Identification of PCR-amplified genetically modified organisms (GMOs) DNA by peptide nucleic acid (PNA) probes in anion-exchange chromatographic analysis

PCR products obtained by selective amplification of transgenic DNA derived from food samples containing Roundup Ready soybean or Bt-176 maize have been analyzed by anion-exchange HPLC. Peptide nucleic acids (PNAs), oligonucleotide analogues known to bind to complementary single-stranded DNA with high affinity and specificity, have been used as specific probes in order to assess the identity of the peaks observed. Two different protocols were adopted in order to obtain single-stranded DNA: amplification with an excess of one primer or digestion of one DNA strand. The single-stranded DNA was mixed with the PNA probe, and the presence of a specific sequence was revealed through detection of the corresponding PNA:DNA peak with significantly different retention time. Advantages and limits of this approach are discussed. The method was tested with reference materials and subsequently applied to commercial samples.     

Different drought-adaptive capacity of a native Patagonian tree species (Nothofagus pumilio) resulting from local adaptation

European Journal of Forest Research - The resistance of different genotypes to abiotic stress may be due to genetic effects and/or to phenotypic plasticity allowing them to acclimate to variable...     

In vitro digestion assay for determination of hidden fumonisins in maize

Hidden fumonisins have received great attention in the last years as they have been frequently found in maize products in addition to the free forms. Several papers have shown that interaction with macromolecular components such as protein and starch is at the base of the phenomenon: although the nature of the interaction (covalent or not) is still not clarified, the occurrence of hidden forms is generally revealed by the application of an alkaline hydrolysis procedure. In this study, an in vitro digestion model has been applied to raw maize to evaluate the possible release of hidden fumonisins under gastrointestinal conditions. Upon digestion of the food matrix, an increased amount of total detectable fumonisins was observed in comparison with the analysis on the nondigested matrix, an amount even higher than that calculated through the application of the hydrolysis procedure. Besides the analytical issues, our data have serious implications, since consumers may be exposed to a systematic higher risk than that estimated by conventional techniques.     

Deamino-oxytocin: inactivation plasma of women in labor

Incubation of deamino-oxytocin with plasma obtained from women in labor reduced the potency of this analog of oxytocin when assayed on an isolated strip of mammary gland taken from a lactating rat. Plasmas of nonpregnant women had no detectable effect on this activity of deamino-oxytocin, and the effect of plasmas from women just previous to the beginning of labor was not significant. The original. activities of the incubated deamino-oxytocin solutions could be restored by treatment with hydrogen peroxide. The inactivation may be caused by reductive cleavage of the disulfide bridge of deamino-oxytocin, this bridge being reformed by oxidation.     

Development of a peptide nucleic acid array platform for the detection of genetically modified organisms in food

Two previously developed platforms, a multiplex polymerase chain reaction (PCR) and a peptide nucleic acid (PNA) array, the former allowing for the simultaneous detection of five transgenes and two endogenous controls in food and feed matrices and the latter for the assessment of the identity of amplified PCR products, were combined in order to develop a PNA array device for the screening of genetically modified organisms (GMOs) in food. PNA probes were opportunely designed, synthesized, and deposited on commercial slides. The length of the probes as well as the distance of the probes from the surface were evaluated and found to be critical points. The most suitable probes were found to be 15-mer PNAs linked to the slide surface by means of two 2-(2-aminoethoxy)ethoxyacetic acids as spacers. The device was tested on a model system constituted by flour samples containing a mixture of standards at known concentrations of transgenic material, in particular Roundup Ready soybean and Bt11, Bt176, Mon810, and GA21 maize: The DNA was amplified using the specific multiplex PCR method and tested on the PNA array. The method proposed was found to be able to correctly identify every GMO present in the tested samples.     

Snow removal and its influence on temperature and N dynamics in alpine soils (Vallée d'Aoste,northwest Italy)

The effect of a lack of snow cover in winter was investigated in two soils, beneath larch and meadow, in NW Italy (Vallée d'Aoste Region). During the late 1980s and early 1990s and 2000s, this region experienced extreme climatic conditions including a low snow pack and lack of snow cover for extended periods with important effects on soil temperature and nutrient dynamics. In particular, the mountain belt in the Alps may be extremely sensitive to these phenomena, in relation to the rise in average snowline projected under a warmer global climate. The study area is located at an elevation of 1450 m asl in the Italian Alps (Mont Mars Natural Reserve). During the winter 2003/04, snow was continuously removed in a treatment plot while a reference plot was maintained undisturbed. Soil temperature was measured at 10 cm depth by data loggers (UTL‐1). Soil N transformations in the topsoil (10 cm depth) were determined by the buried‐bag technique. The removal of the snow cover caused a significant decrease in soil temperature, related to concurrent decreases in air temperature. The lowest soil temperatures recorded were –4.3°C and –4.5°C beneath larch and meadow, respectively, on January 31, 2004. Soil temperature in the undisturbed plots was maintained above the freezing point when the snow cover was present. The snow removal caused significant increases in net ammonification in both soils and net nitrification only under meadow, but did not affect microbial biomass N which decreased in both plots. Our results suggest that the lower temperature reached in the plot without snow favored the production of inorganic N by physical rather than microbial degradation of soil organic matter (SOM). Soil freezing could enhance soil‐aggregate disruption releasing physically protected SOM and fragmentation of OM itself.     

Detection of genetically modified soybean using peptide nucleic acids (PNAs) and microarray technology

Peptide nucleic acid (PNA) microarrays for the detection of Roundup Ready soybeans in food have been prepared. PNA probes are known to be more efficient and selective in binding DNA sequences than the analogous oligonucleotides and are very suitable to be used for diagnostics in food. PNAs of different lengths were carefully designed and synthesized by solid-phase synthesis on an automatic synthesizer adopting the BOC strategy. PNAs were purified by HPLC and characterized by HPLC/MS. The probes were spotted on a functionalized surface to produce a microarray to be hybridized with PCR products. DNA extracted from reference material was amplified using Cy3- and Cy5-labeled primers, and the fluorescent PCR products obtained were hybridized on the microarray. Two protocols were adopted: the hybridization with dsDNA or with ssDNA obtained by digestion with the enzyme lambda exonuclease. The best results were obtained using a 15-mer PNA probe in combination with the ssPCR product derived from enzymatic digestion. The method was applied to the analysis of a sample of certified transgenic soybean flour.     

Detection and quantitation of genetically modified maize (Bt-176 transgenic maize) by applying ligation detection reaction and universal array technology

We have applied the ligation detection reaction (LDR) combined with a universal array approach to the detection and quantitation of the polymerase chain reaction (PCR) amplified cry1A(b) gene from Bt-176 transgenic maize. We demonstrated excellent specificity and high sensitivity. Down to 0.5 fmol (nearly 60 pg) of PCR amplified transgenic material was unequivocally detected with excellent linearity within the 0.1-2.0% range with respect to wild-type maize. We suggest the feasibility of extending the LDR/universal array format to detect in parallel several transgenic sequences that are being developed for food applications.     

Development of a seven-target multiplex PCR for the simultaneous detection of transgenic soybean and maize in feeds and foods

The detection of genetically modified organisms (GMOs) in food and feed is an important issue for all the subjects involved in raw material control, food industry, and distribution. Because the number of GMOs authorized in the EU increased during the past few years, there is a need for methods that allow a rapid screening of products. In this paper, we propose a method for the simultaneous detection of four transgenic maize (MON810, Bt11, Bt 176, and GA21) and one transgenic soybean (Roundup Ready), which allows routine control analyses to be sped up. DNA was extracted either from maize and soybean seeds and leaves or reference materials, and the recombinant DNA target sequences were detected with 7 primer pairs, accurately designed to be highly specific for each investigated transgene. Cross and negative controls were performed to ensure the specificity of each primer pair. The method was validated on an interlaboratory ring test and good analytical parameters were obtained (LOD = 0.25%, Repeatability, (r) = 1; Reproducibility, (R) = 0.9). The method was then applied to a model biscuit made of transgenic materials baked for the purpose and to real samples such as feed and foodstuffs. On account of the high recognition specificity and the good detection limits, this multiplex PCR represents a fast and reliable screening method directly applicable in all the laboratories involved in raw material and food control.     

A microarray platform for parallel detection of five transgenic events in foods: a combined polymerase chain reaction-ligation detection reaction-universal array method

We recently developed a multiplex polymerase chain reaction (PCR) system for the simultaneous detection of four transgenic maize (MON810, Bt176, Bt11, and GA21), one transgenic soybean (Roundup Ready), and two control genes (lectin and zein). Because PCR can lead to ambiguous interpretations due to low specificity, we have developed the ligation detection reaction (LDR) combined with a universal array as a molecular tool to confirm results of PCR analysis. Here, we describe the PCR-LDR-universal array procedure and demonstrate its specificity in revealing the presence of transgenic DNA in experimental samples, raw materials, and commercial foodstuffs.