Diese Arbeit wird von K-INBRE P20GM103418, Kansas Soja Kommission (KSC/PSU-1663), ACS PRF 56629-UNI7 und PSU Polymer Chemie-Gründerfonds, alle SS unterstützt. Wir danken der Universität Videofilmer, Herr Jacob Anselmi, für seine hervorragende Arbeit mit dem Video. Wir danken auch Herrn Roger Heckert und Frau Katha Heckert für ihre großzügige Unterstützung für die Forschung.

1. Synthese und Funktionalisierung von multiparametrischen Magneto-fluoreszierende Nanosensoren (MFnS).
<ol><li>Synthese von superparamagnetischen Eisenoxid-Nanopartikeln (IONPs) <ol><li>zur Vorbereitung der IONP Synthese, bereiten die folgenden 3 Lösungen: Lösung 1: FeCl 3 (0,70 g) und FeCl 2 H 2 O (2 mL), Lösung 2: NH 4 OH (2,0 mL, 13,4 M) H 2 O (15 mL) und Lösung 3: Polyacryl Säure (0,855 g) H 2 O (5 mL).</li>
		<li>Lösung 1 90 µL 2 M Salzsäure (HCl) hinzu und dann mischen Sie mit Lösung 2 sofort beim Aufschütteln bei 875 UPM. Dann fügen Sie Lösung 3. Bereich für 60 min. weiter</li>
		<li>Zentrifuge für 20 min bei 1.620 x g. Zentrifugieren den Überstand für 20 min bei 2.880 x g.</li>
		<li>Reinigen den endgültigen Überstand über Dialyse. Hinzufügen den Überstand auf eine Dialyse-Tasche (MWCO 6 &#8722; 8 K) und legen Sie sie in einen Becher mit Phosphat gepufferte Kochsalzlösung (PBS) (pH = 7,4) und Spin-Bar. Lassen Sie es für 12 h, ersetzen mit frischen Puffer alle 2-3 h einweichen</li>
	</ol></li> <li>Konjugation von Target-spezifische Antikörper auf der Oberfläche des IONPs <ol><li>bereiten die folgenden vier Lösungen: Lösung A: 5 mg 1-Ethyl - 3-(3-Dimethylaminopropyl) Carbodiimide Hydrochlorid (EDC) in 250 µL MES [2-(N- Morpholino) Ethanesulfonic Säure] Puffer (0,1 M, pH = 6,8), Lösung B: 3 mg des N-Hydroxysuccinimide (NHS) in 250 µL MES Puffer (0,1 M, pH = 6,8), Lösung C: 5 µg von IgG1, die E. Coli-mAb in 225 µL des Phosphat-gepufferte Kochsalzlösung (PBS) (pH 7,4) , und Lösung D: 4 mL IONP (5.0 Mmol) in 1 mL PBS (pH 7,4).</li>
		<li>Fügen Sie 2-(N-Morpholino) Ethanesulfonic Säure (MES) Puffer-Lösung A, dann sofort hinzufügen Lösung A Solution D in kleinen Schritten innerhalb von 15 s, invertieren die Lösung nach jeder Zugabe zum Mischen von.</li>
		<li>Beginnen sofort, Lösung B Lösung D in kleinen Schritten über einen Zeitraum von drei Minuten hinzufügen. Mischung durch invertieren die Lösung nach jeder Zugabe.</li>
		<li>Lösung in Schritten von 5 µL Lösung C hinzufügen die Lösung nach jeder Zugabe zum Mischen von invertierenden.</li>
		<li>Reaktion auf weiter für 3 h bei Raumtemperatur und dann weiter die Inkubation über Nacht bei 4 ° C zu ermöglichen.</li>
		<li>Reinigen die resultierende Ab konjugiert IONPs über magnetische Spalte mit PBS (pH = 7,4, Finale [Fe] = 3,5 Mmol), unconjugated Antikörper zu entfernen.
		<ol><li>Der magnetischen Spalte Waschen mit PBS (1 mL). Legen Sie die Spalte auf der Magnetplatte und IONP Lösung hinzufügen.</li>
			<li>Waschen die magnetischen Spalte mit PBS (1 mL) wieder. Entfernen Sie die magnetische Spalte aus magnetischen Platte. Hinzufügen der Spalte PBS und sammeln Sie die Lösung. Shop bei 4 ° C</li></ol></li></ol></li> <li>Kapselung von fluoreszierenden 1,1 &#39; - Dioctadecyl - 3,3,3 &#39;, 3 &#39;-Tetramethylindocarbocyanine Perchlorat (DiI) Farbstoff in die Polyacryl-Säure (PAA)-Beschichtung von der Antikörper konjugiert IONPs mit einem Lösungsmittel-Diffusion-Methode.
	<ol><li>Hinzufügen 4 mL der IONPs, tropfenweise 2.0 µL DiI Farbstoff (2 Mmol) in 100 µL von DMSO mit kontinuierlichen Vermischung bei 1100 u/min.</li>
		<li>Dialyse die resultierende Lösung (MWCO 6-8 KDa, 12 h) gegen PBS, erstellen eine endgültige Lösung der MFnS mit einer endgültigen Eisenkonzentration von [Fe] = 2 Mmol.</li>
	</ol></li> <li>Charakterisierung von MFnS <ol><li>für photometrische Analyse, benutzen Sie einen Platte Reader gekapselte DiI Farbstoff mit Fluoreszenz-Emission bei 595 erkennen nM.</li>
		<li>Dynamische Lichtstreuung <ol><li>legen Sie eine Probe der MFnS Lösung in einem Zetasizer zur Bestimmung der durchschnittlichen Größe und Oberflächenladung der MFnS. 
			Hinweis: Hier die Durchschnittsgröße und Oberflächenladung der funktionalen MFnS erwiesen sich 77.09 nm und-22.3 mV, bzw..</li>
		</ol></li></ol></li></ol> 2. Bakterielle Culturing und Vorbereitung der Lager-Lösung <ol><li>bakterielle Kultivierung <ol><li>Hydrat einen gefriergetrockneten Pellet (E. Coli O157: H7) in 1 mL der Nährbouillon und fügen Sie dann eine zusätzliche 5 mL Brühe.</li>
		<li>Gelten 100 µL dieser Lösung zu einer Nährbodenplatte und Streifen mit Hilfe einer sterilen Impfschlinge, gefolgt von Inkubation bei 37 ° C für 24 h.</li>
		<li>Nach 24 h, wählen Sie eine einzelne Kolonie aus der Agarplatte und eine 15 mL Kultur Brühe hinzufügen. Inkubation bei 37 ° C für ca. 4-6 h, während der Überwachung des optischen Dichte-Wert (Extinktion bei 600 nm).</li>
		<li>Das gewonnene ein optische Dichte-Wert von 0,1 ist Kultivierung zu stoppen, und führen Sie Verdünnungsreihen.</li>
	</ol></li> <li>Verdünnungsreihen <ol><li>Hinzufügen 900 µL Nährbouillon, acht sterile Mikrozentrifugenröhrchen.</li>
		<li>Hinzufügen 100 µL Bakteriensuspension auf das erste Rohr (Verdünnung 10 -1).</li>
		<li>Nehmen 100 µL vom ersten Rohr und fügen Sie es in die zweite, eine Verdünnung von 10 -2 zu erhalten. Dieses Muster für die restlichen Rohre, endend mit einer Endverdünnung von 10 -8 weiter.</li>
		<li>Transfer 100 µL aus jeder Tube Agarplatten zu trennen, und inkubieren Sie für 24 h bei 37 ° c</li>
		<li>Am nächsten Tag zählen die Koloniebildenden Einheiten (KBE) auf jeder Platte. Wählen Sie die Platte mit ~ 100 KBE. Verwenden Sie die entsprechende Verdünnungen für weitere Experimente (100 µL = ~ 100 KBE). 
		Hinweis: Hier war es die 10 -6 Verdünnung, die ~ 100 KBE führten.</li>
	</ol></li></ol> 3. Schnelle Erkennung von E. coli O157: H7 mit MFnS <ol><li>Spike verschiedenen PBS-Lösungen (1 X, pH 7,4, 300 µL) mit steigenden Mengen der 10 -6 bakterielle Lager, wodurch KBE reicht von 1-100. Fügen Sie eine konsistente Menge von MFnS (100 µL) zu diesen Lösungen.</li>
	<li>Schaffen eine Basislösung, die enthält nur PBS (1 X, pH 7,4, 300 µL) und MFnS (100 µL).</li>
	<li>Lösungen für 30 min bei 37 ° C inkubieren und dann auf Raumtemperatur abkühlen lassen.</li>
	<li>Transfer individueller Lösungen für die magnetische Relaxometer (0,47 Tesla) und Datensatzänderungen in Relaxationszeiten (T2) im Verhältnis zu der KBE in jeder Lösung.
	<ol><li>Änderungen in T2 Werte erfassen, beginnen durch die Messung des T2-Wertes der Basislösung, die nur PBS und MFnS enthält.
		<ol><li>Ort die Lösung in der magnetischen Relaxometer. Öffnen Sie die jeweilige Software, wählen Sie die &#34; T2 Entspannung &#34; Einstellung, und drücken Sie &#34; Maßnahme. &#34;</li></ol></li> <li>nach der Auflistung der Baseline T2, T2-Werte der zusätzliche Lösungen, die mit verschiedenen gespickt waren messen Konzentrationen von Bakterien. 
		Hinweis: Die Veränderung der T2 ist äquivalent zum Ausgangswert subtrahiert die Spikes T2 T2.</li>
	</ol></li> <li>Proben aus der magnetischen Relaxometer entfernen und Zentrifugieren der Rohre bei 2880 X g für 10 min.</li>
	<li>Den Überstand abgießen und Aufschwemmen bakterielle Pellets in 100 µL PBS (1 X, pH 7,4).</li>
	<li>Hinzufügen 80 µL jeder Wiederfreisetzung einer 96-Well-Platte und Rekord Fluoreszenz-Intensitäten bei 595 nM. 
	Hinweis: Prüfung wiederholt werden kann mit verschiedenen Lösungsmitteln, einschließlich Seewasser, Milch und andere, wie beschrieben im Abschnitt "Ergebnisse"</li></ol>

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Die nachweisliche Anwendung der dieser Nanotechnologie ist noch nicht von der FDA zugelassen.

Dieses Protokoll wurde entwickelt, um voll funktionsfähige MFnS produzieren so einfach wie möglich. Allerdings gibt es viele wichtige Punkte, die bei denen Änderung des Protokolls möglicherweise nützlich, je nach Ziel des Benutzers. Beispielsweise würde die Verwendung von verschiedenen Antikörpern für die Ausrichtung von vielen anderen Krankheitserregern ermöglichen. Darüber hinaus ist dieses Protokoll nicht beschränkt auf die Verwendung von Antikörpern als targeting Moleküle...

Das dauerhafte auftreten der bakteriellen Kontamination in beiden kommerziell produzierte Lebensmittel und Wasser-Quellen müssen zunehmend schnellen und spezifischen diagnostischen Plattformen geschaffen hat. 1 , 2 einige der häufigeren bakterielle Verunreinigungen für Nahrung und Wasser Kontamination verantwortlich sind aus den Salmonellen, Staphylokokken, Listerien, Vibrio, Shigella, Bacillus und Escherichia Gattungen. 3 , 4 bakterielle Kontamination durch diese Erreger häufig führt zu Symptomen wie Durchfall, Fieber, Cholera und Gastroenteritis. 4 Kontamination von Wasser-Quellen oft hat drastische und nachteilige Auswirkungen auf die Gemeinden ohne Zugang zu ausreichend gefiltertes Wasser und Kontamination von Lebensmitteln führte zu zahlreichen Erkrankungen und Produkt-Rückruf-Bemühungen. 5 , 6
Um das Auftreten von Erkrankungen durch bakterielle Kontamination zu reduzieren, wurden eine Reihe von Maßnahmen, Methoden zu entwickeln, mit denen Wasser und Nahrung effizient vor Verkauf oder Verbrauch gescannt werden können. 3 Techniken wie PCR,1,7,8,9,10 ELISA,11,12 Schleife-vermittelten isothermen Verstärkung) Lampe),13,14 u. a.15,16,17,18,19,20,21, 22,23,24 haben vor kurzem für die Erkennung von verschiedenen Krankheitserregern verwendet worden. Im Vergleich zu traditionellen bakterielle Kultivierung Methoden, sind diese Techniken wesentlich effizienter im Hinblick auf Spezifität und Zeit. Diese Techniken kämpfen noch mit falsch positive und negative, komplexe Verfahren und Kosten. 1 , 3 , 25 es ist aus diesem Grund, dass multiparametric Magneto-fluoreszierende Nanosensoren (MFnS) als eine alternative Methode für bakterielle Erkennung vorgeschlagen werden.
Diese Nanosensoren Paaren eindeutig zusammen magnetische Entspannung und fluoreszierende Modalitäten, so dass für eine Dual-Erkennung-Plattform, die eine schnelle und genaue. Mit E. Coli O157: H7 als eine Verunreinigung der Probe, zeigt die Fähigkeit des MFnS, so wenig wie 1 KBE innerhalb von Minuten erkennen. Erreger-spezifischen Antikörper werden verwendet, um die Spezifität zu erhöhen, und die Kombination von magnetischen und fluoreszierenden Modalitäten ermöglicht die Erkennung und Quantifizierung von bakteriellen Verunreinigungen in beiden Bereichen Low - und High-Kontamination. 16 bei bakteriellen Kontamination werden die Nanosensoren um die Bakterien durch die gezielten Fähigkeiten der Erreger-spezifischen Antikörper schwärmen. Die Bindung zwischen der magnetische Nanosensoren und Bakterien schränkt die Interaktion zwischen der magnetischen Eisenkern und die umliegenden Wasser Protonen. Dies bewirkt eine Erhöhung der T2-Relaxationszeiten von magnetischen Relaxometer aufgenommen. Da die Konzentration von Bakterien in Lösung steigt zu zerstreuen die Nanosensoren mit der erhöhten Anzahl von Bakterien, was zu niedrigeren T2-Werten. Im Gegensatz dazu erhöht Fluoreszenzemission proportional mit der Konzentration von Bakterien durch die gestiegene Zahl der Nanosensoren direkt pathogen gebunden. Zentrifugation der Proben und Isolation des bakteriellen Pellets werden nur die Nanopartikel direkt angeschlossen, die Bakterien, entfernen alle schwebenden Nanosensoren und direkt korrelieren die Fluoreszenz-Emission mit der Anzahl der sparen Bakterien in Lösung. Eine schematische Darstellung dieses Mechanismus ist in Abbildung 1dargestellt.
Diese MFnS-Plattform wurde mit Point-of-Care-Screening im Auge, was zu Low-Cost und tragbaren Eigenschaften entwickelt. MFnS sind bei Raumtemperatur stabil und müssen dann nur in sehr geringen Konzentrationen für präzise Erkennung von bakteriellen Verunreinigungen. Darüber hinaus nach der Synthese, Einsatz von der MFnS ist einfach und erfordert nicht die Verwendung von ausgebildeten Fachleuten auf dem Gebiet. Schließlich ermöglicht dieses Diagnoseplattform für hochgradig anpassbare targeting, ein Mittel, durch welche diese eine Plattform verwendet werden, um Krankheitserreger aller Art in vielen verschiedenen Einstellungen zu erkennen.

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			<td height="21" style="height:21px;width:239px;">Ferrous Chloride Tetrahydrate</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">I90-500</td>
			<td style="width:231px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ferric Chloride Hexahydrate</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td>I88-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ammonium Hydroxide</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">A669S-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Hydrochloric Acid</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">A144S-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Polyacryllic Acid</td>
			<td style="width:156px;">Sigma-Aldrich</td>
			<td style="width:197px;">323667-100G</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:239px;">EDC</td>
			<td style="width:156px;">Thermofisher Scientific</td>
			<td style="width:197px;">22980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">NHS</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td>AC157270250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-E. coli O111 antibody&#160;</td>
			<td style="width:156px;">sera care</td>
			<td>5310-0352</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-E. coli O157:H7 antibody [P3C6]&#160;</td>
			<td style="width:156px;">Abcam</td>
			<td style="width:197px;">ab75244</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">DiI Stain</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">D282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Nutrient Broth</td>
			<td style="width:156px;">Difco</td>
			<td style="width:197px;">233000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Freeze-dried E. coli O157:H7 pellet</td>
			<td style="width:156px;">ATCC</td>
			<td style="width:197px;">700728</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Magnetic Relaxomteter&#160;</td>
			<td style="width:156px;">Bruker</td>
			<td style="width:197px;">mq20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Zetasizer</td>
			<td style="width:156px;">Malvern</td>
			<td style="width:197px;">NANO-ZS90</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Plate Reader&#160;</td>
			<td style="width:156px;">Tecan</td>
			<td style="width:197px;">Infinite M200 PRO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Magnetic Column&#160;</td>
			<td style="width:156px;">QuadroMACS</td>
			<td style="width:197px;">130-090-976</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Centrifuge</td>
			<td style="width:156px;">Eppendorf</td>
			<td style="width:197px;">5804 Series</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Centrifuge (accuSpin Micro 17)</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">13-100-676</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Floor Model Shaking Incubator</td>
			<td style="width:156px;">SHEL LAB</td>
			<td style="width:197px;">SSI5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Analytical Balance</td>
			<td style="width:156px;">Metler Toledo</td>
			<td style="width:197px;">ME104E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Digital Vortex Mixer</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">02-215-370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Open-Air Rocking Shaker</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">02-217-765</td>
			<td></td>
		</tr>
	</tbody>
</table>

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

Enterohemorrhagic Escherichia coli O157: H7 wurde für beide wasserbasierte verbunden und lebensmittelbedingten Erkrankungen, und bleibt eine Bedrohung trotz der Nahrung und Wasser-Screening-Methoden verwendet derzeit. Während konventionelle bakterielle Nachweisverfahren wie Polymerase-Kettenreaktion (PCR) und Enzym-linked Immunosorbentprobe Assays (ELISA) speziell Pathogene Verunreinigungen erkennen können, benötigen sie umfangreiche Probenvorbereitung und lange Wartezeiten. Darüber hinaus sind diese Praktiken fordern anspruchsvolle Laborgeräte und Einstellungen, und müssen von ausgebildeten Fachleuten ausgeführt werden. Hierin wird ein Protokoll für eine einfachere diagnostische Technik vorgeschlagen, die die einzigartige Kombination von magnetischen und fluoreszierenden Parameter in ein Nanopartikel-basierte Plattform bietet. Die vorgeschlagenen multiparametric Magneto-fluoreszierende Nanosensoren (MFnS) erkennt E. Coli O157: H7 Kontamination mit so wenig wie 1 koloniebildenden Einheit in Lösung innerhalb von weniger als 1 h vorhanden. Darüber hinaus die Fähigkeit der MFnS hochfunktionelle in komplexen Medien bleiben wie Milch und Wasser des Sees verifiziert wurde. Weitere Besonderheit Assays dienten auch die Fähigkeit des MFnS, nur zu erkennen, die gezielt Bakterien, auch in Gegenwart von ähnlichen Bakterienarten nachweisen. Die Kopplung der magnetischen und fluoreszierenden Modalitäten ermöglicht die Erkennung und Quantifizierung von Pathogenkontamination in einer Vielzahl von Zusammenschlüssen, seine hohe Leistung in beiden frühen und späten Stadium Fremdkörpererkennung ausstellen. Wirksamkeit, Erschwinglichkeit und Übertragbarkeit von der MFnS machen sie einen idealen Kandidat für Point-of-Care-Screening für bakterielle Verunreinigungen in einer Vielzahl von Einstellungen, von aquatischen Stauseen zu kommerziell verpackten Lebensmitteln.

Der Wirkmechanismus von MFnS wird in Abbildung 1dargestellt. Die Wechselwirkungen zwischen den Magnetic cores der MFnS und die umliegenden Wasserstoffkerne stört das clustering von MFnS um die Oberfläche der bakteriellen Verunreinigungen. Diese Cluster, magnetische Entspannung erhöht die Werte. Mit zunehmender Konzentration der bakteriellen Verunreinigungen clustering verringert, und T2 Wertewandel senkt. Daher ist die Zugabe von einem fluoreszierenden Modalität entscheidend. Mit zunehme...

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

Das übergeordnete Ziel dieses Protokolls ist es, funktionale Nanosensoren für die Portable, kostengünstig, zu synthetisieren und schnelle Erkennung von gezielt pathogene Bakterien durch eine Kombination von magnetischen Entspannung und Fluoreszenz Emission Modalitäten.

Foodborne Krankheitserreger Screening mit Magneto-fluoreszierende Nanosensor: Schnellnachweis von 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 Aufrufe.  Pittsburg State University. Das übergeordnete Ziel dieses Protokolls ist es, funktionale Nanosensoren für die Portable, kostengünstig, zu synthetisieren und schnelle Erkennung von gezielt pathogene Bakterien durch eine Kombination von magnetischen Entspannung und Fluoreszenz Emission Modalitäten.

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

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

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

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

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

Kombination von Klebstoff-tape-basierten Sampling und Fluoreszenz In situ Hybridisierung zum schnellen Nachweis von Salmonellen On Fresh Produce

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

Forschung

Immunologie und Infektion

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

Nach Cell-Schicksal<em&gt; E. coli</em&gt; Nach Infektion mit Phagen

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Live-cell Video Mikroskopie Pilzpathogenentwicklung Phagozytose

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

Rapid-Screening von HIV-Reverse-Transkriptase und Integrase-Inhibitoren

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

Loop-vermittelte isothermen Amplifikation (LAMP) Assays für die artspezifischen Nachweis von<em&gt; Eimeria</em&gt;, Die Hühner infizieren

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

Schnelle molekulare Erkennung und Differenzierung von Influenza-Viren A und B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

Schaffung einer dichten Transposon Insertion Bibliothek verwenden bakterielle Konjugation in Enterobacterial Stämme wie Escherichia Coli oder Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

Schnelle, sichere und einfache manuelle am Krankenbett Nukleinsäure-Extraktion für den Nachweis des Virus in Vollblut-Proben

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

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.

Eine High-Throughput-Plattform für das Screening von 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 ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.