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.     

Survey on housing,management and welfare of dairy cattle in tie-stalls in western Italian Alps

In the Alps, the traditional breeding system for dairy cattle is based on the alternation between a free-ranging period on mountain ranges during the summer, and an indoor period in tie-stalls in the winter. Several welfare issues may arise in tie-stall housing systems. We describe the situation in 47 farms in three villages in western Italy, trying to identify possible relationships among structural and management characteristics, animal health and behaviour traits. A long duration of the grazing period, associated with frequent manure removal during the housing period, are probably key factors for limiting the occurrence of lameness. Teat trauma is more common in narrower stalls. Getting-up behaviour is unnatural in most of the visited farms. Some lack in the farmers’ knowledge of animal behaviour was noted. Some structural and management characteristics are strictly related to geographical constraints. However, circumstances permitting, some expedients may be achieved for improving welfare levels.     

Isolation and identification of two lipid transfer proteins in pomegranate (Punica granatum)

Lipid transfer proteins (LTPs) are a family of low molecular mass (7-9 kDa) polypeptides, the members of which share 35-95% sequence homology. These proteins are widely distributed throughout the plant kingdom and are receiving attention for their biochemical characteristics and biological activity. LTPs are indeed studied in different research fields varying from allergy to food technology, and numerous molecules belonging to this class are progressively being identified and investigated. Proteins from pomegranate juice were fractioned by cation exchange chromatography and analyzed by SDS-PAGE. Two proteins were identified as putative LTPs on the basis of their molecular weights and their electrophoretic behaviors under reducing and nonreducing conditions. Finally, proteins were purified and characterized by mass spectrometry. This analysis confirmed that the two polypeptides are LTPs on the basis of an amino acid sequence common to LTPs from other plant sources and cysteine content. The two proteins, named LTP1a and LTP1b, showed similar molecular masses but different immunological profiles when immunodetected with rabbit antibodies specific for Pru p 3 and human IgE from a patient suffering from pomegranate allergy. The demonstration of the existence of two immunologically unrelated LTPs in pomegranate confirms the variability and the complexity of the plant LTP family. This should be taken into account when the role of these proteins as elicitors of allergies to fruits is investigated and could help to explain the contradictory literature data on pomegranate allergy.     

Real-time Scorpion-PCR detection and quantification of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> on pear leaves and flowers

A specific primer couple (E3–E4) amplifying a single DNA fragment of 111 bp from plasmid pEA29 was designed to identify, detect and quantify Erwinia amylovora by real-time Scorpion-PCR. Specificity of primers and probe was assessed both by means of BLAST analyses and by using genomic DNA from a large number of E. amylovora isolates and other bacteria. In Scorpion-PCR, the limit of detection was of 1 pg of total DNA and a high correlation (r = 0.999) was achieved between target DNA quantity and cycle threshold (Ct). Combining two sequential amplifications with conventional reported primers (PEANT1–PEANT2) and Scorpion primers (E3 Scorpion-E4) the detection limit was of 1 fg (nested Scorpion-PCR). Using serial dilution of the bacterial suspensions the limit of detection was 3.2 × 10⁴ CFU ml⁻¹ in Scorpion-PCR and 2.8 × 10² CFU ml⁻¹ in nested Scorpion-PCR. Real-time PCR combined with effective procedures for DNA extraction enabled the detection and the quantification of the epiphytic population of E. amylovora in the washings of flowers and leaves of artificially inoculated pear. A significant correlation (r = 0.92) was achieved between pathogen CFU on semi-selective media and the corresponding target DNA concentration evaluated by real-time PCR.     

Evaluation of dicarboximide residues in cold-stored grapes exposed to field and post-harvest treatments

Dicarboximide fungicides were used in field treatments as well as in post-harvest fumigations on cold-stored table grapes. They proved to be highly effective against gray mould, a major disease during storage or transport of grapes, and a valuable alternative to sulphur dioxide within one month after harvesting. Residues of the fungicides were determined through gas-chromatographic analysis on grapes field-sprayed with dichlozolinate, iprodione, procymidone or vinclozolin, and on grapes exposed to post-harvest treatments with new smoke formulations of procymidone and vinclozolin. In most cases, residue levels were lower than those allowed by Italian sanitary laws. Post-harvest fumigations caused only a slight increase of residue levels as compared to field applications.     

Comparing geometric morphometrics and outline fitting methods to monitor fish shape variability of Diplodus puntazzo (Teleostea: Sparidae)

The definition of a remote system in the monitoring of fin fish shape variability relies on the appropriate shape analysis tools. In this paper, different methods are proposed and compared. Growth trajectories, computed using geometric morphometrics, allow the pattern of size-related shape change and visualize growth allometry to be analyse. Geometric morphometrics is used to describe shape change in three samples (507 5100 days after hatching) of 119 individuals of the Sparid Diplodus puntazzo. The results are potentially interesting but the method still relies on the manual detection of homologous landmarks (coordinates of points located unambiguously on each specimen’s profile or structure) which are basic for such analysis. In an attempt to proceed towards the remote estimation and the automation of data collection, two alternative approaches are tested. In particular, Elliptic Fourier analysis and Bezier function were applied to the same samples, and results compared with geometric morphometrics. Fourier analysis’ growth curves are very similar to geometric morphometrics’ ones and allows for an appropriate visualisation of global shape changes. At the same time, Fourier methods can be applied to remotely and automatically detected profiles. This method is thus considered as the best candidate in the context of automated fish shape monitoring. The Bezier method performed poorly, both in terms of statistical output and shape visualization.     

Flavonoid glycosides in bergamot juice (Citrus bergamia Risso)

A comprehensive profile of flavonoids in bergamot juice was obtained by a single DAD-ESI-LC-MS-MS course. Eight flavonoids were found for the first time, five of these are C-glucosides (lucenin-2, stellarin-2, isovitexin, scoparin, and orientin 4'-methyl ether), and three are O-glycosides (rhoifolin 4'-O-glucoside, chrysoeriol 7-O-neohesperidoside-4'-O-glucoside, and chrysoeriol 7-O-neohesperidoside). A method is proposed to differentiate chrysoeriol and diosmetin derivatives, which are often indistinguishable by LC-MS-MS. In-depth knowledge of the flavonoid content is the starting point for bergamot juice exploitation in food industry applications.     

Quantification of trans-3,4,5,4'-Tetramethoxystilbene in rat plasma by HPLC: application to pharmacokinetic study

A simple HPLC method was established to quantify trans-3,4,5,4'-tetramethoxystilbene (MR-4 or DMU-212) in rat plasma. Chromatographic separation was obtained with a reversed-phase HPLC column through an 11 min gradient delivery of a mixture of acetonitrile and water at a flow rate of 1.5 mL/min at 50 °C. The limit of quantification was 15 ng/mL. The intra- and interday precisions in terms of relative standard deviation were <9% at all concentrations. Similarly, the accuracy was good, and the bias rates ranged within ±7%. The pharmacokinetic profiles of MR-4 were subsequently assessed in rats using 2-hydroxypropyl-β-cyclodextrin as a dosing vehicle. Upon intravenous administration, MR-4 displayed moderate clearance (46.5 ± 7.6 mL/min/kg) and terminal elimination half-life (154 ± 80 min). However, the absolute oral bioavailability of MR-4 was low (6.31 ± 3.30%). Future investigation on MR-4 as a chemotherapeutic agent should be focused on colorectal cancers.