+H(2) collisions: four center reaction, collision induced dissociation, reactive dissociation, and three-body complex formation. A three-degree-of-freedom reduced dimensionality model has been used for five different geometries of the colliding complex (parallel H, crossed X, collinear L, and two T-shaped geometries T(I) and T(II)), with reactants in selected vibrational states with one diatom vibrationally hot and the other one vibrationally cold. Product probabilities have been calculated using two potential energy surfaces [J. Chem. Phys. 101, 4004 (1994); J. Chem. Phys. 116, 666 (2002)] in order to compare their performance in the dynamics. The regions of the potential energy surfaces responsible of the threshold behavior of the probabilities have been identified. Overall, we have found that the most recent potential energy surface is less anisotropic, provides a smaller propensity for insertion-type processes, and gives lower energy thresholds.

Study of the selective adsorption phenomenon in the 4He/Cu(11α) (with α = 0, 3, 5, 7) elastic scattering: the critical kinematic effect

In a recent paper [Surf. Sci. 251/252 (1991) 369] we have presented a new analysis of the selective adsorption phenomenon within the close-coupling formalism, applied to the 4He/Cu(110) elastic scattering. That analysis is extended here to the series of stepped surfaces 4He/Cu(11α) (α = 3, 5, 7). For incident energies from 5 to 90 meV, internal and external widths are obtained as a function of the face index α. Special attention is focussed on the critical kinematic effect, i.e., an enhancement of resonance widths at certain values of the scattering geometry (modulus and angles of the wave vector of the incident atom). At these incident conditions, a remarkable loss of the typical (Lorentzian or Fano) behavior is exhibited by the line shapes of the diffraction intensities. These features are shown in detail for the 4He/Cu(115) system, where the incident conditions are suitable for an experimental verification. A comparison between experimental and theoretical results at 21 meV is also presented.