Detection and quantification of genetically modified organisms using very short, locked nucleic acid TaqMan probes

Many countries have introduced mandatory labeling requirements on foods derived from genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (PCR) based upon the TaqMan probe chemistry has become the method mostly used to support these regulations; moreover, event-specific PCR is the preferred method in GMO detection because of its high specificity based on the flanking sequence of the exogenous integrant. The aim of this study was to evaluate the use of very short (eight-nucleotide long), locked nucleic acid (LNA) TaqMan probes in 5'-nuclease PCR assays for the detection and quantification of GMOs. Classic TaqMan and LNA TaqMan probes were compared for the analysis of the maize MON810 transgene. The performance of the two types of probes was tested on the maize endogenous reference gene hmga, the CaMV 35S promoter, and the hsp70/cryIA(b) construct as well as for the event-specific 5'-integration junction of MON810, using plasmids as standard reference molecules. The results of our study demonstrate that the LNA 5'-nuclease PCR assays represent a valid and reliable analytical system for the detection and quantification of transgenes. Application of very short LNA TaqMan probes to GMO quantification can simplify the design of 5'-nuclease assays.     

Nature conservation in Italy: the existing national parks and other protected areas

This paper briefly reviews the present status of the Italian National Parks and other protected areas. Nature conservation is still rather weak in Italy and the likely causes of this are pointed out and discussed. Inadequate boundaries, hostility of park inhabitants, speculation by promoters, insufficient support by the State and by the Regional Governments badly affect all National and Natural Parks. This may depend partly on historical factors and partly on the lack of State interest and public opinion on nature preservation. On the other hand, there are a few signs that this situation is slowly becoming better and the management of the Parks has noticeably improved during the last five years. The activity of associations such as the World Wildlife Fund has been of particular importance in stimulating public opinion and in protecting valuable areas.     

Assay of the set of all Sudan azodye (I, II, III, IV, and Para-Red) contaminating agents by liquid chromatography-tandem mass spectrometry and isotope dilution methodology

A high-throughput mass spectrometric method is presented for the simultaneous detection of Sudan I, II, III, IV, and Para-Red azodyes in foodstuff. The method is based on the use of deuterium-labeled internal standards and atmospheric pressure chemical ionization (APCI) in a triple-quadrupole instrument. The gas-phase breakdown pattern of each labeled and unlabeled analogue displays the naphthoic moiety as a common fragment. The search for the parents of this common species (parent ion scans) allows, by flow injection and in a single run, the evaluation of the presence of each polluting species spiked in typical foodstuffs. A detailed assay of each azodye was performed by LC-APCI and isotope dilution method, through the multiple reaction monitoring approach, using deuterium-labeled internal standards. Sudan dyes can be quantified above the threshold of 10 ppb except for Sudan Para-Red, for which the limit of quantification was 20 ppb, likely due to the different ionization efficiency.     

Physicochemical Properties of Oat Varieties and Their Potential for Breadmaking

The breadmaking potential of six oat varieties was compared with and related to their physicochemical properties. The most significant differences in the bread characteristics were found in the crumb structure. The varieties Buggy, Energie, and Zorro resulted in good bread quality with an even gas‐cell distribution characterized by a high number of relatively small pores. In contrast, Typhon, Ivory, and Nord 08/311 each had a large hole in the center of the crumb and accordingly poor quality. Breads differed little in specific volume, bake loss, and density. Rheological analysis revealed positive effects of low batter resistance to deformation on oat bread quality. On the basis of the physicochemical characterization, protein and fat contents were identified as key factors responsible for differences observed in bread quality, provided that starch damage and water‐hydration capacity were low. Additionally, high setback and final viscosity, as determined by Rapid Visco Analyser (RVA) analysis, positively affected oat bread quality. High α‐amylase activity was found to influence negatively the breadmaking performance of oats. Overall, protein, fat, dietary fiber content, starch pasting properties, and α‐amylase activity were responsible for the breadmaking properties of oat varieties.     

Soil enzymatic activities and microbial biomass in an integrated agroforestry chronosequence compared to monoculture and a native forest of Colombia

Large tracts of land in South American native forests have been converted to monoculture for livestock production which could negatively affect soils and environmental quality. A proposed management alternative is to use agroforestry systems, but little information is available on how they affect the soils. The objective was to assess the effect of a silvopastoral chronosequence in a tropical region of Colombia on soil microbiological and physico/chemical properties. The systems (three replications) were: monoculture grass conventional pasture (CP), native forest (F), and a silvopastoral system (SS) chronosequence with ages of 3 to 6 (SS₃), 8 to 10 (SS₈), or 12 to 15 (SS₁₂) years. Soil responses to these land management were determined by measuring soil chemical (total C and pH), physical (penetrometer resistance and bulk density), and microbiological properties (activities of ß-glucosidase, urease, and alkaline and acid phosphatase and microbial biomass). Because of differences in soil texture across management treatments, microbiological properties were normalized on organic C content basis. SS₁₂ showed the highest microbial biomass and enzyme activities on a per unit C basis and was consistently and significantly different from CP. Additionally, microbiological to C ratios were significantly affected by SS establishment age (P?相似文献   

Sweet Pepper (Capsicum annuum L.) Organic Seedling Production: The Role of Compost,Cultivar, and Protein Hydrolyzate

In the past decade, the increasing environmental concerns regarding the use of non-renewable resources, such as peat, for growing media production has led to a search for alternative materials as substrate components. Until now, compost represents the most investigated one. Despite the critical aspects related to compost use (i.e., its high pH, salinity, and potential content of toxic elements), partial peat substitution with this material represents an achievable objective. In this study, green compost was evaluated as a component of peat substrates for organic sweet pepper (Capsicum annuum L.) seedling production. Three compost rates (CR) were compared: 0 (peat without compost), 30, and 60% of compost v/v. Two pepper cultivars, one derived from a local (medium-Adriatic) germplasm (L) and a commercial hybrid (H), were tested. Furthermore, organic fertilizers based on hydrolyzed proteins, of animal (A) and vegetal (V) origin, were compared. Substrates were analyzed for characterization (pH, EC, nutrient contents, heavy metals). Seedling performances were evaluated in terms of size, biomass, nutrient uptake, and toxic element contents. Indices able to assess seedling resistance to transplant stress were also calculated. Results showed that CR60 treatment was able to produce seedlings with the same biomass of control. CR30, instead, assured the same chance to resist the transplanting stress of CR0. The performances of fertilizer A were significantly higher than those of fertilizer V. Local germplasm (L) showed the highest resistance to transplant stress, while H was more performing in nitrogen uptake. The risk of seedling contamination by toxic elements resulted negligible.     

On the chemical composition and nutritional value of pleurotus taxa growing on umbelliferous plants (apiaceae)

Unpublished data on the chemical composition and nutritional value of Pleurotus mushrooms, growing on Umbelliferous plants (Apiaceae), are here reported. Cultivated basidiomata of four different Pleurotus taxa were analyzed in order to evaluate the composition in lipids, sugars, nitrogen, water, vitamins, ashes, and energetic values. The results showed that Pleurotus mushrooms are suitable in every type of diet thanks to their low caloric content, gastronomic value, vitamins, and mineral salt contents. The presence of a high content of vitamin B(12) and riboflavin in Pleurotus nebrodensis is noteworthy.     

Quantification of the biocontrol agent Pseudomonas fluorescens Pf153 in soil using a quantitative competitive PCR assay unaffected by variability in cell lysis- and DNA-extraction efficiency

Although often neglected, variability in cell lysis efficiency and DNA extraction yield represents the major hurdles of any polymerase chain reaction (PCR)-based quantification protocol in soil and other natural environments. In this study we developed a technique that minimizes the effects of these constraints, providing at the same time a reliable internal control to distinguish between PCR-inhibition and negative results. We used Pseudomonas fluorescens Pf153, a root-colonizing bacterium that shows biocontrol activity against tobacco and cucumber black root rot, as the target organism for PCR quantification. Prior to DNA extraction, the genetically engineered, cognate reference strain P. fluorescens CHA0/c2 was inoculated in a reference soil. CHA0/c2 in the reference soil and Pf153 in the soil sample were lysed in parallel and afterward the lysates were mixed in known proportions. CHA0/c2 carries the plasmid pME6031-cmp2 that contains an allelic variant (competitor) of the Pf153 specific sequence Pf153_2. In a quantitative competitive PCR (QC-PCR) assay the competitor allows the quantification of the target strain down to 0.66 Pf153 CFU/mg soil. Processing the reference strain in the same way as Pf153 enables the exact quantification of the target strain in biocontrol assays performed in natural soil, overcoming differences in DNA extraction efficiency and PCR amplification from different soil environments. This technique is easily adaptable to other Pseudomonas strains simply by replacing the competitor used here with one derived from a SCAR-marker which is specific for the strain of choice.     

Characterisation of thirteen cowpea landraces from Umbria (Italy). Strategy for their conservation and promotion

Thirteen landraces of cowpea (Vigna unguiculata (L.) Walp. subsp. unguiculata cv. gr. Unguiculata) have been morphologically and agronomically characterised as part of a plan for safeguarding and protecting plant biodiversity typical of Lake Trasimeno (Umbria, central Italy). Sixty plants per accession were scored for plant vigour, growth habit, flower colour, days to flowering, days to first mature pod, pod length, total number of pods produced, seed colour pattern, 100 seed weight, total seed yield and virus susceptibility. With the exception of vigour and growth habit, all landraces appeared to be well differentiated from one another for all the characters considered. Strategies and problems related to their in situ conservation are discussed.