Massimo Luzzana

Detection of enterotoxigenic Staphylococcus aureus isolates in raw milk cheese

Aim:  To develop an easy, rapid and efficient DNA extraction procedure for Staphylococcus aureus detection with a low number of steps and removing completely the PCR inhibitors, applicable to raw milk cheese samples, and to compare phenotypical and genotypical method to detect Staph. aureus isolates and staphylococcal enterotoxins (SEs) production.

Continuous Determination of the Oxygen Dissociation Curve for Whole Blood

We report here the development of a new method that allows continuous determination of the oxygen dissociation curve for microsamples (600 mul) of whole blood under conditions of pH, pCO2, methemoglobin concentration, and 2,3-diphosphoglycerate content closely approaching those found in the circulatory system. The method consists of gradually oxygenating a blood sample by adding H2O2 in the presence of catalase (EC 1.11.1.6), to produce the reaction H2O2 leads to H2O + 1/2 O2. Because the total oxygen content of blood can be derived from the known rate of H202 addition and the pO2 is determined in the liquid phase by an oxygen electrode, the two functions (total O2 content) and (% oxygen saturation) vs. pO2 are simple to calculate. pCO2 and pH are controlled by adding base simultaneously with the gradual oxygenation of blood. The method described thus avoids the direct measurement of oxygen saturation of whole blood.

A horizontal apparatus for isoelectric protein purification in a segmented immobilized pH gradient

A modification of the previously described apparatus (Faupel et al. (1987) J. Biochem. Biophys. Methods 15, 147-162), for recycling isoelectric focusing in a segmented immobilized pH gradient, is here reported. The most important improvements are: (1) a horizontal, vs. the previously vertical assembly; (2) a reduction of the thickness of the central flow chamber to 6 mm, vs. the previous 3 cm length and (3) the introduction, at both gel extremities of each Immobiline segment, of polypropylene filters, thus efficiently blocking the gel in situ. The advantages are: (i) the spontaneous removal of air bubbles, which in the vertical apparatus tend to accumulate in the ceiling of the flow chamber and to obstruct the flow of electric current; (ii) a more efficient hydraulic flow with a reduced chance of heating the liquid stream in the flow chamber, due to its reduced length along the separation path and (iii) a reduced risk of gel detachment from the tube walls, due to osmotic swelling caused by focused protein zones in the gel phase and by the fixed Immobiline charges in the polyacrylamide matrix.

Fractionation of carrier ampholytes for isoelectric focusing.

A simple method for fractionating synthetic carrier ampholytes is reported, based on the principle of continuous-flow isoelectric focusing in gel-stabilized layers. An 8% ampholyte solution, encompassing the pH range 3-9.5, is separated into 12 fractions in a chamber filled with Sephadex G-100 by a continuous-flow technique. We are thus able to obtain ampholytes of narrow pH range, encompassing approximately 2 pH units, whose resolving power is comparable with that obtained with commercial Ampholine covering similar pH ranges.

The functional properties of sickle cell blood

It is known that whole blood from patients with sickle cell anemia has a decreased oxygen affinity [ 1,2]. Purified hemoglovin S solutions, however, are normal [3,4]. A systematic study of the effect of 2,3-DPG and of other known allosteric regulators of oxygen affinity of HbS in whole blood seems desirable, indeed essential, to understand the reason for the altered oxygen affinity of the hemoglobin S molecule in the intact red cell environment. A more complete understanding of the functional properties of SSblood would also be important to assess the physiological significance of such an alteration, and to evaluate the therapeutic effect of cyanate, administration.

Development of DNA Extraction and PCR Amplification Protocols for Detection of Mycoplasma bovis Directly from Milk Samples

Cremonesi, P., Vimercati, C., Pisoni, G., Perez, G., Miranda Ribera, A., Castiglioni, B., Luzzana, M., Ruffo, G. and Moroni, P., 2007. Development of DNA extraction and PCR amplification protocols for detection of Mycoplasma bovis directly from milk samples. Veterinary Research Communications, 31(Suppl. 1), 225–227

Enzymatic reactions for the determination of sugars in food samples using the differential pH technique.

All ATP coupled reactions, when performed at neutral or moderately alkaline pH, produce an acidification of the reaction mixture. The detection of small pH changes--0.1 mpH (1 mpH = 10(-3) pH)--in a constant buffering capacity solution makes it possible to quantify, over a wide concentration range (1-1500 mmol L(-1)), various analytes with very high precision and accuracy. Glucose, fructose, glycerol and gluconic acid can be analysed in less than 1 min with a single step reaction. Wine samples were analysed using the hexokinase reaction for glucose + fructose (sugars undergoing fermentation) and compared against an established method, showing excellent performance over the whole range of concentrations (R = 0.9994). Increased sensitivity in some applications can be obtained by cycling reactions, e.g. a kinase reaction followed by a phosphatase reaction. in a one step analysis, as required for lactulose assay in milk, a useful indicator of heat treatment damage. A sensitivity well below 0.1 mmol L(-1) in the original milk sample has been demonstrated.

Identification of Enterotoxin Genes in Staphylococcus aureus Isolates from Bovine and Caprine Milk

Among the main microorganisms responsible for bovine, ovine and caprine mastitis, Staphylococcus aureus is important, not only with respect to the animal disease, but also because of its potential impact on public health and food safety. In fact, some Staphylococcus aureus strains can produce enterotoxins which could cause food poisoning through consumption of milk or dairy products, particularly cheeses manufactured with raw milk. Inasmuch as milk of infected animals is considered the main source of enterotoxigenic S. aureus of animal origin, the importance of an efficient screening process for the presence of enterotoxigenic strains is required. The aim of this study was to develop an assay using a multiplex PCR technique to verify the presence of genes encoding for classical (from A to D) and recently described (G, H, I, J, L) staphylococcal enterotoxins in Staphylococcus aureus strains isolated from bovine and caprine milk.

Surface-activated chemical ionization and cation exchange chromatography for the analysis of enterotoxin A

Surface-activated chemical ionization (SACI) has been widely used in recent years for the analysis of different compounds (e.g. peptides, street drugs, amino acids). The main benefits of this technology are its high sensitivity and its effectiveness under different chromatographic conditions [i.e. ion exchange chromatography and reversed-phase (RP) chromatography]. Here we used SACI in conjunction with quadrupole time-of-flight mass spectrometry to analyze enterotoxin A, which is produced by Staphylococcus aureus, in milk matrix using both RP and ion exchange chromatographies. SACI had increased sensitivity as compared with electrospray ionization. Moreover, the higher quantitation efficiency of this technique, mainly in terms of limit of detection (0.01 ng/ml), limit of quantitation (0.05 ng/ml), linearity range (0.05-50 ng/ml), matrix effect, accuracy (intraday and interday accuracy errors were 9.2% and 10.3%, respectively) and precision (intraday and interday precision errors were 5.3% and 12.8%, respectively), is shown and discussed.

Analysis of genetic polymorphisms in Staphylococcus aureus strains isolated from bovine milk

C. Vimercati1, P. Cremonesi2, B. Castiglioni3, P. Boettcher3, M. Luzzana2, G. Ruffo1 and P. Moroni1,∗ 1Department of Animal Pathology, Hygiene and Veterinary Public Health, University of Milano, Italy; 2Department of Biomedical Sciences and Technologies, University of Milano, Italy; 3Institute of Agricultural Biology and Biotechnology, Italian National Research Council, Milano, Italy ∗Correspondence: E-mail: paolo.moroni@unimi.it