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.     

Isotope ratios of lead in Italian wines by inductively coupled plasma mass spectrometry

Lead and its isotope ratios (IRs) in 83 Italian wines from 42 different administrative provinces produced on laboratory scale in 2000 were measured by inductively coupled plasma mass spectrometry. Lead had a median of 19.0 microg/L, ranging from 10.0 up to 149 microg/L and was higher in the samples from northwestern Italy. The values of the IRs were (mean +/- standard deviation) 1.171 +/- 0.011 for (206)Pb/(207)Pb, 2.071 +/- 0.017 for (208)Pb/(206)Pb, 2.425 +/- 0.014 for (208)Pb/(207)Pb, 17.84 +/- 0.32 for (206)Pb/(204)Pb, 15.24 +/- 0.31 for (207)Pb/(204)Pb, and 37.31 +/- 0.52 for (208)Pb/(204)Pb. These ratios agree with those in the literature for several European regions. (206)Pb/(207)Pb distinguished the northwestern from the northeastern and southern Italian wines, and the southern samples were distinguishable only from the northwestern ones. (208)Pb/(206)Pb distinguished the northwestern from the southern Italian samples. Nevertheless, the lead IRs do not seem to be a very effective tool for the origin authentication of wines from different Italian regions.     

Determination of choline in milk, milk powder, and soy lecithin hydrolysates by flow injection analysis and amperometric detection with a choline oxidase based biosensor

A fast-response and interference-free amperometric biosensor based on choline oxidase immobilized onto an electropolymerized polypyrrole film for flow injection determination of choline in milk, milk powder, and soy lecithin hydrolysates is described. The sensor displayed an Imax value of 1.9 +/- 0.2 microA and an apparent Michaelis-Menten constant, k'M, equal to 1.75 +/- 0.07 mM. Detection limits of 0.12 microM could be obtained. Because even a slight deterioration of the anti-interference membrane can adversely affect measurement accuracy, a real time monitoring of the biosensor selectivity has been achieved by a dual Pt electrode flow-through cell where the enzyme modified electrode is coupled to an enzyme-free electrode in a parallel configuration. Finally, bracketing technique (alternate injections of sample and standards) allows a two-point calibration to be performed in real-time, correcting for any drift in sensor response.     

Rapid quantitative assessment of the adulteration of virgin olive oils with hazelnut oils using Raman spectroscopy and chemometrics

The authentication of extra virgin olive oil and its adulteration with lower-priced oils are serious problems in the olive oil industry. In addition to the obvious effect on producer profits, adulteration can also cause severe health and safety problems. A number of techniques, including chromatographic and spectroscopic methods, have recently been employed to assess the purity of olive oils. In this study Raman spectroscopy together with multivariate and evolutionary computational-based methods have been employed to assess the ability of Raman spectroscopy to discriminate between chemically very closely related oils. Additionally, the levels of hazelnut oils used to adulterate extra virgin olive oil were successfully quantified using partial least squares and genetic programming.     

Survey of polychlorinated dibenzo-p-dioxins (PCDDs), Polychlorinated Dibenzo-p-furans (PCDFs), polychlorinated biphenyls (PCBs), and mineral components in Italian citrus cold-pressed essential oils

Investigation of PCDDs, PCDFs, PCBs, Al, As, Pb, Ba, Co, Cu, Cr, Fe, Mn, Cd, Ag, Sn, Zn, and Hg contents in 60 samples of cold-pressed essential oils produced in Calabria and Sicily in 2003-2005 was carried out. PCDDs, PCDFs, and PCBs were analyzed by HRGC-HRMS techniques using U.S. EPA 1613/94 and U.S. EPA 1668/A (1999) analytical methods. Mineral components were determined through GFAAS techniques; Hg content was determined by FI-M/H-AAS. The results of this study showed that essential oil contamination was due to a widespread pollution, typical background of rural areas, with relatively higher concentrations of PCDDs compared to PCDFs and little presence of PeCDF. Congeners OCDD, HpCDF, and OCDF were found at high concentrations. Regarding mineral components, mean values of Cr, Fe, and Ni were in agreement with data reported in the literature. Concentrations of As and Pb were below the maximum limits accepted by the current legislation. Finally, none of the samples analyzed were contaminated with Hg.     

Studies on the phytotoxicity of acid smuts

Experiments were carried out to investigate, under controlled conditions, the effects of acid smut emitted by oil-fired power plants. The foliar response of 31 selected plant species was ranked and marked differences in sensitivity were observed. Anatomical features of the foliar lesions were described. The toxicity of smuts was largely dependent on their acidity and size. Toxicity was related to temperature but was completely independent of relative humidity in the range from 50 to 90%. No interactive effects were observed between acid smuts and some commercial fungicides, or treatments with asymptomatic concentrations of So₂ in fumigation chambers. Some growth parameters were negatively affected by acid smuts in radish.     

Evaluation of a method for assaying sulfonamide antimicrobial residues in cheese: hot-water extraction and liquid chromatography-tandem mass spectrometry

Several sulfonamide antimicrobials (SAAs) are largely used in veterinary medicine. A rapid, specific, and sensitive procedure for determining 12 SAAs in cheese is presented. The method is based on the matrix solid-phase dispersion technique followed by liquid chromatography (LC)-tandem mass spectrometry (MS) equipped with an electrospray ion source. Target compounds were extracted from Mozzarella, Asiago, Parmigiano, Emmenthal, and Camembert cheese samples by 6 mL of water modified with 10% methanol and heated at 120 degrees C. The addition of methanol to hot water served to improve remarkably extraction yields of the most lipophilic SAAs, that is, sulfadimethoxine and sulfaquinoxaline. After acidification and filtration, 100 microL of the aqueous extract was injected in the LC column. MS data acquisition was performed in the multireaction monitoring mode, selecting two precursor-to-product ion transitions for each target compound. Methanol-modified hot water appeared to be an efficient extractant, because absolute recovery ranged between 67 and 88%. Using sulfamoxole as surrogate analyte, recovery of the 12 analytes spiked in the five types of cheese considered at the 50 ng/g level ranged between 75 and 105% with RSD not higher than 11%. Statistical analysis of the mean recovery data showed that the extraction efficiency was not affected by the type of cheese analyzed. This result indicates this method could be applied to other cheese types not considered here. The accuracy of the method was determined at three spike levels, that is, 20, 50, and 100 ng/g, and varied between 73 and 102% with relative standard deviations ranging between 4 and 12%. On the basis of a signal-to-noise ratio of 10, limits of quantification were estimated to be <1 ng/g.     

Genetic immunization with the immunodominant antigen P48 of Mycoplasma agalactiae stimulates a mixed adaptive immune response in BALBc mice

A DNA vaccine against contagious agalactia was developed for the first time, encoding the P48 of Mycoplasma agalactiae. Specific immune responses elicited in BALB/c mice were evaluated. Both total IgG and IgG1 were detected in mice vaccinated with pVAX1/P48. Proliferation of mononuclear cells of the spleen, levels of gamma interferon, interleukin-12, and interleukin-2 mRNAs were enhanced in immunized animals. Results indicate that pVAX1/P48 vaccination induced both T_h1 and T_h2 immune responses. Nucleic acid immunization could be a new strategy against M. agalactiae infections and may be potentially used to develop vaccines for other Mycoplasma diseases.