RESUMEN
Tetracyclines are extensively used in veterinary medicine. For the detection of tetracycline residues in animal products, a broad array of methods is available. Luminescent bacterial biosensors represent an attractive inexpensive, simple and fast method for screening large numbers of samples. A previously developed cell-biosensor method was subjected to an evaluation study using over 300 routine poultry samples and the results were compared with a microbial inhibition test. The cell-biosensor assay yielded many more suspect samples, 10.2% versus 2% with the inhibition test, which all could be confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Only one sample contained a concentration above the maximum residue limit (MRL) of 100 microg kg(-1), while residue levels in most of the suspect samples were very low (<10 microg kg(-1)). The method appeared to be specific and robust. Using an experimental set-up comprising the analysis of a series of three sample dilutions allowed an appropriate cut-off for confirmatory analysis, limiting the number of samples and requiring further analysis to a minimum.
Asunto(s)
Antibacterianos/análisis , Técnicas Biosensibles , Residuos de Medicamentos/análisis , Carne/análisis , Músculo Esquelético/química , Aves de Corral , Tetraciclinas/análisis , Animales , Antibacterianos/química , Antibacterianos/metabolismo , Técnicas Biosensibles/economía , Residuos de Medicamentos/química , Residuos de Medicamentos/metabolismo , Residuos de Medicamentos/normas , Escherichia coli/genética , Escherichia coli/metabolismo , Unión Europea , Contaminación de Alimentos , Inspección de Alimentos/economía , Inspección de Alimentos/métodos , Inspección de Alimentos/normas , Límite de Detección , Luciferasas de la Bacteria/genética , Luciferasas de la Bacteria/metabolismo , Operón/efectos de los fármacos , Operón/genética , Proteínas Represoras/metabolismo , Reproducibilidad de los Resultados , Tetraciclinas/química , Tetraciclinas/metabolismo , Factores de Tiempo , Drogas Veterinarias/análisis , Drogas Veterinarias/química , Drogas Veterinarias/metabolismoRESUMEN
A bioluminescent Escherichia coli K-12 strain for the specific detection of the tetracycline group of antibiotics is described. A sensor plasmid, containing five genes from bacterial luciferase operon of Photorhabdus luminescens inserted under the control of tetracycline-responsive elements of the transposon Tn10, was constructed. Usage of the full-length luciferase operon in the sensor resulted in tetracycline-dependent light production without additions, i.e., self-luminescent phenotype, since all the substrates were intrinsically produced by the recombinant organism. The time needed for optimal induction of light emission was 90 min. Maximal induction of approximately 100-fold over uninduced levels by using 20 ng of tetracycline, and picomole sensitivities for the seven different tetracyclines tested, were obtained without added Mg2+ ions. The higher the pH and the magnesium ion concentration in the assay medium the higher was the amount of membrane-impermeable tetracycline-Mg2+ chelate complex. In consequence, by adjusting the pH and the Mg2+ ion concentration, the sensitivity of the assay can be modified for different analytical purposes. Different non-tetracycline antibiotics did not cause induction of light emission.
Asunto(s)
Antibacterianos/análisis , Técnicas Biosensibles , Escherichia coli/genética , Tetraciclina/análisis , Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Concentración de Iones de Hidrógeno , Mediciones Luminiscentes , Magnesio/química , Fenotipo , Plásmidos , Relación Estructura-Actividad , Tetraciclina/farmacologíaRESUMEN
A new method for studying the action of membranolytic agents by simple measurement of light emitted from cells is described. It is based on the expression of the click beetle (Pyrophorus plagiophthalamus) luciferase gene (lucGR) in Escherichia coli, Bacillus subtilis and Spodoptera frugiperda cells in order to make them bioluminescent. The diffusion of the substrate for luciferase enzyme through the cell membranes is very low at physiological pH, and therefore a change in membrane permeability is seen as a change of in-vivo luminescence of cells. The cells used in this study represent different membrane structures, and thus allow a comparison of the reactions of the different membranes towards membranolytic agents in a real-time measurement. The dose-response data correlated well with target cell viable count. In addition, the time course of light emission as a consequence of permeabilizing compound is dose-dependent. The action of the compounds on prokaryotic and eukaryotic cells was found to be highly dependent on the permeabilizer used.
Asunto(s)
Permeabilidad de la Membrana Celular/efectos de los fármacos , Animales , Antibacterianos/farmacología , Bacillus subtilis/metabolismo , Bacillus subtilis/ultraestructura , Escarabajos , Digitonina/farmacología , Escherichia coli/metabolismo , Escherichia coli/ultraestructura , Luciferina de Luciérnaga , Concentración de Iones de Hidrógeno , Luciferasas/genética , Luciferasas/metabolismo , Mediciones Luminiscentes , Meliteno/farmacología , Peso Molecular , Polimixina B/farmacología , Spodoptera , Factores de TiempoRESUMEN
A bioluminescent assay based on the bacterial luciferase reaction has been developed for the determination of total lactate dehydrogenase and heart-specific lactate dehydrogenase isoenzyme-1 activity in serum. The lactate dehydrogenase-catalyzed reaction was measured in both directions, but NADH formation (lactate----pyruvate) is recommended because it allows the use of optimal reaction conditions. Internal calibration with a known amount of NADH accounts for possible interference from samples when both NADH formation and consumption are followed. The bioluminescent method is sensitive, has good precision, and is readily automated. Serum lactate dehydrogenase isoenzyme-1 was immunochemically isolated and the activity was assayed by bioluminescence. A good correlation between the bioluminescent assays and the conventional spectrophotometric procedure used as reference was obtained.
Asunto(s)
L-Lactato Deshidrogenasa/sangre , Humanos , Isoenzimas , Cinética , Luciferasas , Mediciones Luminiscentes , Métodos , Miocardio/enzimología , NAD/análisis , Oxidación-Reducción , Espectrofotometría UltravioletaRESUMEN
In this study the application of gel filtration for purification of heterogeneous DNA is described. The fractionation of partial restriction enzyme digests of bacterial chromosomal DNA on a Sephacryl S-1000 -column is easy and rapid. Simultaneously intact chromosomal DNA and low molecular weight substances are eliminated in the run. The method is also applicable to the purification of plasmid DNA, as has been previously reported (3). Thus we are able to get pure DNA with yields over 80%.
Asunto(s)
ADN Bacteriano/aislamiento & purificación , Resinas Acrílicas , Cromatografía en Gel/métodos , Escherichia coli , Genes Bacterianos , Geobacillus stearothermophilus , PlásmidosRESUMEN
A method for incorporating into proteins a nonradioactive Eu3+ label, which exhibits fluorescence of a long decay time in the presence of suitable ligands, is described. As an example of the use of this label the method has been developed to work as a sensitive protease assay. By hydrolyzing the Eu3+-labeled casein, bound to an insoluble matrix (Sepharose 4B or Affi-Gel 10), with proteases and measuring the Eu3+ released with a pulsed time-resolved fluorometer it was possible to detect as low as 2.5, 1.0, or 1.0 ng of alpha-chymotrypsin, trypsin, or subtilisin, respectively.
Asunto(s)
Quimotripsina/metabolismo , Europio , Subtilisinas/metabolismo , Tripsina/metabolismo , Animales , Bacillus/enzimología , Bovinos , Cinética , Microquímica , Páncreas/enzimología , Espectrometría de Fluorescencia/métodos , PorcinosRESUMEN
A new method for extracting pyridine nucleotides from tissue samples at room temperature that allows the simultaneous extraction of both the oxidized and reduced nucleotide when using a 70% buffered ethanol solution as the extractant has been developed. The extraction efficiencies for NAD+ and NADH were 91 and 102%, respectively. The extraction method was followed by a combined bioluminescent assay of both nucleotides. A bacterial bioluminescent system, which included luciferase and low levels of a NADH-specific oxidoreductase, was used to produce a constant light intensity directly proportional to the amount of NADH in the tissue extract sample. When the NADH had been measured, the NAD+ present in the extract was enzymatically converted to NADH by the addition of alcohol dehydrogenase, after which the second increase in light level was recorded. The sensitivity of the bioluminescent assay presented here is 5 X 10(-14) mol NADH or NAD+ per assay.