Name: combination of adhesive-tape-based sampling and fluorescence in situ hybridization for rapid detection of salmonella on fresh produce
Uploaded: 2010-10-18T17:00:00
Description: Watch this Scientific Journal Video about Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce at JoVE.com

La financiación de este trabajo fue proporcionado por un premio del Fondo de crecer Iowa Valores de BFBS.

1. Superficie de muestreo con cinta adhesiva estéril <ol><li> Seleccione una cinta a utilizar para el muestreo. Disponibles en el comercio Hongos-Tape o póngase en contacto-Es cintas de muestreo son estériles y envasadas especialmente para la facilidad de uso. Sin embargo, hemos encontrado que la transparencia (ópticamente transparente) cinta de la oficina de genéricos también se puede utilizar. </li><li> Use un marcador permanente para dibujar 1 cm 2 plazas en el lado no pegajosa de una pieza de 10 c...

<ol>
	<li>Almeida, C., Azevedo, N. F., Fernandes, R. M., Keevil, C. W., Vieira, 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=Fluorescence+in+situ+hybridization+method+using+a+peptide+nucleic+acid+probe+for+the+identification+of+Salmonella+spp.+in+a+broad+spectrum+of+samples.">Fluorescence in situ hybridization method using a peptide nucleic acid probe for the identification of Salmonella spp. in a broad spectrum of samples.</a> Appl. Environ. Microbiol. 76, 4476-4485 (2010).</li><li>Barnetson, R. S., Milne, L. J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+sampling+for+Candida+with+adhesive+tape.">Skin sampling for Candida with adhesive tape.</a> Br. J. Dermatol. 88, 487-491 (1973).</li><li>Bisha, B., Brehm-Stecher, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+adhesive-tape-based+sampling+of+tomato+surfaces+combined+with+rapid+fluorescence+in+situ+hybridization+for+Salmonella+detection.">Simple adhesive-tape-based sampling of tomato surfaces combined with rapid fluorescence in situ hybridization for Salmonella detection.</a> Appl. Environ. Microbiol. 75, 1450-1455 .</li><li>Bisha, B., Brehm-Stecher, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-through+imaging+cytometry+for+characterization+of+Salmonella+subpopulations+in+alfalfa+sprouts,+a+microbiologically+complex+food+system.">Flow-through imaging cytometry for characterization of Salmonella subpopulations in alfalfa sprouts, a microbiologically complex food system.</a> Biotechnol. J. 4, 880-887 (2009).</li><li>Edwards, R. W., Hartman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+collecting+fungus+specimens+from+infected+surfaces.">A simple technique for collecting fungus specimens from infected surfaces.</a> Lloydia. 15, 39-39 (1952).</li><li>Evancho, G. M., Sveum, W. H., Moberg, L. J., Frank, J. F., Pouch Downes, F., Ito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiological+monitoring+of+the+food+processing+environment.">Microbiological monitoring of the food processing environment.</a> Compendium of Methods for the Microbiological Examination of Foods. , (2001).</li><li>Fung, D. Y. C., Lee, C. Y., Kastner, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesive+tape+method+for+estimating+microbial+load+on+meat+surfaces.">Adhesive tape method for estimating microbial load on meat surfaces.</a> J. Food. Prot. 43, 295-297 (1980).</li><li>Kutter, S., Hartmann, A., Schmid, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonization+of+barley+(Hordeum+vulgare)+with+Salmonella+enterica+and+Listeria+spp.">Colonization of barley (Hordeum vulgare) with Salmonella enterica and Listeria spp.</a> FEMS Microbiol. Ecol. 56, 262-271 (2006).</li><li>La Cono, V., Urz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+in+situ+hybridization+applied+on+samples+taken+with+adhesive+tape+strips.">Fluorescent in situ hybridization applied on samples taken with adhesive tape strips.</a> J. Microbiol. Meth. 55, 65-71 (2003).</li><li>Lakshmanan, C., Schaffner, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+and+controlling+microbiological+contamination+of+beverage+dispensers+in+university+foodservice+operations.">Understanding and controlling microbiological contamination of beverage dispensers in university foodservice operations.</a> Food Prot. Trends. 26, 27-31 (2005).</li><li>Langvad, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+rapid+method+for+qualitative+and+quantitative+study+of+the+fungal+flora+of+leaves.">A simple and rapid method for qualitative and quantitative study of the fungal flora of leaves.</a> Can. J. Microbiol. 26, 666-670 (1980).</li><li>Nordentoft, S., Christensen, H., Wegener, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+fluorescence-labelled+oligonucleotide+probe+targeting+23S+rRNA+for+in+situ+detection+of+Salmonella+serovars+in+paraffin-embedded+tissue+sections+and+their+rapid+identification+in+bacterial+smears.">Evaluation of a fluorescence-labelled oligonucleotide probe targeting 23S rRNA for in situ detection of Salmonella serovars in paraffin-embedded tissue sections and their rapid identification in bacterial smears.</a> J. Clin. Microbiol. 35, 2642-2648 (1997).</li></ol>

Métodos sencillos y rápidos para la detección de patógenos en las superficies de los productos pueden ayudar a mitigar las enfermedades transmitidas por los alimentos, proporcionando información oportuna y procesable. Adhesivo a base de cinta de métodos de muestreo se han utilizado en el medio ambiente, clínica y microbiología de los alimentos desde la década de 1950 e implican apremiantes de &quot;Scotch&quot; al estilo de la cinta a la superficie para la eliminación de microorganismos, seguido por el examen ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Fungi-Tape sampling tape</td>
<td>&nbsp;</td>
<td>Scientific Device Laboratory, Des Plaines, IL</td>
<td>745</td>
<td>http://www.scientificdevice.com/</td>
</tr>
<tr>
<td>Con-Tact-It sampling tape</td>
<td>&nbsp;</td>
<td>Birko Corporation, Denver, CO</td>
<td>&nbsp;</td>
<td>http://www.birkocorp.com/</td>
</tr>
<tr>
<td>Clear office tape, generic</td>
<td>&nbsp;</td>
<td>Various suppliers</td>
<td>&nbsp;</td>
<td>Should be optically clear, have low intrinsic fluorescence</td>
</tr>
<tr>
<td>Food surface</td>
<td>&nbsp;</td>
<td>Local grocery</td>
<td>&nbsp;</td>
<td>Tomatoes (red tomatoes on the vine, not waxed or oiled) used here</td>
</tr>
<tr>
<td>Trypticase Soy Broth</td>
<td>&nbsp;</td>
<td>Difco, Sparks, MD</td>
<td>211768</td>
<td>For non-selective liquid surface miniculture enrichment</td>
</tr>
<tr>
<td>Xylose-lysine-Tergitol 4 agar base</td>
<td>&nbsp;</td>
<td>Difco, Sparks, MD</td>
<td>223420</td>
<td>For Salmonella-selective agar (XLT-4)</td>
</tr>
<tr>
<td>Xylose-lysine-Tergitol 4 agar supplement</td>
<td>&nbsp;</td>
<td>Difco, Sparks, MD</td>
<td>235310</td>
<td>For Salmonella-selective agar (XLT-4)</td>
</tr>
<tr>
<td>Formalin solution </td>
<td>&nbsp;</td>
<td>Sigma-Aldrich, St. Louis, MO</td>
<td>HT5011</td>
<td>10% solution, neutral, buffered (cell fixative)</td>
</tr>
<tr>
<td>Absolute ethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich, St. Louis, MO</td>
<td>E7023</td>
<td>Molecular biology grade (pre-hybridization dehydration)</td>
</tr>
<tr>
<td>1.5 ml microcentrifuge tubes</td>
<td>&nbsp;</td>
<td>Various suppliers</td>
<td>&nbsp;</td>
<td>RNase- and DNase-free</td>
</tr>
<tr>
<td>Microscope slides and cover slips</td>
<td>&nbsp;</td>
<td>Thermo Fisher Scientific, Waltham, MA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NaCl solution</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich, St. Louis, MO</td>
<td>S5150</td>
<td>Molecular biology grade, 5M solution (hybridization buffer component)</td>
</tr>
<tr>
<td>Tris-EDTA buffer solution (100X concentrate)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich, St. Louis, MO</td>
<td>T9285</td>
<td>1M Tris [pH 8.0], 0.1M EDTA (hybridization buffer component)</td>
</tr>
<tr>
<td>Sodium dodecyl sulfate solution</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich, St. Louis, MO</td>
<td>L4522</td>
<td>10% solution in 18 megohm water (hybridization buffer component)</td>
</tr>
<tr>
<td>Sal3 and Salm-63 oligonucleotide probes</td>
<td>&nbsp;</td>
<td>Integrated DNA Technologies, Coralville, IA</td>
<td>&nbsp;</td>
<td>5&#x2019;-labeled with 6-carboxyfluorescein (FAM) or Texas Red (for microscopy) or Cy5 (for cytometry), HPLC-purified</td>
</tr>
<tr>
<td>Variable speed microcentrifuge</td>
<td>&nbsp;</td>
<td>Various suppliers</td>
<td>&nbsp;</td>
<td>Use rotor diameter to calculate RPM needed for RCF values described in protocol</td>
</tr>
<tr>
<td>CoverWell perfusion chamber </td>
<td>&nbsp;</td>
<td>Grace Bio-Labs Inc., Bend, OR</td>
<td>PC1R-2.0</td>
<td>Non-sterile</td>
</tr>
<tr>
<td>Gel loading pipette tips (FS MultiFlex)</td>
<td>&nbsp;</td>
<td>Thermo Fisher Scientific, Waltham, MA</td>
<td>05-408-151</td>
<td>Long, thin tips for easy access to small sampling ports and maneuverability within chamber</td>
</tr>
<tr>
<td>Aluminum heat block or precision-controlled heating station</td>
<td>&nbsp;</td>
<td>Various suppliers</td>
<td>&nbsp;</td>
<td>Eppendorf Thermomixer R dry block heating and cooling shaker used here</td>
</tr>
<tr>
<td>Bambino mini hybridization oven</td>
<td>&nbsp;</td>
<td>Boekel Scientific, Feasterville, PA</td>
<td>Model 230300</td>
<td>Slides are placed in 50 ml polypropylene centrifuge tubes for hybridization, heat transfer not direct</td>
</tr>
<tr>
<td>Slide Moat slide hybridizer</td>
<td>&nbsp;</td>
<td>Boekel Scientific, Feasterville, PA</td>
<td>Model 240000</td>
<td>Provides rapid, direct transmission of heat through glass slide</td>
</tr>
<tr>
<td>Vectashield H-1200 mounting medium with 4&#x2019;,6-diamidino-2-phenylindole (DAPI)</td>
<td>&nbsp;</td>
<td>Vector Laboratories, Inc., Burlingame, CA</td>
<td>H-1200</td>
<td>Minimizes quenching of fluorescence during microscopy, provides DAPI counterstain</td>
</tr>
<tr>
<td>Fluorescence microscope</td>
<td>&nbsp;</td>
<td>Various suppliers </td>
<td>&nbsp;</td>
<td>Leitz Laborlux S used here</td>
</tr>
<tr>
<td>Digital camera</td>
<td>&nbsp;</td>
<td>Various suppliers</td>
<td>&nbsp;</td>
<td>Canon PowerShot A640 camera used here</td>
</tr>
<tr>
<td>Image acquisition software</td>
<td>&nbsp;</td>
<td>Various suppliers</td>
<td>&nbsp;</td>
<td>Axiovision software v. 4.6 (Carl Zeiss) used</td>
</tr>
<tr>
<td>Adobe Photoshop</td>
<td>&nbsp;</td>
<td>Adobe Inc.</td>
<td>&nbsp;</td>
<td>For minimal processing of images (overlay of images taken in different channels)</td>
</tr>
<tr>
<td>Flow cytometer</td>
<td>&nbsp;</td>
<td>Various suppliers</td>
<td>&nbsp;</td>
<td>FACSCanto flow cytometer (BD Biosciences, San Jose, CA) with red (647 nm) excitation used</td>
</tr>
<tr>
<td>Flow cytometry analysis software</td>
<td>&nbsp;</td>
<td>Various suppliers</td>
<td>&nbsp;</td>
<td>FlowJo software v. 8.7.1 (Tree Star, Inc.) used</td>
</tr>		</tbody>
		</table>
		</div>

combination of adhesive-tape-based sampling and fluorescence in situ hybridization for rapid detection of salmonella on fresh produce

Este protocolo describe un método sencillo para que el adhesivo, la cinta basada en el muestreo de tomate y otras superficies de los productos frescos, seguido por fluorescencia en cinta de hibridación in situ (FISH) para un rápido cultura independiente de la detección de Salmonella spp. Cintas cargadas de células también puede ser colocado boca abajo en agar selectivo para la fase sólida de enriquecimiento antes de la detección. Por otra parte, bajo volumen de enriquecimiento líquido (miniculture superficie del líquido) se puede realizar en la superficie de la cinta en el no selectivo caldo, seguidos por el pescado y el análisis mediante citometría de flujo. Para comenzar, cinta adhesiva estéril se pone en contacto con productos frescos, se aplica presión leve, y de retirar la cinta, de extraer físicamente los microbios presentes en estas superficies. Las cintas están montadas pegajoso hacia arriba en portaobjetos de vidrio y las células de la muestra se fijan con formol al 10% (30 min) y deshidratados mediante una serie gradual de etanol (50, 80, y 95%, 3 min cada concentración). A continuación, las células cargadas las cintas se han manchado con un tampón que contiene un cóctel de ADN de Salmonella objetivo de la sonda y se hibridaron durante 15 - 30 min a 55 ° C, seguido de una breve aclaración en un tampón de lavado para eliminar la sonda no unido. Adherente, PESCADO marcado células se contrastados con el ADN tinte 4 &#39;,6-diamidino-2-phenylindole (DAPI) y los resultados se ven usando la microscopía de fluorescencia. Para la fase sólida de enriquecimiento, las células cargadas las cintas se colocan boca abajo sobre una superficie de agar selectivo adecuado y se incuban para permitir el crecimiento in situ de microcolonias Salmonella, seguidos por el pescado y la microscopia como se describió anteriormente. Para miniculture superficie del líquido, las células cargadas las cintas se colocan lado pegajoso hacia arriba y una cámara de perfusión de silicona se aplica para que la cinta y la forma del portaobjetos del microscopio la parte inferior de una cámara hermética en la que un pequeño volumen (≤ 500 l) de tripticasa de soja caldo (TSB) se introduce. Los puertos de entrada están cerrados y las cámaras se incuban a 35 - 37 ° C, permitiendo que el crecimiento basado en la amplificación de extraer la cinta-microbios. Después de la incubación, los puertos de entrada son sin sellar, las células se separan y se mezclan con la espalda vigorosa y pipeteo adelante, cosechadas por centrifugación y se fija en 10% neutros formalina tamponada. Finalmente, las muestras se hibridan y se examina a través de citometría de flujo para revelar la presencia de Salmonella spp. Como se describe aquí, nuestra &quot;cinta-FISH&quot; enfoque puede proporcionar un muestreo simple y rápida y la detección de Salmonella en las superficies de tomate. También hemos utilizado este enfoque para el muestreo de otros tipos de productos frescos, como la espinaca y chiles jalapeños.

Watch this Scientific Journal Video about Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce at JoVE.com

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

Este protocolo describe un simple adhesivo cinta enfoque basado en el muestreo de tomate y otras superficies de los productos frescos, seguido por la rápida detección de células enteras de<em&gt; Salmonella</em&gt; Mediante fluorescencia<em&gt; In situ</em&gt; Hibridación (FISH).

Combinación de adhesivo basado en cinta de muestreo y de fluorescencia In situ La hibridación para la detección rápida de Salmonella En los productos frescos

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Research

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Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

Flujo de ácido nucleico cerrado Citometría de fluorescencia In situ</emHibridación> (LNA flujo-FISH): un método para la detección de bacterias pequeñas de ARN

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

Investigación

Inmunología e Infección

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Hibridación<em&gt; In situ</em&gt; De las glándulas salivales, los ovarios y embriones de los mosquitos vectores

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescente<em&gt; In situ</em&gt; La hibridación en los cromosomas mitóticos de mosquitos

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Método de muestreo hisopo para la Detección de norovirus humano en superficies

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

La neuraminidasa Ensayo de inhibición basado en la fluorescencia para evaluar la susceptibilidad de los virus de la influenza a El Inhibidor de la neuraminidasa clase de antivirales

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

Muestreo no invasivo de revestimiento mucoso líquido para la cuantificación In Vivo de la vía aérea superior mediador inmune niveles

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Aislamiento de Salmonella typhimurium-que contienen fagosomas de los macrófagos

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

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

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

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 ...

Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells

Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells

Fluorescencia cuantitativa en hibridación situ (FISH) e inmunofluorescencia (IF) de productos genéticos específicos en células infectadas por KSHV

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout ...

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Un método rápido para imágenes de fluorescencia multiespectral de secciones de tejido congelado

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample ...