Detection of food-borne pathogens with DNA arrays on disk.

A DNA oligonucleotide array for duplex pathogen detection on a DVD platform is developed. The assay involves hybridization of PCR products and optical detection using compact disc technology. Different DNA array constructions for attachment of synthetic oligonucleotides on to DVD surface are evaluated, finding that streptavidin-biotin coupling method yielded the highest sensitivity in combination with enzymatic signal amplification. Issues of importance for the DNA array construction such immobilized probes design, PCR product labeling strategy and composition of the hybridization buffer were addressed. The methodology was proved scoring single nucleotide polymorphisms with high selectivity. The assay capability was also demonstrated by the identification of two pathogenic microorganisms in powder milk samples. In fifty minutes, the DVD-array system identifies Salmonella spp. and Cronobacter spp. (previously named Enterobacter sakazakii) precise and simultaneously with a sensitivity of 10(0) and 10(2) cfu/mL, respectively, in infant milk. Results were in good agreement with those obtained by quantitative real-time PCR.

Application of pressurized fluid extraction to determine cadmium and zinc in plants.

A procedure for the determination of Cd and Zn in plants is proposed. The metals are extracted by pressurized fluid extraction (PFE). Operational conditions are: pressure 1500 psi, temperature 75 degrees C, static time 5 min, flush volume 35%, purge time 60s, cycles 1 and 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) 0.01M at pH 4.5 as extracting solution. Determination of Zn is carried out by flame atomic absorption spectroscopy and depending on the concentration level, Cd content is determined by flame or electrothermal atomic absorption spectroscopy. Certified samples of Virginia tobacco leaves, tea leaves, spinach leaves, poplar leaves, a commercial spinach sample (Spinacea oleracea) and genetically modified Arabidopsis thaliana were analysed by the proposed procedure and also by microwave acid digestion and extraction with HCl-Triton X-100. Confidence intervals for Cd and Zn content obtained by the proposed procedure overlap with the certified values. The other procedures, however, provide inaccurate results for Cd. Recoveries obtained for a confidence level of 95% are 96+/-6% and 95+/-5% for Zn and Cd, respectively. Reproducibility of Zn by the proposed procedure is 7% (n=8), similar to the other tests and the detection limit is 2.6 microg. For Cd reproducibility is 8.5% (n=8), better than with HCl-Triton X-100 and similar to acid digestion, the detection limit is 3.5 ng of Cd.