Diese Arbeit wurde durch die National Institutes of Health (NIH) unter dem Förder RC3 AI089106 unterstützt. MDR-TB Nukleinsäuren wurden durch das Kinderhilfswerk der Vereinten Nationen Entwicklungsprogramm Fund / UN / Weltbank / Weltgesundheitsorganisation Sonderprogramm für Forschung und Ausbildung in Tropical Diseases (TDR), Genf, Schweiz zur Verfügung gestellt. Wir danken Dr. Tom Shinnick der US Centers for Disease Control and Prevention zur Orientierung auf die spezifischen Gene und Mutationen in der Prototypen-Test enthalten.

Für Labore, die universelle PCR-Anweisungen nicht befolgen, ist es operativ effizienter, mehrere Verstärkung Microarrays und Dichtungen pro Substrat enthalten und waschen für alle Verstärkungsmicroarrays gleichzeitig in einem Großbehälter, wie hier beschrieben. Verbrauchsmaterialformate sind für die Durchführung nach der Amplifikation Microarray-Waschschritte in einem völlig abgedichtet, geschlossene Amplikon-Test zur Verfügung, wie an anderer Stelle berichtet, 30,33. 1. Setup- <ol><li> Extrahieren und zu reinigen Nukleinsäuren aus der Probe unter geeigneten Bedingungen die biologische Sicherheit mit einer Methode der Wahl. Voraussetzung ist, dass Nukleinsäuren ausreichender Reinheit für die asymmetrische, Multiplex-PCR-Amplifikation. </li><li> Bereiten Arbeitsbereich durch Abwischen Oberflächen und Geräte mit Dekontaminierung Lösungen. </li><li> Zeigen Amplifikationsreagenzien auf Eis oder kalten Block. </li><li> Beschriften Sie eine sterile 1,5-ml-Mikrozentrifugenröhrchen für die Verstärkung Master-Mix, und kennzeichnen eine sterile 0,2-ml-Mikro tuwerden für jede Probe. </li><li> Nach Reagenzien werden aufgetaut, kurz Wirbel und sammeln Inhalt auf den Boden des Röhrchens mit einem puls Spin in einer Minizentrifuge. Vortexen nicht Taq-Polymerase. Flick vorsichtig das Rohr zu mischen und folgen mit einem Impuls-Spin-in Mini-Zentrifuge. </li><li> Bereiten Sie eine Verstärkung Master-Mix mit den pro-Probenreaktion in Tabelle 1 dargestellt Bände. Um mögliche Ungenauigkeiten Pipettieren Konto, bereiten mindestens eine weitere Reaktionsvolumen als die Gesamtzahl der Proben verarbeitet werden. Beachten Sie, dass die Master-Mix enthält zusätzliche Taq-Polymerase, die über das, was mit der Muliplex-PCR-Puffer vorgesehen ist. Fügen Sie nur die Verstärkung / Hemmung Steuerung an den Master-Mix, nachdem alle anderen Reagenzien kombiniert werden, und die Aktien Reagenzröhrchen den Speicher zurück. </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Reagens </td><td> Per-Probenvolumen (ul) </td><td> &lt;strong&gt; Finale [] </td></tr><tr><td> Mulitplex-PCR-Puffer mit HotStar Taq Plus- </td><td> 25 </td><td> 1x </td></tr><tr><td> Rinderserumalbumin (BSA) </td><td> 0,55 </td><td> 0,6 mg / ml </td></tr><tr><td> Formamid </td><td> 3.8 </td><td> 7,6% </td></tr><tr><td> Zusätzliche Taq-Polymerase </td><td> 0,8 </td><td> Einheiten / ul (4 Einheiten insgesamt) </td></tr><tr><td> MDR-TB-Primer-Gemisch </td><td> 15,75 </td><td> - </td></tr><tr><td> RNase-freie H 2 O </td><td> 2.1 </td><td> - </td></tr><tr><td> Verstärkung / Hemmung Steuer </td><td> 1 </td><td> 5,0 fg / ul </td></tr><tr><td> Gesamt </td><td> 49 </td><td></td></tr></tbody></table> Tabelle 1. MDR-TB Verstärkung Microarray-Master-Mix-Zusammensetzung. <ol start="7"><li> Wirbel die Master-Mix und sammeln Inhalt auf den Boden des Röhrchens mit einem Impuls-Spin in einer Minizentrifuge. </li><li> Kombinieren Sie 49 ul des Master-Mix und 1 ul von M. tuberculosis-DNA mit jedem der Probenröhrchen von Schritt 1.4, oben. Für eine externe, nicht-Template-Kontrolle (NTC), verwenden Sie 1 ul der Molekularbiologie Wasser anstelle des M. Tuberkulose DNA-Probe. Vortex-Spin-und Impuls die Röhre (n), wenn Sie fertig. </li></ol> 2. Legen Amplification Microarrays <ol><li> In 48 ul jeder Master-Mix / Probe auf die Mitte der jeweiligen Mikroarrays, wobei Sie darauf achten, den Microarray selbst nicht berühren. </li><li> Legen Sie ein Deckglas auf dem Microarray-Dichtung und Dichtung, man aufpassen, nicht zu stoppen keine Luftblasen in der Microarray-Kammer. Luftblasen können sich negativ auf Verstärkungseffizienzund kann zu Artefakten oder Rauschen in dem nachfolgenden Bild-Mikroarray zu erzeugen. </li></ol> 3. Thermal Cycling <ol><li> Sobald alle Microarrays sind mit Probe werden Substrate auf der flachen Block Thermocycler geladen. Stellen Sie sicher, dass der beheizte Deckel Option auf &quot;Aus&quot;. Ausstattung von Akonni Biosystems wird bereits für die Verwendung präqualifiziert werden. Ansonsten ist es sehr wichtig, um zu überprüfen, dass die flache Block Thermocycler (n) bieten (n) einheitliche, konsistente Heizung in allen Mikroarray-Substraten. </li><li> Öffnen Sie den Thermocycler-Software, wählen Sie das entsprechende Programm, geben Sie &quot;50 ul&quot; für das Reaktionsvolumen, und starten Sie die Sicht. Die hier beschriebene Wärmezyklus-Protokoll besteht aus: <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Thermal Cycling Schritte </td></tr><tr><td> 1 </td><td> 88 ° C </td><td> 5 min </td></tr><tr><td> 2 </td><td> 88 ° C </td><td> 30 Sek. </td></tr><tr><td> 3 </td><td> 55 ° C </td><td> 1 min </td></tr><tr><td> 4 </td><td> 65 ° C </td><td> 30 Sek. </td></tr><tr><td> 5 </td><td> Wiederholen Sie die Schritte (2-4) für 50 Zyklen </td></tr><tr><td> 6 </td><td> 65 ° C </td><td> 3 min </td></tr><tr><td> 7 </td><td> 55 ° C </td><td> 3 Stunden </td></tr></tbody></table></li><li> Die Gesamtzahl der thermischen Zyklen und 55 ° C nach der Amplifikation Haltezeit unabhängige Variable sind, dass der Benutzer zu ändern, wie benötigt oder für den spezifischen Experiment geeignet. Da der MDR-TB-Master-Mix schafft asymmetrisch (überwiegend Einzelstrang) Amplikons, ist es meist hilfreich, mehr Wärmezyklen, als es sonst für einen herkömmlichen, exponentielle PCR-Amplifikation-Protokoll verwendet werden, umfassen. </li><li> Wenn der Temperaturwechsel und Hybridisierung Programm abgeschlossen ist, beenden Sie das Programm, entfernen Sie die Verstärkung Microarrays, schließen Sie die Software, und schalten Sie den Thermocycler (n).</li></ol> 4. Waschen und Trocknen <ol><li> Zum Waschen bis zu 24 Substrate gleichzeitig werden mindestens 250 ml 1x SSPE - 0,1% Triton X-100 Waschpuffer, und zwei Behälter mit mindestens 250 ml deionisiertem oder Milli-Q-Wasser jeweils. Der Waschpuffer kann im Voraus erstellt werden. </li><li> Das Deckglas und die Dichtung von jedem Verstärkung Microarray Kammer mit Flach-End-Zange vorsichtig entfernen. Dieser Schritt ist der Punkt, an dem es das größte Risiko von schädlichen physikalisch-Gel-Element-Arrays. Verwenden Sie geeignete PCR-Kontaminationskontrollen, um die Ausbreitung zu verhindern amplifizierten Nukleinsäuren in der Arbeitsumgebung. </li><li> Zeigen Microarray-Substraten in einer Histologie Objektträger-Halter, und setzen Sie alle Folien in den Waschkorb mit der Waschpuffer. Bedecken Sie den Behälter mit einem Deckel. </li><li> Waschen Mikroarrays für 10 min bei Raumtemperatur unter leichtem Schütteln. </li><li> Tauchen Sie den Microarrays jeweils drei Mal in zwei aufeinanderfolgenden Waschungen VE-oderMilli-Q-Wasser und der Luft trocknen. </li></ol> 5. Imaging Die asymmetrische MDR-TB-Primer-Gemisch erzeugt Cy3-markierten Amplikons. Gel-Element-Microarrays kann mit jedem Standard-Microarray-Imager in der Lage, Bild Cy3 abgebildet werden. Die folgende Bildverfahren ist spezifisch für die MDR-TB-Master-Mix, Dx2100 Feld tragbare Imager und automatisierte Analyse-Software von Akonni Verfügung gestellt. <ol><li> Stellen Sie sicher, dass das Ethernet-Kabel vom Scanner an den Computer angeschlossen ist. </li><li> Schalten Sie den Scanner. Der Netzschalter befindet sich auf der Rückseite des Gerätes befindet. Blaue LEDs auf der Vorderseite der Abbildungs ​​leuchtet, wenn Imager eingeschaltet ist. </li><li> Den Computer einzuschalten. </li><li> Auf dem Computer-Desktop, doppelklicken Sie auf das Software-Symbol, um die automatisierte Bildanalyse-Software zu starten. </li><li> Warten Sie auf die Software, um eine Verbindung mit dem Imager-Kamera zu etablieren. Sobald die Verbindung hergestellt ist, wird die Schaltfläche Analysieren zur Verfügung, und dieMeldung &quot;Verbindung erfolgreich&quot; wird in der unteren linken Ecke des Bildschirms angezeigt. </li><li> Legen Sie die getrockneten Mikroarray-Verstärkung mit der Mikroarray abgewandten den Benutzer und Richtung der Objektivlinse. Wenn das Mikroarray mit nicht gegen der Linse und der Kamera ausgerichtet ist, wird die automatisierte Analyse-Software nicht, um ein Raster auf dem Fluoreszenzbild zu platzieren. </li><li> Schließen Sie die Tür. Eine Vorschau Bild wird auf dem Computer-Bildschirm verfügbar sein. </li><li> Wählen Sie das MDR-TB-Analyse Skript aus dem Dropdown-Menü und geben Sie die gewünschte Belichtungszeit (in Millisekunden). Ein typischer Ausgangspunkt für Gel-Element Verstärkung Microarrays 100-500 ms. </li><li> Gesättigte Pixel auf dem Vorschaubild in rot angezeigt. Einstellen der Belichtungszeit, um die Anzahl von gesättigten Bildelementen innerhalb einzelner Flecken zu minimieren. </li><li> Klicken Sie auf Analysieren, um das Fluoreszenzbild zu erwerben und zu analysieren. Die Software wird automatisch vergeben einenMDR-TB-Assay-spezifischen Gitter auf das Bild, zu extrahieren und integrierte Intensität der Hintergrundwerte und berichten Arzneimittelresistenz und Genotypisierung Ergebnisse. Die automatisierte Analyse dauert in der Regel ein paar Sekunden. Für anspruchsvolle Bilder, die Kratzer, Staub, fluoreszierende Artefakte oder physische Schäden an den Microarray-Funktionen enthalten, muss die Software bis zu 1 min, um die Analyse abzuschließen. </li><li> Sobald die Analyse abgeschlossen ist, klicken Sie auf Speichern, wählen Sie den Zielordner, geben Sie einen Dateinamen ein, und klicken Sie auf OK. Basiswert Microarray-Daten werden als. Xls-Datei gespeichert und können für die Offline-Analysen exportiert werden, wenn gewünscht. </li><li> Sobald die Analyse abgeschlossen ist, entfernen die Verstärkung aus der Microarray-Imager, und klicken Sie auf Neu, um die nächste Analyse einzuleiten. </li><li> Beim Sammeln von Daten abgeschlossen ist, schließen Sie die Software, den Computer herunterfahren, und schalten Sie den Bildwandler. </li></ol>

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Microbiol. 45, 2802-2807 (2007).</li><li>Chandler, D. P., Bryant, L., 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=Integrated+amplification+microarrays+for+infectious+disease+diagnostics.">Integrated amplification microarrays for infectious disease diagnostics.</a> Microarrays. 1, 107-124 (2012).</li><li>Vincent, V., Rigouts, L., 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=The+TDR+Tuberculosis+Strain+Bank:+a+resource+for+basic+science,+tool+development+and+diagnostic+services.">The TDR Tuberculosis Strain Bank: a resource for basic science, tool development and diagnostic services.</a> Int. J. Tuberc. Lung Dis. 16, 24-31 (2012).</li><li>Golova, J. B., Chernov, B. K., 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=Non-volatile+copolymer+compositions+for+fabricating+gel+element+microarrays.">Non-volatile copolymer compositions for fabricating gel element microarrays.</a> Anal. Biochem. 421, 526-533 (2012).</li><li>Cooney, C. G., Sipes, D., Thakore, N., Holmberg, R., Belgrader, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+plastic,+disposable+microfluidic+flow+cell+for+coupled+on-chip+PCR+and+microarray+detection+of+infectious+agents.">A plastic, disposable microfluidic flow cell for coupled on-chip PCR and microarray detection of infectious agents.</a> Biomed. Microdevices. 14, 45-53 (2012).</li><li>Lane, S., Evermann, J., Loge, F., Call, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplicon+secondary+structure+prevents+target+hybridization+to+oligonucleotide+microarrays.">Amplicon secondary structure prevents target hybridization to oligonucleotide microarrays.</a> Biosens. Bioelectron. 20, 728-735 (2004).</li><li>El-Hajj, H. H., Marras, S. A. E., 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=Use+of+sloppy+molecular+beacon+probes+for+identification+of+Mycobacterial+species.">Use of sloppy molecular beacon probes for identification of Mycobacterial species.</a> J. Clin. Microbiol. 47, 1190-1198 (2009).</li><li>Boehme, C. C., Nabeta, P., 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+molecular+detection+of+tuberculosis+and+rifampin+resistance.">Rapid molecular detection of tuberculosis and rifampin resistance.</a> N. Engl. J. Med. 363, 1005-1015 (2010).</li></ol>

Die Autoren erklären, keine finanziellen Interessen konkurrieren.

Das Ausmaß der mit einer Multiplex-Amplifikation Mikroarray wird letztendlich von der Effizienz der Multiplex-PCR asymmetrisch, nicht das Mikroarray bestimmt wird. Nach unserer Erfahrung können 10-12 einzigartigen Primerpaare leicht zu einer Verstärkung Microarray-Format gemultiplext werden. Herkömmliche Primer-und Sondendesign Kriterien daher, neue Tests, mit Ausnahme, dass man muss auch mögliche Wechselwirkungen zwischen Lösungsphase Nukleinsäuren und Microarray-Sonden immobilisiert, der thermische Wirkungsgrad...

Frühe Erkennung und Fall schnelle Behandlung gelten als die wirksamsten Kontrollstrategien zur Mycobacterium tuberculosis (MTB) Getriebe 1 zu reduzieren, und es gibt jetzt einen breiten Konsens in der TB-Community, die ein Point of Care (POC) oder in der Nähe von POC-Test, um gleichzeitig zu diagnostizieren TB und Arzneimittelresistenz (DR) benötigt. Technologien wie Cepheid Genexpert und andere Nukleinsäure-Amplifikation Tests reduzieren die Zeit bis zur Diagnose für viele TB-Patienten und bieten eine schnelle Auslesen der angibt, Resistenz gegen Rifampicin oder ausgewählte Mutationen eine Resistenz gegen andere erste oder zweite Linie 2 Drogen. Obwohl Echtzeit-und isothermen Nucleinsäureamplifikation Tests sind für die Medikamentenresistenz Mutationen, die zu MDR-TB führen, zu identifizieren, ist das Spektrum der Mutationen erkennen sie oft nicht ausreichend, um eine individuelle medikamentöse Behandlung, die der Arzneimittel-Resistenz-Profil des Patienten zu gestalten, und technische Zwänge im Zusammenhangum optisches Übersprechen oder die Komplexität der chemischen Verstärkung und Berichts Mai 3-7 die Anzahl der Loci oder Mutationen, die erfasst werden, zu begrenzen. So sind Detektionstechnologien mit höherer Kapazität erforderlich, um Multiplexing bekannt Lücken im MDR-TB POC Diagnostik zu adressieren. Microarrays und die WHO-endorsed Hain Linie Sondentests können die &quot;multiple Gen, mehrere Mutationen&quot; Herausforderung der Diagnose von MDR-TB 8-29 ehen. Leider sind diese Hybridisierung-basierte Multiplex-Detektionsplattformen verwenden mehrstufigen, kompliziert und Open-Amplikon Protokolle, die eine umfassende Ausbildung und Kenntnisse in molekularen Techniken erfordern. Die Verstärkung Microarray-30 wurde entwickelt, um einige dieser Microarray-Work-Flow und Betriebs Anliegen anzusprechen. Die Vereinfachung fluidischen Grundsätze sind zu ergänzen, zu hybridisieren und erkennen Nukleinsäure-Targets in einem einzigen mikrofluidischen Kammer. Der Benutzer stellt die Nukleinsäure-Amplifikation und master mischen in einem fluidischen Kammer mit einer Pipette und startet den Temperaturwechsel-Protokoll. Für die hier gezeigte Stapelverarbeitung Verfahren werden Mikroarrays anschließend in Lösung gewaschen, getrocknet und abgebildet. Diese Studie veranschaulicht die Funktionalität von einem Verstärkungsmikroarray unter Verwendung eines MDR-TB Microarray-Test rpoB (30 Mutationen) katG (2 Mutationen), inhA (4 Mutationen), rpsL (2 Mutationen), embB (1-Mutation), IS1245, IS6110 und eine interne Verstärkung und Hybridisierungskontrolle. Mindestens ein angepaßtes Paar von Mikroarray-Sonden (Wildtyp (WT) und Single-Nukleotid-Mutante (MU)) für jede Mutation von Interesse enthalten. Gereinigte Nukleinsäuren von multiresistenter M. Tuberkulose sind von der TDR-Tuberkulose-Stamm Bank-31. Gel-Element-Microarrays auf Glassubstraten durch Copolymerisation im Wesentlichen hergestellt wie an anderer Stelle 32, mit der Ausnahme, dass wir 4% Monomer und 0,05 mM jede Sonde in der polymerization Mischung. Arrays werden mit einem 50-ml-Dichtung vor der Verwendung umgeben. Nach dem thermischen Zyklus, Hybridisierung und die Waschschritte werden Amplifikation Mikroarrays auf ein tragbares Analyse Akonni abgebildet. Hintergrund-korrigiert, integrierte Signalintensitäten von der rohen erhalten. Tif Bilder mit einem festen Kreis-Algorithmus. Lärm für jedes Gel Element als das Dreifache der Standardabweichung der lokalen Stelle Hintergründe berechnet. Gen-Targets werden in der Regel als nachweisbar Signal-Rausch-Verhältnis (SNR)-Werte ≥ 3. Um die Anwesenheit oder Abwesenheit eines spezifischen Mutation in jedem Gen oder Codon zu bestimmen, wird eine Diskriminanzanalyse Verhältnis aus den SNR-Werte als (WT-MU) / (WT + MU) berechnet. Diskriminanzanalyse Verhältnisse &lt;0 deuten auf eine Medikamentenresistenz Mutation an der Stelle, während die Verhältnisse&gt; 0 sind bezeichnend für die Wildtyp-Sequenz.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;">MDR-TB amplification microarrays, with applied gasket and pre-cut cover slips</td>
			<td>Akonni Biosystems</td>
			<td>Inquire</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Multiplex PCR kit, containing 2X PCR buffer with HotStar Taq plus</td>
			<td>Qiagen</td>
			<td>#206143</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Taq polymerase</td>
			<td>Qiagen</td>
			<td>#201207</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">RNAse-free water</td>
			<td>Qiagen</td>
			<td>#206143&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Formamide</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#BP227-500</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">20 mg mL-1 non-acetlyated bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>#3B6917</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">5X concentrated MDR-TB primer mix</td>
			<td>Akonni Biosystems</td>
			<td>Inquire</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">500 fg uL-1 amplification and inhibition control</td>
			<td>Akonni Biosystems</td>
			<td>Inquire</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">20X SSPE</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#BP1328-4</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Triton X-100</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#BP151-500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Disinfecting Spray&#160;</td>
			<td>Current Technologies, Inc.</td>
			<td>#BRSPRAY128</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">70% Isopropyl Alcohol</td>
			<td>Decon Labs, Inc.</td>
			<td>#8416</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Equipment</td>
			<td>Company&#160;</td>
			<td>Catalog Number&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Microarray imager, with automated image and data analysis software</td>
			<td>Akonni Biosystems</td>
			<td>100-20011</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Thermal cycler with flat block insert</td>
			<td>Akonni Biosystems</td>
			<td>100-10021</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">High-throughput wash station and slide holder</td>
			<td>ArrayIt</td>
			<td>HTW</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Dissecting forceps</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#10-300</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Mini Vortexer</td>
			<td>VWR</td>
			<td>#3365040</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Mini-centrifuge</td>
			<td>VWR</td>
			<td>#93000-196</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Airbrush Compressor Kit</td>
			<td>Central Pneumatic</td>
			<td>#95630</td>
			<td></td>
		</tr>
	</tbody>
</table>

demonstrating a multi-drug resistant mycobacterium tuberculosis amplification microarray

Vereinfachung der Microarray-Workflow ist ein notwendiger erster Schritt für die Erstellung von MDR-TB-Microarray-basierte Diagnostik, die routinemäßig in der unteren-Ressource-Umgebungen eingesetzt werden kann. Ein Verstärkungsmicroarray kombiniert asymmetrische PCR-Amplifikation Zielgrößenauswahl, Zielkennzeichnung, und Microarray-Hybridisierung in einer einzigen Lösung und in einem einzigen mikrofluidischen Kammer. Eine Batch-Verarbeitungsverfahren ist mit einem 9-plex asymmetrische Master-Mix und Low-Density-Gel Element Microarray zur Genotypisierung multiresistenter Mycobacterium-Tuberkulose (MDR-TB) demonstriert. Das hier beschriebene Protokoll kann in 6 Stunden abgeschlossen sein und korrekte Genotypisierung mit mindestens 1000 Zelläquivalente von genomischer DNA. Mit On-Chip Waschschritten durchführbar ist, die in einer völlig geschlossenen Amplikon Verfahren und System führt. Das Ausmaß der mit einer Multiplex-Amplifikation Mikroarray wird schließlich durch die Anzahl der Primerpaare, die in einem einzigen Master-m kombiniert werden können, eingeschränktix und trotzdem die gewünschte Empfindlichkeit und Spezifität Leistungsmetriken, anstatt die Anzahl von Sonden, die auf dem Array immobilisiert sind. Ebenso kann die Gesamtanalysezeit verkürzt oder verlängert werden, je nach dem speziellen Verwendungszweck Fragestellung und gewünschte Nachweisgrenze. Dennoch ist die allgemeine Vorgehensweise deutlich Microarray-Workflow optimiert für den Endanwender, indem die Anzahl der manuell intensive und zeitaufwendige Verarbeitungsschritte und bietet eine vereinfachte biochemischen und Mikrofluidik-Pfad für die Übersetzung von Microarray-basierte Diagnostik in der klinischen Routine.

Qualitative Bildanalyse Einblick in Quellen von Lärm oder experimentelle Variabilität, die herausfordern, um in Datentabellen durch automatisierte Bildanalyse-Software erzeugt identifizieren. So kann es sinnvoll sein, um visuell festzustellen, dass 1) Alle Elemente Gel intakt und unbeschädigt sind, 2) die globalen Grund frei von Artefakten, die einzelnen Fluoreszenzsignal-Rausch-Verhältnis-Werten (SNR) beeinträchtigen könnte, 3) es gibt keine Beweise für Blasenbildung oder ungleichmäßige Amplifikation / Hybridi...

Watch this Scientific Journal Video about Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray at JoVE.com

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Ein Verstärkungsmikroarray kombiniert asymmetrischen PCR-Amplifikation und Mikroarray-Hybridisierung in einer einzigen Kammer, die Mikroarray-Arbeitsprozess deutlich vereinfacht für den Endbenutzer. Vereinfachung der Microarray-Workflow ist ein notwendiger erster Schritt für die Erstellung von Microarray-basierte Diagnostik, die routinemäßig in der unteren-Ressource-Umgebungen eingesetzt werden kann.

Der Nachweis eines multiresistenten<em&gt; Mycobacterium tuberculosis</em&gt; Verstärkung Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

demonstrating-multi-drug-resistant-mycobacterium-tuberculosis

Research

JoVE Journal

Immunology and Infection

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

12.6K Aufrufe.  Akonni Biosystems, Inc..  Ein Verstärkungsmikroarray kombiniert asymmetrischen PCR-Amplifikation und Mikroarray-Hybridisierung in einer einzigen Kammer, die Mikroarray-Arbeitsprozess deutlich vereinfacht für den Endbenutzer. Vereinfachung der Microarray-Workflow ist ein notwendiger erster Schritt für die Erstellung von Microarray-basierte Diagnostik, die routinemäßig in der unteren-Ressource-Umgebungen eingesetzt werden kann. 

Serie: Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

Antimikrobielle Empfindlichkeitsprüfung von Mycobacterium tuberculosis-Komplexes für die First und Second Line Drugs von Bouillon Verdünnung in einem Mikrotiterplatten-Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

Forschung

Immunologie und Infektion

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

Probenvorbereitung von<em&gt; Mycobacterium tuberculosis</em&gt; Extrakte für Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

Wachstum von Mycobacterium tuberculosis Biofilme

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Eine experimentelle Modell zum Studium Tuberkulose-Koinfektion auf Natur Malaria Übertragung von Mycobacterium tuberculosis Und Plasmodium berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section1. The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&#38;E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been extensively employed for the examination of DNA, RNA, and protein within tissue. Laser capture microscopy (LCM) is the most commonly used method, but a new milling technique, mesodissection, is recently available. We demonstrate RNA extraction from mesodissected formalin fixed paraffin embedded tissue slides of Mycobacterium tuberculosis granulomas.

A Novel Microdissection Ansatz zur Wiederherstellung<em&gt; Mycobacterium tuberculosis</em&gt; Spezifische Transkripte aus Formalin fixierten und in Paraffin eingebetteten Lung Granulome

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

Ein 3D-humanem Lungengewebe Modell für funktionelle Studien auf<em&gt; Mycobacterium tuberculosis</em&gt; Infektions

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

System zur Wirksamkeit und Zytotoxizität Screening von Inhibitoren Targeting intrazellulärer<em&gt; Mycobacterium tuberculosis</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

Ein Hochdurchsatz-Assay unterstützte Wirksamkeit von Medikamenten gegen Makrophagen agierten zur Bewertung<em&gt; Mycobacterium tuberculosis</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Analyse der 18FDG PET/CT Imaging als Werkzeug für Studium Mycobacterium-Tuberkulose -Infektion und Behandlung bei nicht-menschlichen Primaten

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Mycobacterium Tuberculosis in Mäusen mit Reporter Enzym Fluoreszenz Imaging

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...