An 8-hour system for Salmonella detection with immunomagnetic separation and homogeneous time-resolved fluorescence PCR.

We describe a system consisting of rapid sample enrichment and homogeneous end-point PCR analysis that enables the detection of Salmonella in various food matrices in 8 h. Sample preparation starts with 6 h enrichment step in supplemented broth, after which Salmonella cells are collected with immunomagnetic particles. The particles are washed and dispensed to ready-to-use PCR reaction vessels, which contain dried assay-specific reagents and an internal amplification control. PCR is performed with a novel instrument platform utilising the sensitive label technology of time-resolved fluorometry. Qualitative assay results are automatically interpreted and available in 45 min after sample addition. The overall accuracy, sensitivity and specificity of the Magda CA Salmonella system were 99.1%, 98.4% and 100.0%, respectively, based on the evaluation of 107 samples (beef, pork, poultry and ready-to-eat meals) artificially contaminated with sub-lethally injured Salmonella cells.

An automated PCR platform with homogeneous time-resolved fluorescence detection and dry chemistry assay kits.

We have developed a novel instrument platform, GenomEra, for small-scale analysis of nucleic acids. The platform combines a rapid thermal cycler, an integrated time-resolved fluorescence measurement unit, and user-friendly software for the analysis of results. Disposable low-cost plastic reaction vessels are designed specifically for the instrument and contain all of the assay-specific reagents in dry form. The appropriate assay protocol is specified on barcodes printed under the vessels and is automatically initiated by the software. Detection is based on the use of sequence-specific probes labeled with intrinsically fluorescent europium or terbium chelates and complementary quencher probes, which enable sensitive, homogeneous closed-tube assays without the risk of carryover contamination. The detection limit of the instrument (background + 3 SD) is approximately 20 pmol/L for both chelates with a dynamic range of nearly four orders of magnitude. The functionality of the platform is demonstrated with a dual-label homogeneous polymerase chain reaction (PCR) assay for the detection of Salmonella using a Magda CA Salmonella assay kit. An internal amplification control is included in each reaction to eliminate false negative results caused by PCR inhibition. Qualitative assay results are automatically interpreted by the software and are available 45 min after sample addition.

Synthesis, characterization, and application of Eu(III), Tb(III), Sm(III), and Dy(III) lanthanide chelate nanoparticle labels.

Preparation and characterization of europium(III), terbium(III), samarium(III), and dysprosium(III) polystyrene nanoparticle labels with lanthanide-specific fluorescence properties has been presented. Emulsion copolymerization of styrene and acrylic acid was used to synthesize uniform-sized nanoparticles approximately 45 nm in diameter. Europium(III) and samarium(III) lanthanides were chelated with 2-naphthoyltrifluoroacetone and trioctylphosphine oxide to dye the spherical particles, whereas terbium(III) and dysprosium(III) chelate complexes contained a newly synthesized ligand, 4-(2,4,6-tridecyloxyphenyl)pyridine-2,6-dicarboxylic acid. The fluorescence properties of the four lanthanides-including a wide Stokes shift, a narrow emission peak, and long fluorescence lifetime-were retained despite the incorporation into the nanoparticles. Furthermore, the nanoparticles, containing more than 1000 lanthanide chelates, were detectable at label concentrations 3 orders of magnitude lower than the corresponding soluble lanthanide chelate labels. The applicability of the labels prepared was demonstrated by a heterogeneous sandwich-type immunoassay for human prostate-specific antigen, where the lowest limits of detection of 1.6, 2.4, 10.1, and 114.2 ng/L were achieved using europium(III), terbium(III), samarium(III), and dysprosium(III) nanoparticles, respectively. The spectral and functional properties of the lanthanide-embedded polystyrene nanoparticles developed here suggest that the technology is applicable for high-sensitivity multicolor assays.

Generic lanthanide fluoroimmunoassay for the simultaneous screening of 18 sulfonamides using an engineered antibody.

Sulfa antibiotics (sulfonamides) are used in veterinary and human medicine for therapeutic and prophylactic purposes. Veterinary use can result in foodstuffs derived from animals being contaminated with residual sulfonamides. Current sulfonamide-screening methods (mainly based on bacterial growth inhibition) are slow and inaccurate, since sensitivities of bacteria to different sulfonamides vary a lot. Therefore, a rapid immunoassay that was able to detect at least 18 different sulfonamides at the MRL level (100 microg/kg) from food samples in a single reaction was developed. The assay was reproducible and adequately accurate for screening purposes. The presence of sulfonamide metabolites did not cause major assay interference. We also demonstrated reliable detection of sulfonamides from a panel of meat, milk, and serum samples with the assay.

An all-in-one dry chemistry immunoassay for the screening of coccidiostat nicarbazin in poultry eggs and liver.

An automated immunoassay for the detection of nicarbazin residues in poultry eggs and liver was developed. The assay was based on a novel all-in-one dry chemistry concept and time-resolved fluorometry. The analyte specific antibody was immobilized into a single microtiter well and covered with an insulation layer, on top of which the label was dried in a small volume. The extracted sample was added automatically to the dry microtiter well, and the result was available within 18 min. Due to the rapidity and simplicity, the quantitative immunoassay could also be used as a high throughput screening method. The analytical limit of detection for the assay was calculated as 0.1 ng mL(-)(1) (n = 12) and the functional limit of detection as 3.2 ng g(-)(1) for egg (n = 6) and 11.3 ng g(-)(1) for liver (n = 6) samples. The sample recovery varied from 97.3 to 115.6%. Typically, the intra-assay variations were less than 10%, and interassay variations ranged between 8.1 and 13.6%.

Rapid time-resolved fluoroimmunoassay for the screening of narasin and salinomycin residues in poultry and eggs.

Anticoccidial drugs are extensively used in the poultry industry to control the infection of the single-cell protozoa of the genus Eimeria. The most commonly used coccidiostats in poultry are the polyether ionophores such as narasin and salinomycin. This paper presents a rapid and simple method for the screening of residues of these two coccidiostatic compounds in poultry and eggs. The method is based on time-resolved fluoroimmunoassay. Sample preparation of eggs consists only of one extraction and evaporation step, and a solid phase extraction step is needed only for the muscle sample preparation. Mean recoveries were 91.0% from muscle tissue and 81.1% from eggs for both narasin and salinomycin. The performance of the assay was evaluated only for narasin because salinomycin had a cross-reactivity of 100% in the assay, and the recoveries of the compounds were not significantly different (P >0.05). The limits of detection [mean + 3 x standard deviation (SD)] of narasin were 0.56 and 0.28 microg/kg, and the limits of quantification (mean + 9 x SD) were 1.80 and 0.57 microg/kg for muscle and eggs, respectively. The coefficients of variation (CV) of the interassay precision of the method, evaluated by five replicate analyses of muscle samples spiked with 2 microg/kg of narasin and egg samples spiked with 1 microg/kg of narasin, were 4.1 and 6.4%, respectively. The CVs of intra-assay precision tests, determined by 10 replicate analyses at the above-mentioned concentration levels, were 3.8 and 4.5%, respectively.

One-step quantitative thyrotropin assay for the detection of hypothyroidism in point-of-care conditions.

OBJECTIVES: Different screening strategies for early diagnosis of hypothyroidism have been discussed increasingly. We demonstrate the applicability of a miniaturized microparticle assay format for rapid and quantitative determination of increased thyrotropin (TSH) concentrations in serum. DESIGN AND METHODS: Porous microparticles were used as solid phase for a noncompetitive, one-step, kinetic immunoassay with varying incubation times and time-resolved fluorescence detection. RESULTS: The analytical (mean of zero + 3 SD) and functional (CV <15%) detection limits were 1.5 and 6.0 mIU/L for 2-min, 0.5 and 1.5 mIU/L for 7-min, and 0.2 and 0.5 mIU/L for 15-min assays, respectively. A good correlation was found with the Chiron Diagnostics ACS:180 assay (slopes 0.885-1.051, y-intercepts < +/- 0.20 mIU/L, S(y logical or, bar below x) 0.98, n = 20). CONCLUSION: The kinetic TSH assay provides reproducible and quantitative information on thyroid status within minutes and is applicable for the detection of hypothyroidism in point-of-care (POC) conditions.