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. METHODS AND RESULTS: A total of 33 bovine and caprine raw milk cheese samples were analysed by means of both classic microbiological and molecular techniques. All samples were positive for Staph. aureus contamination. The DNA extraction protocol optimized was found to achieve a detection limit of 100 CFU g(-1) for Staph. aureus. None of the samples tested with immunological assays contained SEs but in 14 of 33 samples a mixture of se positive (sea, sec, sed, seg, sel, sej) isolates were identified. CONCLUSIONS: Staphylococcus aureus is a food-borne pathogen mainly detected in finished dairy products. The rapid and efficient detection of Staph. aureus isolates from dairy products is essential for consumer safety. The direct detection of pathogens from food is possible with careful attention to sample preparation and nucleic acid amplification optimization. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that raw milk cheese samples can be tested for Staph. aureus contamination with a rapid, simple and reproducible procedure.

Technical note: Improved method for rapid DNA extraction of mastitis pathogens directly from milk.

Efficient control against bovine mastitis requires sensitive, rapid, and specific tests to detect and identify the main bacteria that cause heavy losses to the dairy industry. Molecular detection of pathogenic microorganisms is based on DNA amplification of the target pathogen. Therefore, efficient extraction of DNA from pathogenic bacteria is a major step. In this study, we aimed to develop a specific, sensitive, and rapid method to extract DNA directly from the main gram-positive bacteria known to cause bovine mastitis (Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis) found in milk samples. The DNA extraction method is based on the lysing and nuclease-inactivating properties of the chaotropic agent, guanidinium thiocyanate, together with the nucleic acid-binding properties of the silica particles. An efficient protocol consisting of 6 basic steps (3 of which were done twice) was developed and applied directly to milk samples. Absence of PCR inhibitors and DNA quality were evaluated by PCR amplification of the species-specific DNA sequences of the target bacteria. The level of sensitivity achieved in our experiments is applicable to milk sample analysis without sample enrichment.

A microdialysis technique for continuous subcutaneous glucose monitoring in diabetic patients (part 1).

The performances and the stability of a novel subcutaneous glucose monitoring system have been evaluated. GlucoDay (A. Menarini I.F.R. S.r.l, Florence Italy) is a portable instrument provided with a micro-pump and a biosensor coupled to a microdialysis system capable of recording the subcutaneous glucose level every 3 min. Long and short term stability of the biosensor are discussed and the results of some critical in vitro and in vivo (on rabbits) experiments are reported. A linear response up to 30 mM has been found for in vivo glucose concentration. The sensitivity referred to blood glucose is better than 0.1 mM and the zero current is typically below the equivalent of 0.1 mM. In the accuracy study a mean bias of 2.7 mg/dl and a correlation coefficient equal to 0.9697 have been found. At room temperature, an excellent membrane stability assures good performances up to 6 months from the first use.

A microdialysis technique for continuous subcutaneous glucose monitoring in diabetic patients (part 2).

The aim of this study was to evaluate the reproducibility, the accuracy and the reliability of a continuous subcutaneous glucose measuring system. The GlucoDay system (A. Menarini I.F.R. S.r.l.-Florence, Italy) is a portable instrument provided with a micro-pump and a biosensor, coupled to a microdialysis system (see part 1). This instrument has demonstrated high reliability coupled with a low degree of invasivity. The profiles of glucose monitoring allow to achieve an excellent knowledge of the real variation of glucose in diabetic patients. The reproducibility study showed a bias lower than 10% between instruments. The accuracy study showed a difference from the reference method lower than 15%.

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.

Apolipoprotein E genotyping by capillary electrophoretic analysis of restriction fragments.

We present the genotyping of apolipoprotein (apo) E by means of restriction fragment analysis of amplified genomic DNA by high-performance capillary electrophoresis and a replaceable non-gel-sieving matrix. This procedure streamlines the genotyping of apo E in large-scale population studies because of the automation and speed of capillary electrophoresis.

Simultaneous automated determination of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities in whole blood.

We report a potentiometric fully automated method for determining red cell glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase activities and the glucose 6-phosphate dehydrogenase/6-phosphogluconate dehydrogenase index using 25 microliters of whole blood. No sample pre-treatment (e.g., preparation of the haemolysate) is needed and the measurements are performed at pH 8.0 and 37 degrees C under the conditions recommended by the ICSH committee. The reproducibility was constantly good, with within-run CV of 1.0% (glucose 6-phosphate dehydrogenase) and 5.9% (glucose 6-phosphate dehydrogenase/6-phosphogluconate dehydrogenase) for activities in glucose 6-phosphate dehydrogenase non-deficient adults, and of 2.3% (glucose 6-phosphate dehydrogenase, G6PD) and 2.5% (glucose 6-phosphate dehydrogenase/6-phosphogluconate dehydrogenase) for G6PDMediterranean heterozygotes. Linearity was observed up to an activity of 2800 U/l of glucose 6-phosphate dehydrogenase. Results of glucose 6-phosphate dehydrogenase activity (U/l) in whole blood (y) correlated well with those obtained with the previously described monostarter assay, performed at pH 9.2 (y = 0.60x + 37; n = 80; r = 0.991). Results of 6-phosphogluconate dehydrogenase (U/l) in whole blood (y) correlated well with those obtained by the ICSH recommended method (x) (y = 0.779x - 44; n = 23; r = 0.991). Reference intervals are reported for glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase/6-phosphogluconate dehydrogenase index relatively to normal, beta- and alpha-thalassaemic glucose 6-phosphate dehydrogenase non-deficient adults, to glucose 6-phosphate dehydrogenase deficient adult males and to G6PDMediterranean non-thalassaemic heterozygotes. We demonstrate that the diagnostic sensitivity of the glucose 6-phosphate dehydrogenase/6-phosphogluconate dehydrogenase index in detecting the G6PDMediterranean heterozygotes is superior to that of the glucose 6-phosphate dehydrogenase activity alone.

Solid phase purification and SSCP analysis of amplified genomic DNA by capillary electrophoresis.

Detection and identification of point mutations in genomic DNA has proven increasingly important in biomedical research. A variety of methods for the analysis of single base substitutions have been proposed among which Single Strand Conformational Polymorphism (SSCP) quickly gained success due to its simplicity. In this work we present an analytical on-line tool which combines the ease of solid phase purification of amplified genomic DNA, the simplicity of SSCP and the significant potential advantages offered by capillary electrophoresis (CE).

Anion-exchange HPLC analysis of biotinylated oligonucleotides.

Biotinylated oligonucleotides combined with streptavidin-coated magnetic beads are commonly used in current molecular biology. Their quality and the level of incorporated biotin are essential for yielding good results in either solid-phase DNA sequencing or solid-phase purification procedures. This paper presents a very simple analytical test using anion-exchange HPLC and avidin to ascertain the quality of biotinylated oligonucleotides and to predetermine their ability to bind to avidin, which is a prerequisite for functionality in some solid-phase methods.

Fluorescence-based automated DNA sequencing by limited primer labeling.

The applicability of automated DNA sequencing systems to sequencing strategies that require a large number of primers is limited by the necessity for expensive fluorescence-labeled oligonucleotides. Here we present a simple procedure that allows the use of unlabeled oligonucleotides to perform fluorescence-based DNA sequencing. This method is based on a limited primer extension that incorporates three deoxynucleotides, one of which carries a fluorescent moiety. The elongated fluorescent primer is then used in a standard T7 sequencing reaction. This labeling procedure is both economical and straightforward and offers a valid alternative to current fluorescence-labeling protocols. Results of this method with different DNA templates demonstrate the reliability of the protocol.

A patient-side technique for real-time measurement of acetylcholinesterase activity during monitoring of eptastigmine treatment.

Rapid and reliable measurement of acetylcholinesterase (AChE) activity is of crucial importance to the pharmacodynamic monitoring of anticholinesterase drugs. A new assay has been developed to measure AChE from 10 microliter samples of capillary blood. AChE activity was calculated from the change in pH of the reaction medium caused by the hydrolysis of acetylcholine and measured with a highly sensitive differential pH apparatus (CL-10, Eurochem, Rome, Italy). Interference by butyrylcholinesterase was eliminated by a specific inhibitor, quinidine sulfate. The assay lasts 1 min. The coefficient of variation (CV) for replicated measurements was 2.8% (3267 U/L, n = 33). Linearity ranged from 0 to 10,000 U/L. The correlation coefficient between the new technique and Ellman's colorimetric method on washed erythrocytes was r = 0.987 (y = 1.299x - 63, n = 29). The correlation coefficient between assays on capillary and venous samples was r = 0.979 (y = 0.974x + 174, n = 47). A cross-laboratory validation study was performed in 10 centers using glycerol-stabilized hemolysates with normal and reduced AChE activity. Samples were assayed in triplicate. The within- and between-laboratory CVs for samples with normal AChE activity (6,018 U/L) were 2.2 and 8.1%, respectively. The new method was applied to a double-blind, placebo-controlled multicenter study of eptastigmine in Alzheimer patients and proved to be a simple, noninvasive, rapid, and reliable method for pharmacodynamic monitoring of this drug.

Mixed-mode fluorescent DNA sequencing.

Fluorescence-based, automated DNA sequences represent one of the major advances in recent molecular biology. Two main technologies have been developed in this field: the single-label/four-lane system and the four-label/one-lane system. The following present the use of single-label-sequencing chemistry, which resembles traditional radioactive DNA sequencing, using the four-label system ABI 373A that expands its flexibility and obtains data that are immediately interpretable without software manipulation. This method has been named mixed-mode fluorescent DNA sequencing. Here we show one of its possible applications in molecular genetic analysis.