Este trabajo es apoyado por P20GM103418 K INBRE, Kansas soja Comisión (KSC/PSU 1663), ACS PRF 56629-UNI7 y PSU polímero química Inicio fondo todos SS. Agradecemos al camarógrafo de la Universidad, el Sr. Jacob Anselmi, por su destacada labor con el video. También agradecemos al Sr. Roger Heckert y Sra. Katha Heckert por su generoso apoyo para la investigación.

1 síntesis y funcionalización de nanosensores multi-paramétrico de Magneto-fluorescente (MFnS).
<ol><li>Síntesis de óxido de hierro superparamagnético nanopartículas (IONPs) <ol><li>para preparar síntesis IONP, preparar las 3 siguientes soluciones: solución 1: FECLAS 3 0,70 g y FECLAS 2 H 2 O (2 mL), solución 2: NH 4 OH (2,0 mL, 13,4 M) en H 2 O (15 mL) y solución 3: ácido poliacrílico (0,855 g) H 2 O (5 mL).</li>
		<li>Agregar 90 μl de 2 M de ácido clorhídrico (HCl) solución 1 y luego mezcle inmediatamente con solución 2 mientras Vortex a 875 rpm. A continuación Agregar solución 3. Continuar con un vórtex durante 60 minutos</li>
		<li>Centrifugar 20 min a 1.620 x g. Centrifugar el sobrenadante por 20 min a 2.880 x g.</li>
		<li>Purifica el sobrenadante final mediante diálisis. Añadir el sobrenadante a una bolsa de diálisis (MWCO 6 &#8722; 8 K) y colóquelo en un vaso de precipitados que contiene tamponada fosfato salino (PBS) (pH = 7,4) y el bar de la vuelta. Deje reposar 12 h, reemplazando con el almacenador intermediario fresco cada 2-3 h.</li>
	</ol></li> <li>Conjugación de anticuerpos específicos del destino a la superficie de IONPs <ol><li>preparar las siguientes cuatro soluciones: solución A: 5 mg de 1-etil - 3-(3-dimetilaminopropil) carbodiimida clorhidrato (EDC) en 250 μl MES [2-(N- buffer de morfolino) (n-2-hidroxietilpiperazina-N'-2-ácido ácido] (0.1 M, pH = 6.8), solución B: 3 mg de N-Hydroxysuccinimide (NHS) en 250 μl de tampón MES (0.1 M, pH = 6.8), solución C: 5 μg de IgG1, el mAb de e. coli, en 225 μL de tampón fosfato salino (PBS) (pH 7.4) y solución D: 4 mL de IONP (5,0 mmol) en 1 mL de PBS (pH 7.4).</li>
		<li>Añadir 2 (N-morfolino) (n-2-hidroxietilpiperazina-N'-2-ácido ácido (MES) del almacenador intermediario para la solución A, entonces inmediatamente agregue solución A la solución D en incrementos pequeños dentro de los 15 s, invertir la solución después de cada adición para la mezcla de.</li>
		<li>Comenzar inmediatamente a agregar solución B solución d en pequeños incrementos durante un período de tres minutos. Mezclar por inversión la solución después de cada adición.</li>
		<li>Agregar solución C solución en incrementos de 5 μl, invirtiendo la solución después de cada adición para la mezcla de.</li>
		<li>Permitir que la reacción continúe durante 3 h a temperatura ambiente y luego continuar la incubación a 4 ° C durante la noche.</li>
		<li>Purificar las resultantes IONPs conjugado con Ab vía columna magnética con PBS (pH = 7.4, final [Fe] = 3,5 mmol) para eliminar los anticuerpos sin conjugar.
		<ol><li>Lave la columna magnética con PBS (1 mL). Coloque la columna en la placa magnética y agregar solución IONP.</li>
			<li>Lavar la columna magnética con PBS (1 mL) otra vez. Retire la columna magnética placa magnética. Añadir PBS a la columna y recoger la solución. Conservar a 4 ° C</li></ol></li></ol></li> <li>encapsulado de 1,1 fluorescente &#39; - Dioctadecyl - 3,3,3 &#39;, 3 &#39;-tinte Tetramethylindocarbocyanine perclorato (DiI) en el recubrimiento de poliacrílico (PAA) el ácido de las IONPs de anticuerpo conjugado con una método de difusión del solvente.
	<ol><li>A 4 mL de las IONPs, añadir gota a gota 2.0 μL de colorante DiI (2 mmol) en 100 μl de DMSO con mezcla continua a 1100 rpm.</li>
		<li>Dializarse la solución resultante (MWCO 6-8 KDa, 12 h) contra PBS, creando una solución de MFnS final con una concentración final de hierro [Fe] = 2 mmol.</li>
	</ol></li> <li>Caracterización de MFnS <ol><li>para el análisis espectrofotométrico, usar un lector de placas para detectar encapsulado DiI tinte con la emisión de fluorescencia a 595 nM.</li>
		<li>Dispersión dinámica de luz <ol><li>Coloque una muestra de la solución de MFnS en zetasizer para determinar el tamaño promedio y la carga superficial de los MFnS. 
			Nota: Aquí, el tamaño promedio y la carga superficial de MFnS funcional fueron encontrados para ser 77.09 nm y-22.3 mV, respectivamente.</li>
		</ol></li></ol></li></ol> 2. Bacteriana cultivo y preparación de solución Stock <ol><li><ol>cultivo bacteriano <li>hidratar un pellet liofilizado (e. coli O157: H7) en 1 mL de caldo nutritivo y luego añadir un adicional 5 mL de caldo.</li>
		<li>Aplicar 100 μl de esta solución en una placa de agar y raya utilizando un asa de inoculación estéril, seguido por incubación a 37 ° C por 24 h.</li>
		<li>Después de 24 h, seleccionar una colonia aislada de la placa de agar y añadirlo a una 15 mL de caldo de cultivo. Incubar a 37 ° C durante 4-6 h, mientras el valor de densidad óptica (absorbancia a 600 nm).</li>
		<li>Una vez que se obtiene un valor de densidad óptica de 0.1, dejar de cultivar y realizar diluciones seriadas.</li>
	</ol></li> <li>Diluciones seriadas <ol><li>Agregar 900 μl de caldo nutritivo a los ocho tubos de microcentrífuga estéril.</li>
		<li>Agregar 100 μl de la suspensión bacteriana en el primer tubo (dilución 10 -1).</li>
		<li>Tomar 100 μl del primer tubo y añade a la segunda, para obtener una dilución de 10 -2. Continuar este patrón para los tubos restantes, terminando con una dilución final de 10 -8.</li>
		<li>Transferir 100 μl de cada tubo para separar las placas de agar e incubar por 24 h a 37 ° C.</li>
		<li>Al día siguiente, contar las colonias formando unidades (UFC) en cada placa. Seleccione la placa con ~ 100 UFC. Utilizar las diluciones correspondientes para experimentos adicionales (100 μl = ~ 100 unidades formadoras de colonias). 
		Nota: Aquí es la dilución 10 -6 que dio lugar a la UFC de ~ 100.</li>
	</ol></li></ol> 3. Rápida detección de E. coli O157: H7 con MFnS <ol><li>Spike diversas soluciones de PBS (1 X, pH 7,4, 300 μL) con cantidades crecientes de la población bacteriana de 10 -6, resultando en UFC oscila entre 1-100. Agregue una cantidad consistente de MFnS (100 μL) a estas soluciones.</li>
	<li>Crear una solución de referencia que contiene solamente PBS (1 X, pH 7,4, 300 μL) y MFnS (100 μL).</li>
	<li>Incubar soluciones durante 30 min a 37 ° C y luego déjelos enfriar a temperatura ambiente.</li>
	<li>Transferencia de soluciones individuales a la relaxometer magnética (Tesla 0,47) y grabamos cambios en tiempos de relajación (T2) en relación con el UFC en cada solución.
	<ol><li>Registrar cambios en los valores de T2, comenzar midiendo el valor de T2 de la solución de referencia que contiene solamente PBS y MFnS.
		Relaxometer <ol><li>lugar la solución en el magnético. Abra el software respectivo, seleccione la &#34; T2 relajación &#34; programación y oprima &#34; medida. &#34;</li></ol></li> <li>siguiendo la colección de la línea de base de T2, medir los valores de T2 de las soluciones adicionales que se añadieron varios concentraciones de bacterias. 
		Nota: El cambio en T2 es equivalente a base T2 restada de la T2 claveteados.</li>
	</ol></li> <li>Extraer muestras de la relaxometer magnética y centrifugar los tubos a 2880 x g durante 10 minutos</li>
	<li>Decantar el sobrenadante y resuspender el pellet bacteriano en 100 μl de PBS (1 X, pH 7.4).</li>
	<li>Añadir 80 μl de cada resuspensión a una placa de 96 pocillos e intensidades de fluorescencia récord a 595 nM. 
	Nota: La prueba puede repetirse utilizando diferentes solventes como agua, leche y otros, como se describe en la sección de resultados</li></ol>

<ol>
	<li>Law, J. W., Ab Mutalib, N. S., Chan, K. G., Lee, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+methods+for+the+detection+of+foodborne+bacterial+pathogens:+principles,+applications,+advantages+and+limitations.">Rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations.</a> Front Microbiol. 5, 770 (2014).</li><li>Pandey, P. K., Kass, P. H., Soupir, M. L., Biswas, S., Singh, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contamination+of+water+resources+by+pathogenic+bacteria.">Contamination of water resources by pathogenic bacteria.</a> AMB Express. 4, 51 (2014).</li><li>Zhao, X., Lin, C. W., Wang, J., Oh, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+rapid+detection+methods+for+foodborne+pathogens.">Advances in rapid detection methods for foodborne pathogens.</a> J Microbiol Biotechnol. 24 (3), 297-312 (2014).</li><li>Heithoff, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraspecies+variation+in+the+emergence+of+hyperinfectious+bacterial+strains+in+nature.">Intraspecies variation in the emergence of hyperinfectious bacterial strains in nature.</a> PLoS Pathog. 8 (4), e1002647 (2012).</li><li>Ishii, S., Sadowsky, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+in+the+Environment:+Implications+for+Water+Quality+and+Human+Health.">Escherichia coli in the Environment: Implications for Water Quality and Human Health.</a> Microbes Environ. 23 (2), 101-108 (2008).</li><li>Chiou, C. S., Hsu, S. Y., Chiu, S. I., Wang, T. K., Chao, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vibrio+parahaemolyticus+serovar+O3:K6+as+cause+of+unusually+high+incidence+of+food-borne+disease+outbreaks+in+Taiwan+from+1996+to+1999.">Vibrio parahaemolyticus serovar O3:K6 as cause of unusually high incidence of food-borne disease outbreaks in Taiwan from 1996 to 1999.</a> J Clin Microbiol. 38 (12), 4621-4625 (2000).</li><li>Zhou, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR+methods+for+the+rapid+detection+and+identification+of+four+pathogenic+Legionella+spp.+and+two+Legionella+pneumophila+subspecies+based+on+the+gene+amplification+of+gyrB.">PCR methods for the rapid detection and identification of four pathogenic Legionella spp. and two Legionella pneumophila subspecies based on the gene amplification of gyrB.</a> Appl Microbiol Biotechnol. 91 (3), 777-787 (2011).</li><li>Chen, J., Tang, J., Liu, J., Cai, Z., Bai, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evaluation+of+a+multiplex+PCR+for+simultaneous+detection+of+five+foodborne+pathogens.">Development and evaluation of a multiplex PCR for simultaneous detection of five foodborne pathogens.</a> J Appl Microbiol. 112 (4), 823-830 (2012).</li><li>LeBlanc, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Switching+gears+for+an+influenza+pandemic:+validation+of+a+duplex+reverse+transcriptase+PCR+assay+for+simultaneous+detection+and+confirmatory+identification+of+pandemic+(H1N1)+2009+influenza+virus.">Switching gears for an influenza pandemic: validation of a duplex reverse transcriptase PCR assay for simultaneous detection and confirmatory identification of pandemic (H1N1) 2009 influenza virus.</a> J Clin Microbiol. 47 (12), 3805-3813 (2009).</li><li>Mahony, J. B., Chong, S., Luinstra, K., Petrich, A., Smieja, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+novel+bead-based+multiplex+PCR+assay+for+combined+subtyping+and+oseltamivir+resistance+genotyping+(H275Y)+of+seasonal+and+pandemic+H1N1+influenza+A+viruses.">Development of a novel bead-based multiplex PCR assay for combined subtyping and oseltamivir resistance genotyping (H275Y) of seasonal and pandemic H1N1 influenza A viruses.</a> J Clin Virol. 49 (4), 277-282 (2010).</li><li>Alvarez, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+recognition+of+influenza+A/H1N1/2009+antibodies+in+human+serum:+a+simple+virus-free+ELISA+method.">Specific recognition of influenza A/H1N1/2009 antibodies in human serum: a simple virus-free ELISA method.</a> PLoS One. 5 (4), e10176 (2010).</li><li>Huang, C. J., Dostalek, J., Sessitsch, A., Knoll, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-range+surface+plasmon-enhanced+fluorescence+spectroscopy+biosensor+for+ultrasensitive+detection+of+E.+coli+O157:H7.">Long-range surface plasmon-enhanced fluorescence spectroscopy biosensor for ultrasensitive detection of E. coli O157:H7.</a> Anal Chem. 83 (3), 674-677 (2011).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+visual+detection+of+highly+pathogenic+Streptococcus+suis+serotype+2+isolates+by+use+of+loop-mediated+isothermal+amplification.">Rapid visual detection of highly pathogenic Streptococcus suis serotype 2 isolates by use of loop-mediated isothermal amplification.</a> J Clin Microbiol. 51 (10), 3250-3256 (2013).</li><li>Han, F., Wang, F., Ge, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+potentially+virulent+Vibrio+vulnificus+strains+in+raw+oysters+by+quantitative+loop-mediated+isothermal+amplification.">Detecting potentially virulent Vibrio vulnificus strains in raw oysters by quantitative loop-mediated isothermal amplification.</a> Appl Environ Microbiol. 77 (8), 2589-2595 (2011).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+pathogenic+bacteria+and+screening+of+phage-derived+peptides+using+microcantilevers.">Rapid detection of pathogenic bacteria and screening of phage-derived peptides using microcantilevers.</a> Anal Chem. 86 (3), 1671-1678 (2014).</li><li>Banerjee, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparametric+Magneto-fluorescent+Nanosensors+for+the+Ultrasensitive+Detection+of+Escherichia+coli+O157:H7.">Multiparametric Magneto-fluorescent Nanosensors for the Ultrasensitive Detection of Escherichia coli O157:H7.</a> ACS Infect Dis. 2 (10), 667-673 (2016).</li><li>Shelby, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+magnetic+relaxation+nanosensors:+an+unparalleled+&amp;#34;spin&amp;#34;+on+influenza+diagnosis.">Novel magnetic relaxation nanosensors: an unparalleled &amp;#34;spin&amp;#34; on influenza diagnosis.</a> Nanoscale. 8, 19605-19613 (2016).</li><li>Bui, M. P., Ahmed, S., Abbas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Digit+Pathogen+and+Attomolar+Detection+with+the+Naked+Eye+Using+Liposome-Amplified+Plasmonic+Immunoassay.">Single-Digit Pathogen and Attomolar Detection with the Naked Eye Using Liposome-Amplified Plasmonic Immunoassay.</a> Nano Lett. 15 (9), 6239-6246 (2015).</li><li>Farnleitner, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+enzymatic+detection+of+Escherichia+coli+contamination+in+polluted+river+water.">Rapid enzymatic detection of Escherichia coli contamination in polluted river water.</a> Lett Appl Microbiol. 33 (3), 246-250 (2001).</li><li>Huh, Y. S., Lowe, A. J., Strickland, A. D., Batt, C. A., Erickson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface-enhanced+Raman+scattering+based+ligase+detection+reaction.">Surface-enhanced Raman scattering based ligase detection reaction.</a> J Am Chem Soc. 131 (6), 2208-2213 (2009).</li><li>Jayamohan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+sensitive+bacteria+quantification+using+immunomagnetic+separation+and+electrochemical+detection+of+guanine-labeled+secondary+beads.">Highly sensitive bacteria quantification using immunomagnetic separation and electrochemical detection of guanine-labeled secondary beads.</a> Sensors (Basel). 15 (5), 12034-12052 (2015).</li><li>Kaittanis, C., Naser, S. A., Perez, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step,+nanoparticle-mediated+bacterial+detection+with+magnetic+relaxation.">One-step, nanoparticle-mediated bacterial detection with magnetic relaxation.</a> Nano Lett. 7 (2), 380-383 (2007).</li><li>Meeker, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+Photothermal+and+Antibiotic+Killing+of+Biofilm-Associated+Staphylococcus+aureus+Using+Targeted+Antibiotic-Loaded+Gold+Nanoconstructs.">Synergistic Photothermal and Antibiotic Killing of Biofilm-Associated Staphylococcus aureus Using Targeted Antibiotic-Loaded Gold Nanoconstructs.</a> ACS Infect Dis. 2 (4), 241-250 (2016).</li><li>Wang, Y., Ye, Z., Si, C., Ying, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subtractive+inhibition+assay+for+the+detection+of+E.+coli+O157:H7+using+surface+plasmon+resonance.">Subtractive inhibition assay for the detection of E. coli O157:H7 using surface plasmon resonance.</a> Sensors (Basel). 11 (3), 2728-2739 (2011).</li><li>Zhao, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+bioassay+for+single+bacterial+cell+quantitation+using+bioconjugated+nanoparticles.">A rapid bioassay for single bacterial cell quantitation using bioconjugated nanoparticles.</a> Proc Natl Acad Sci U S A. 101 (42), 15027-15032 (2004).</li></ol>

La aplicación demostrada de esta nanotecnología aún no es aprobada por la FDA.

Este protocolo ha sido diseñado para producir MFnS completamente funcional como simplemente como sea posible. Sin embargo, hay muchos puntos claves en el que alteración del Protocolo puede ser útil, dependiendo de la finalidad del usuario. Por ejemplo, el uso de diferentes anticuerpos permitiría atacar muchos otros patógenos. Además, este Protocolo no se limita a la utilización de anticuerpos como dirigidos a moléculas. Cualquier molécula que tiene afinidad de Unión específicos...

La ocurrencia persistente de contaminación bacteriana en los alimentos comercialmente producidos y fuentes de agua ha creado la necesidad de plataformas diagnóstico cada vez más rápidas y específicas. 1 , 2 algunos de los más comunes contaminantes bacterianos responsables de contaminación de alimentos y agua son de los géneros Salmonella, Staphylococcus, Listeria, Vibrio, Shigella, Bacillus y Escherichia. 3 , 4 contaminación bacteriana por estos patógenos a menudo resultados en síntomas tales como fiebre, cólera, gastroenteritis y diarrea. 4 contaminación de fuentes de agua a menudo tiene efectos drásticos y nocivos en las comunidades sin acceso a agua suficientemente filtrada y contaminación de los alimentos ha llevado a un gran número de enfermedades y los esfuerzos de recuperación de producto. 5 , 6
Con el fin de reducir la ocurrencia de enfermedades causadas por contaminación bacteriana, ha habido una serie de esfuerzos para desarrollar métodos con que agua y comida pueden eficientemente analizará antes de la venta o consumo. 3 técnicas como PCR,1,7,8,9,10 ELISA,11,(12 ) amplificación isotérmica mediada lazo LÁMPARA),13,14 entre otros,15,16,17,18,19,20,21, 22,23,24 se han utilizado recientemente para la detección de diferentes patógenos. En comparación con bacterias tradicionales métodos de cultivo, estas técnicas son mucho más eficientes en cuanto a la especificidad y el tiempo. Sin embargo, estas técnicas aún batallan con los falsos positivos y negativos, procedimientos complejos y los costos. 1 , 3 , 25 es por este motivo que nanosensores de magneto-fluorescente multiparamétrico (MFnS) se propusieron como un método alternativo para la detección de bacteria.
Estos nanosensores únicamente par juntos relajación magnética y modalidades fluorescentes, lo que permite una plataforma de doble detección rápida y precisa. Utilizando e. coli O157: H7 como un contaminante de la muestra, se demuestra la capacidad de MFnS para detectar tan poco como 1 UFC en minutos. Anticuerpos patógenos específicos se utilizan para aumentar la especificidad, y permite la combinación de modalidades magnéticas y fluorescentes para la detección y cuantificación de contaminantes bacterianos en dos rangos de baja y alta contaminación. 16 en el caso de contaminación bacteriana, los nanosensores que pululan alrededor de las bacterias debido a las capacidades de segmentación de los anticuerpos específicos del patógeno. La unión entre las bacterias y nanosensores magnético limita la interacción entre el núcleo de hierro magnético y los protones de agua circundantes. Esto provoca un aumento en los tiempos de relajación T2, según lo registrado por un relaxometer magnético. Medida que aumenta la concentración de bacterias en solución, los nanosensores dispersan con el aumento del número de bacterias, lo que resulta en valores más bajos de la T2. Por el contrario, emisión de fluorescencia se incrementará en proporción a la concentración de bacterias, debido al mayor número de nanosensores vinculado directamente al patógeno. Centrifugación de las muestras y el aislamiento de los pellets bacterianos, sólo conservará las nanopartículas conectadas directamente a las bacterias, la eliminación de cualquier flotante nanosensores y correlacionar directamente la emisión de fluorescencia con el número de bacterias presentes en la solución. Una representación esquemática de este mecanismo se representa en la figura 1.
Esta plataforma de MFnS ha sido diseñada con proyección del punto de atención en la mente, dando por resultado características portables y de bajo costo. MFnS son estables a temperatura ambiente y sólo son necesarios en concentraciones muy bajas para la precisa detección de contaminantes bacterianos. Además, después de la síntesis, el uso de los MFnS es simple y no requiere el uso de profesionales en el campo. Por último, esta plataforma de diagnóstico permite orientación altamente personalizable, proporcionar un medio por que esta una plataforma puede usarse para detectar patógenos de todo tipo, en muchos entornos diferentes.

<table><tbody><tr><td>Ferrous Chloride Tetrahydrate</td><td>Fisher Scientific</td><td>I90-500</td><td></td></tr><tr><td>Ferric Chloride Hexahydrate</td><td>Fisher Scientific</td><td>I88-500</td><td></td></tr><tr><td>Ammonium Hydroxide</td><td>Fisher Scientific</td><td>A669S-500</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Fisher Scientific</td><td>A144S-500</td><td></td></tr><tr><td>Polyacryllic Acid</td><td>Sigma-Aldrich</td><td>323667-100G</td><td></td></tr><tr><td>EDC</td><td>Thermofisher Scientific</td><td>22980</td><td></td></tr><tr><td>NHS</td><td>Fisher Scientific</td><td>AC157270250</td><td></td></tr><tr><td>Anti-E. coli O111 antibody&amp;nbsp;</td><td>sera care</td><td>5310-0352</td><td></td></tr><tr><td>Anti-E. coli O157:H7 antibody [P3C6]&amp;nbsp;</td><td>Abcam</td><td>ab75244</td><td></td></tr><tr><td>DiI Stain</td><td>Fisher Scientific</td><td>D282</td><td></td></tr><tr><td>Nutrient Broth</td><td>Difco</td><td>233000</td><td></td></tr><tr><td>Freeze-dried E. coli O157:H7 pellet</td><td>ATCC</td><td>700728</td><td></td></tr><tr><td>Magnetic Relaxomteter&amp;nbsp;</td><td>Bruker</td><td>mq20</td><td></td></tr><tr><td>Zetasizer</td><td>Malvern</td><td>NANO-ZS90</td><td></td></tr><tr><td>Plate Reader&amp;nbsp;</td><td>Tecan</td><td>Infinite M200 PRO</td><td></td></tr><tr><td>Magnetic Column&amp;nbsp;</td><td>QuadroMACS</td><td>130-090-976</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804 Series</td><td></td></tr><tr><td>Centrifuge (accuSpin Micro 17)</td><td>Fisher Scientific</td><td>13-100-676</td><td></td></tr><tr><td>Floor Model Shaking Incubator</td><td>SHEL LAB</td><td>SSI5</td><td></td></tr><tr><td>Analytical Balance</td><td>Metler Toledo</td><td>ME104E</td><td></td></tr><tr><td>Digital Vortex Mixer</td><td>Fisher Scientific</td><td>02-215-370</td><td></td></tr><tr><td>Open-Air Rocking Shaker</td><td>Fisher Scientific</td><td>02-217-765</td><td></td></tr></tbody></table>

foodborne pathogen screening using magneto-fluorescent nanosensor: rapid detection of e. coli o157:h7

Enterohemorrágica Escherichia coli O157: H7 se ha ligado a ambas transmitidas por el agua y enfermedades transmitidas por los alimentos y sigue siendo una amenaza a pesar de los métodos de investigación de alimentos y agua utilizada en la actualidad. Mientras que los métodos convencionales de detección bacteriana, tales como reacción en cadena de polimerasa (PCR) y análisis enzima-ligado del inmunosorbente (ELISA) detectan específicamente a contaminantes patógenos, requieren preparación de la muestra extensos y largos períodos de espera. Además, estas prácticas exigen instrumentos de laboratorio sofisticado y configuración y deben ser ejecutadas por profesionales capacitados. Aquí, se propone un protocolo para una técnica de diagnóstico más simple que ofrece una combinación única de parámetros magnéticos y fluorescentes en una plataforma basada en nanopartículas. La propuesta nanosensores de magneto-fluorescente multiparamétrico (MFnS) pueden detectar contaminación de e. coli O157: H7 con tan poco como 1 unidad formadora de colonias presente en solución en menos de 1 h. Además, la capacidad de MFnS permanecer altamente funcional en medios complejos tales como leche y agua del lago ha sido verificada. Análisis de especificidad adicional también fueron utilizados para demostrar la capacidad de MFnS para detectar sólo las bacterias objetivo específico, incluso en presencia de especies bacterianas similares. El apareamiento de las modalidades magnéticas y fluorescentes permite la detección y cuantificación de la contaminación por patógenos en una amplia gama de las concentraciones, exhibiendo su alto rendimiento en la detección de contaminación de ambas etapa temprana y tardía. La eficacia, la asequibilidad y la portabilidad de los MFnS hacen un candidato ideal para punto de atención detección de bacterias contaminantes en una amplia gama de ajustes, de reservorios acuáticos a alimentos envasados comercialmente.

El mecanismo de acción de MFnS está representado en la figura 1. El agrupamiento de MFnS alrededor de la superficie de contaminantes bacterianas interfiere con las interacciones entre la magnetic cores de los MFnS y los núcleos de hidrógeno circundante. Como resultado de este agrupamiento, magnética relajación aumentan de valores. A medida que aumenta la concentración de contaminantes bacterianos, agrupamiento reduce, y el cambio en los valores de T2 disminuye. Por lo tanto, la adici?...

Watch this Scientific Journal Video about Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7 at JoVE.com

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

El objetivo general del presente Protocolo es sintetizar funcional nanosensores para el portable, eficiente, y rápida detección de específicamente las bacterias patógenas a través de una combinación de relajación magnética y las modalidades de emisión de fluorescencia.

Patógenos de transmisión alimentaria proyección utilizando Magneto-fluorescente Nanosensor: Detección rápida de e. Coli O157: H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and ...

foodborne-pathogen-screening-using-magneto-fluorescent-nanosensor

Research

JoVE Journal

Immunology and Infection

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

7.6K vistas.  Pittsburg State University. El objetivo general del presente Protocolo es sintetizar funcional nanosensores para el portable, eficiente, y rápida detección de específicamente las bacterias patógenas a través de una combinación de relajación magnética y las modalidades de emisión de fluorescencia.

Video: Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

La detección de bacterias mediante ADNzimas fluorogénicos

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

Investigación

Inmunología e Infección

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

Detección óptica de<em&gt; E. coli</em&gt; Bacterias por mesoporosos Silicon Biosensores

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Bioingeniería

Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine, a siderophore recognized by Yersinia enterocolitica, using a succinyl moiety as a linker are described.
Magnetic nanoparticles (MNP) of magnetite (Fe3O4) were prepared by solvothermal method and coated with SiO2 (MNP@SiO2) using the St&#246;ber process followed by functionalization with APTES (MNP@SiO2@NH2). Then, feroxamine was conjugated with the MNP@SiO2@NH2 by carbodiimide coupling to give MNP@SiO2@NH2@Fa. The morphology and properties of the conjugate and intermediates were examined by eight different methods including powder X-Ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and energy dispersive X-Ray (EDX) mapping. This exhaustive characterization confirmed the formation of the conjugate. Finally, in order to evaluate the capacity and specificity of the nanoparticles, they were tested in a capture bacteria assay using Yersinia enterocolitica.

This work describes protocols for the preparation of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine using a succinyl moiety as a linker. A deep structural characterization description and a capture bacteria assay using Y. enterocolitica for all the intermediate nanoparticles and the final conjugate are also described in detail.

Síntesis de nanopartículas magnéticas funcionalizadas, su conjugación con el Siderophore Feroxamine y su Evaluación para la Detección de Bacterias

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with ...

Química

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

Detección rápida homogénea de Ensayos Biológicos Usando el Sistema de modulación magnética biosensores

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Biología

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

Desarrollo de un Biosensor de ADN electroquímico para la detección de un patógeno de transmisión alimentaria

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

Nanosensores para detectar actividad proteasa en Vivo para el diagnóstico no invasivo

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Una plataforma de alto rendimiento para la detección de Salmonella spp. /Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System

Polymerase chain reaction (PCR) is a traditional method employed for the amplification of a target gene that has played an important role in biomolecular diagnostics. However, traditional PCR is very time-consuming because of the low-temperature variation efficiency. This work proposes a continuous-flow-PCR (CF-PCR) system based on a microfluidic chip. The amplification time can be greatly reduced by running the PCR solution into a microchannel placed on heaters set at different temperatures. Moreover, as capillary electrophoresis (CE) is an ideal way to differentiate positive and false-positive PCR products, a CE system was built to achieve efficient separation of the DNA fragments. This paper describes the process of amplification of Escherichia coli (E. coli) by the CF-PCR system built in-house and the detection of the PCR products by CE. The results demonstrate that the target gene of E. coli was successfully amplified within 10 min, indicating that these two systems can be used for the rapid amplification and detection of nucleic acids.

Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System

This protocol describes how to build a continuous-flow-polymerase chain system based on a microfluidic chip and how to build a capillary electrophoresis system in the lab. It presents a simple method for the analysis of nucleic acids in the lab.

Amplificación de Escherichia coli en un chip microfluídico de PCR de flujo continuo y su detección con un sistema de electroforesis capilar

Polymerase chain reaction (PCR) is a traditional method employed for the amplification of a target gene that has played an important role in biomolecular ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

La detección de vivo<em&gt; Escherichia coli</em&gt; O157: H7 células por PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Colorimetric Detection of Bacteria Using Litmus Test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

La detección colorimétrica de bacterias mediante prueba de fuego

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...

Patógenos de transmisión alimentaria proyección utilizando Magneto-fluorescente Nanosensor: Detección rápida de e. Coli O157: H7

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