Questo lavoro è stato sostenuto dal National Institutes of Health (NIH) in concessione RC3 AI089106. Acidi nucleici MDR-TB sono stati forniti dal / Mondo del Programma delle Nazioni Unite Programma di sviluppo Fund / Nazioni Unite Banca / Organizzazione Mondiale della Sanità Speciale per la Ricerca e la Formazione in malattie tropicali (TDR), Ginevra, Svizzera. Ringraziamo il Dr. Tom Shinnick dei Centri statunitensi per il Controllo e la Prevenzione delle malattie per indicazioni sui geni e mutazioni specifiche da includere nel test del prototipo.

Per i laboratori che seguono le precauzioni universali della PCR, è operativamente più efficace per includere diversi microarrays di amplificazione e guarnizioni per substrato e lavare tutti i microarrays amplificazione simultaneamente in un contenitore di massa, come descritto qui. Formati di consumo sono disponibili per l&#39;esecuzione di post-amplificazione passi microarray di lavaggio in modo del tutto sigillato, prova amplicone chiuso, come riportato altrove 30,33. 1. Setup <ol><li> Estrarre e purificare acidi nucleici dal campione in appropriate condizioni di biosicurezza con un metodo di scelta. Il requisito è che gli acidi nucleici essere di purezza sufficiente per asimmetrica, multiplex PCR. </li><li> Preparare lavoro pulendo le superfici e le attrezzature con soluzioni decontaminanti. </li><li> Mettere reagenti di amplificazione in ghiaccio o piastra fredda. </li><li> Etichettare una sterile provetta da microcentrifuga 1,5 ml per il master mix di amplificazione, e etichettare una sterile 0,2 ml microcentrifuga tuessere per ciascun campione. </li><li> Dopo reagenti vengono scongelate, brevemente vortice e raccolgono contenuto al fondo della provetta con un impulso-spin in una mini-centrifuga. Non vortice Taq polimerasi. Flick delicatamente il tubo per mescolare e seguire con un impulso-spin in mini-centrifuga. </li><li> Preparare una master mix di amplificazione utilizzando i volumi ogni campione di reazione mostrati in Tabella 1. Per tenere conto di eventuali imprecisioni pipettaggio, preparare almeno un altro volume di reazione rispetto al numero totale di campioni in lavorazione. Si noti che il master mix contenente ulteriori Taq polimerasi, al di là di quanto previsto con il tampone PCR muliplex. Aggiungere solo il controllo di amplificazione / inibizione per il mix master dopo tutti gli altri reagenti sono combinati, ei tubi stock reagenti restituiti allo stoccaggio. </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Reagente </td><td> Per-Sample Volume (ml) </td><td> &lt;strong&gt; Finale [] </td></tr><tr><td> Mulitplex PCR Buffer con HotStar Taq Plus. </td><td> 25 </td><td> 1x </td></tr><tr><td> Albumina di siero bovino (BSA) </td><td> 0.55 </td><td> 0,6 mg / ml </td></tr><tr><td> Formammide </td><td> 3.8 </td><td> 7,6% </td></tr><tr><td> Taq polimerasi aggiuntive </td><td> 0.8 </td><td> unità / ml (4 unità totale) </td></tr><tr><td> MDR-TB miscela di primer </td><td> 15.75 </td><td> - </td></tr><tr><td> RNase-free H 2 O </td><td> 2.1 </td><td> - </td></tr><tr><td> Amplificazione / Inibizione controllo </td><td> 1 </td><td> 5,0 fg / pl </td></tr><tr><td> Totale </td><td> 49 </td><td></td></tr></tbody></table> Tabella 1. MDR-TB amplificazione microarray maestro di composizione della miscela. <ol start="7"><li> Vortex il master mix e raccogliere contenuti al fondo della provetta con un impulso-spin in una mini-centrifuga. </li><li> Combina 49 ml di master mix e 1 ml di M. tubercolosi DNA per ciascuna delle provette per campioni dal punto 1.4, sopra. Per un esterno, nessun controllo template (NTC), usare 1 ml di acqua per biologia molecolare al posto del M. tubercolosi campione di DNA. Vortex e impulso-spin tubo (s) una volta terminato. </li></ol> 2. Caricare Amplificazione Microarray <ol><li> Aggiungere 48 ml di ogni master mix / campione sul centro dei rispettivi microarrays, facendo attenzione a non toccare il microarray stessa. </li><li> Posizionare un vetrino sulla parte superiore della guarnizione di microarray e di tenuta, facendo attenzione a non intrappolare bolle d&#39;aria nella camera di microarray. Le bolle d&#39;aria possono influenzare negativamente l&#39;efficienza di amplificazionee possono creare artefatti o rumore nell&#39;immagine microarray successivo in. </li></ol> 3. Ciclismo termica <ol><li> Una volta che tutti i microarrays vengono caricati con campione, posto substrati sul termociclatore blocco piatto. Assicurarsi che l&#39;opzione coperchio riscaldato è &quot;Off&quot;. Attrezzature ottenute da Akonni Biosystems sarà già prequalified per l&#39;uso. In caso contrario, è molto importante verificare che il termociclatore blocco piatto (s) fornire (s) uniforme, riscaldamento coerente in tutti i substrati di microarray. </li><li> Aprire il software termociclatore, selezionare il programma appropriato, immettere &quot;50 microlitri&quot; per il volume di reazione e avviare la corsa. Il protocollo cicli termici qui descritto è costituito da: <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Cicli termici Steps </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 sec </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 sec </td></tr><tr><td> 5 </td><td> Ripetere i passaggi (2-4) per 50 cicli </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 ore </td></tr></tbody></table></li><li> Il numero totale di cicli termici e 55 ° C dopo l&#39;amplificazione tempo di mantenimento sono variabili indipendenti che l&#39;utente può modificare, se necessario o opportuno per l&#39;esperimento specifico. Poiché il master mix MDR-TB crea asimmetrici amplificati (prevalentemente a singolo filamento), di solito è utile includere più cicli termici che altrimenti potrebbero essere utilizzati per un esponenziale protocollo di amplificazione PCR convenzionale. </li><li> Quando il programma ciclismo e ibridazione termico è completa, terminare il programma, rimuovere i microarrays amplificazione, chiudere il software, e spegnere il termociclatore (s).</li></ol> 4. Wash and Dry <ol><li> Per il lavaggio fino a 24 supporti contemporaneamente, preparare almeno 250 ml 1x PESS - 0,1% Triton X-100 tampone di lavaggio, e due contenitori con almeno 250 ml di acqua deionizzata o grado Milli-Q ciascuno. Il tampone di lavaggio può essere preparato in anticipo. </li><li> Rimuovere con cautela la polizza di copertura e la guarnizione di ogni amplificazione camera di microarray usando pinze flat-end. Questo passo è il punto in cui c&#39;è il maggior rischio di danneggiare fisicamente array di elementi gel. Utilizzare adeguati controlli di contaminazione PCR per prevenire la diffusione degli acidi nucleici amplificati all&#39;interno dell&#39;ambiente di lavoro. </li><li> Mettere substrati microarray in un supporto istologia diapositive e inserire tutte le diapositive nel cestino di lavaggio contenente il tampone di lavaggio. Coprire il contenitore con un coperchio. </li><li> Lavare microarray per 10 minuti a temperatura ambiente con agitazione. </li><li> Immergere i microarrays tre volte in ciascuna delle due lavaggi successivi di deionizzata oAcqua di grado Milli-Q, e aria secca. </li></ol> 5. Imaging Il asimmetrica miscela di innesco MDR-TB genera ampliconi Cy3-etichettati. Microarrays elementi Gel può essere ripreso con qualsiasi imager microarray livello in grado di immagini Cy3. La seguente procedura di imaging è specifica per il mix MDR-TB maestro, campo dx2100 imager portatile e software di analisi automatizzata fornito da Akonni. <ol><li> Assicurarsi che il cavo ethernet dalla termocamera è collegata al computer. </li><li> Accendere la termocamera. L&#39;interruttore di alimentazione si trova sul pannello posteriore dello strumento. LED blu sulla parte anteriore della termocamera si accendono quando imager è acceso. </li><li> Accendere il computer. </li><li> Sul desktop del computer, fare doppio clic sull&#39;icona del software per lanciare il software automatizzato di analisi delle immagini. </li><li> Attendere che il software per stabilire una connessione con la fotocamera imager. Una volta stabilita la connessione, il pulsante Analizza diventa disponibile, e ilmessaggio &quot;Connection Successful&quot; apparirà nell&#39;angolo in basso a sinistra dello schermo. </li><li> Inserire il microarray di amplificazione essiccato con il microarray rivolto verso l&#39;utente, e verso la lente obiettivo. Se il microarray è correttamente orientato rispetto alla lente e telecamera, il software di analisi automatizzata mancherà di inserire una griglia l&#39;immagine di fluorescenza su. </li><li> Chiudere lo sportello di accesso. Una anteprima immagine sarà disponibile sullo schermo del computer. </li><li> Selezionare lo script analisi MDR-TB dal menu a tendina e inserire il tempo di esposizione desiderato (in millisecondi). Un tipico punto di partenza per elementi gel microarrays amplificazione è 100-500 msec. </li><li> Pixel saturi verranno visualizzati l&#39;immagine di anteprima in rosso. Regolare il tempo di esposizione per ridurre al minimo il numero di pixel saturi all&#39;interno dei singoli punti. </li><li> Fare clic su Analizza per acquisire e analizzare l&#39;immagine di fluorescenza. Il software metterà automaticamente unMDR-TB griglia specifica-test sull&#39;immagine, estrarre intensità integrata e valori di fondo, e riferire i risultati resistenza ai farmaci e genotipizzazione. L&#39;analisi automatizzata in genere richiedere alcuni secondi. Per le immagini stimolanti che contengono graffi, polvere, artefatti a fluorescenza o danni fisici alle caratteristiche microarray, il software può richiedere fino a 1 minuto per completare l&#39;analisi. </li><li> Una volta completata l&#39;analisi, fare clic su Salva, selezionare la cartella di destinazione, digitare un nome di file e fare clic su OK. Dati di microarray sottostanti vengono salvati come file xls. E possono essere esportati per analisi off-line, se lo si desidera. </li><li> Una volta completata l&#39;analisi, rimuovere il microarray di amplificazione dal imager, e fare clic su Nuovo per avviare l&#39;analisi successiva. </li><li> Al termine la raccolta dei dati, chiudere il software, spegnere il computer e spegnere l&#39;imager. </li></ol>

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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>

Gli autori dichiarano interessi finanziari concorrenti.

Il grado di multiplazione con un microarray amplificazione è infine dettata dall&#39;efficienza del multiplex PCR asimmetrica, non microarray. Nella nostra esperienza, 10-12 coppie di primer uniche possono essere facilmente multiplexing in un formato di amplificazione microarray. Criteri di progettazione di primer e sonde convenzionali applicano a nuovi saggi, salvo che uno deve anche considerare potenziali interazioni tra acidi nucleici soluzione monofase e sonde immobilizzate microarray, l&#39;efficienza termica del ...

Rilevamento caso precoce e un trattamento rapido sono considerate le strategie di controllo più efficaci per ridurre Mycobacterium tuberculosis (MTB) trasmissione 1, e ora c&#39;è un ampio consenso nella comunità TB che un punto di assistenza (POC) o in prossimità POC test per diagnosticare contemporaneamente TB ed è necessaria resistenza ai farmaci (DR). Tecnologie come GeneXpert di Cefeidi e di altri test di amplificazione degli acidi nucleici riducono il tempo di diagnosi per molti pazienti affetti da tubercolosi, e di fornire una rapida read-out che indica la resistenza alla rifampicina o mutazioni selezionate che conferiscono resistenza agli altri prima o farmaci di seconda linea 2. Sebbene i test di amplificazione in tempo reale e di acido nucleico isotermica sono progettati per identificare le mutazioni di resistenza ai farmaci che portano a MDR-TB, lo spettro di mutazioni che scoprono è spesso insufficiente per progettare un regime individualizzato farmaco corrispondente al profilo farmacoresistenza del paziente, e vincoli tecnici connessidi ottica cross-talk o la complessità di amplificazione e di reporting chimiche 3-7 maggio limitare il numero di loci o mutazioni che vengono rilevati. Così, tecnologie di rilevazione con una maggiore capacità di multiplexing sono necessari per colmare le lacune note nella diagnostica POC MDR-TB. Microarray e le Hain saggi sonda linea OMS-omologati possono affrontare il &quot;gene multipla, mutazioni multiple&quot; sfida di diagnosticare MDR-TB 8-29. Purtroppo, questi, piattaforme di rilevamento multiplex a base di ibridazione-usano più fasi, complicato, e protocolli open-ampliconi che richiedono una formazione significativa e competenza in tecniche molecolari. Il microarray di amplificazione 30 è stato progettato per affrontare alcuni di questi microarray flusso di lavoro e le preoccupazioni operative. I principi fluidici di semplificazione sono per amplificare, ibridare, e individuare gli obiettivi di acido nucleico all&#39;interno di una singola camera di microfluidica. L&#39;utente introduce i mas acidi e amplificazione dell&#39;acido nucleicoter miscela in una camera fluidico con una pipetta e attiva il protocollo di cicli termici. Per il metodo di elaborazione batch mostrato qui, microarrays sono successivamente lavati in soluzione bulk, essiccati, e ripreso. Questo studio dimostra la funzionalità di un microarray di amplificazione mediante test di MDR-TB microarray per rpoB (30 mutazioni), katG (2 mutazioni), inhA (4 mutazioni), rpsL (2 mutazioni), embB (1 mutazione), IS1245, IS6110 , e un amplificazione interna e controllo ibridazione. Almeno una coppia di sonde microarray (di tipo selvatico (WT) e single-nucleotide mutanti (MU)) è incluso per ogni mutazione di interesse. Acidi nucleici purificati da multi-farmaco resistente M. tubercolosi sono dal ceppo TDR Tubercolosi Bank 31. Microarrays elemento Gel sono realizzati su substrati di vetro per copolimerizzazione essenzialmente come descritto altrove 32, tranne che usiamo 4% monomero e 0,05 mm ciascuna sonda nel polymerizmiscela zione. Array sono circondati da una guarnizione 50 ml prima dell&#39;uso. Dopo cicli termici, di ibridazione e di lavaggio gradini, microarrays amplificazione vengono esposte su un analizzatore portatile Akonni. , Intensità di segnale integrati Sfondo correzione sono ottenuti dal greggio. Immagini tif utilizzando un algoritmo cerchio fisso. Rumore per ciascun elemento gel è calcolato come tre volte la deviazione standard degli sfondi posto locale. Obiettivi del gene sono in genere considerati rilevabile per il rapporto segnale-rumore (SNR) valori ≥ 3. Per determinare la presenza o l&#39;assenza di una specifica mutazione in ogni gene o codone, un rapporto discriminante è calcolato dai valori SNR (WT-MU) / (WT + MU). Rapporti discriminanti &lt;0 sono indicativi di un farmaco-resistenza mutazione al locus, mentre i rapporti&gt; 0 sono indicativi della sequenza wild-type.

<table border="0" cellpadding="0" cellspacing="0">
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			<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>
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			<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>
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			<td height="22" style="height:22px;">Taq polymerase</td>
			<td>Qiagen</td>
			<td>#201207</td>
			<td></td>
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			<td height="22" style="height:22px;">RNAse-free water</td>
			<td>Qiagen</td>
			<td>#206143&#160;</td>
			<td></td>
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			<td height="22" style="height:22px;">Formamide</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#BP227-500</td>
			<td></td>
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			<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>
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			<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>
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			<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>
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			<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>
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		<tr height="22">
			<td height="22" style="height:22px;">Mini Vortexer</td>
			<td>VWR</td>
			<td>#3365040</td>
			<td></td>
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		<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>
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</table>

demonstrating a multi-drug resistant mycobacterium tuberculosis amplification microarray

La semplificazione del flusso di lavoro microarray è un primo passo necessario per la creazione di sistemi diagnostici basati su microarray-MDR-TB che possono essere utilizzati di routine in ambienti inferiore di risorse. Un microarray di amplificazione combina asimmetrica PCR, selezione del formato di destinazione, l&#39;etichettatura di destinazione e microarray ibridazione all&#39;interno di un&#39;unica soluzione e in una sola camera microfluidica. Un metodo di elaborazione batch è dimostrato con un 9-plex master mix asimmetrico e bassa densità elemento gel microarray per la genotipizzazione multi-droga Mycobacterium tuberculosis resistenti (MDR-TB). Il protocollo qui descritto può essere completato in 6 ore e fornire corretta genotipizzazione con almeno 1.000 equivalenti cellulari di DNA genomico. Incorporando on-chip fasi di lavaggio è fattibile, che si tradurrà in un metodo amplicone interamente chiuso e sistema. Il grado di multiplazione con un microarray amplificazione è in definitiva limitata dal numero di coppie di primer che possono essere combinati in un unico master mix e ancora ottenere una sensibilità desiderata e metriche di performance specificità, piuttosto che il numero di sonde che sono immobilizzati sulla matrice. Allo stesso modo, il tempo di analisi totale può essere accorciato o allungato in funzione dell&#39;uso specifico previsto, domanda di ricerca, e limiti desiderati di rilevamento. Tuttavia, l&#39;approccio generale semplifica notevolmente workflow microarray per l&#39;utente finale, riducendo il numero di fasi di elaborazione manualmente intensivo e richiede tempo, e fornisce un percorso biochimico e microfluidica semplificata per tradurre diagnostici basati su microarray nella pratica clinica di routine.

Analisi di immagine qualitativa può fornire indicazioni in fonti di rumore sperimentale o variabilità che sono difficili da individuare in tabelle di dati generati dal software automatizzato di analisi di immagine. Quindi, può essere utile verificare visivamente che: 1) tutti gli elementi gel sono integro e non danneggiato, 2) lo sfondo globale è esente da artefatti fluorescenti che possono influenzare il segnale individuo a valori di rumore (SNR), 3) non vi è alcuna prova di formazione di bolle o non uniforme di a...

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

Un microarray amplificazione combina asimmetrica amplificazione PCR e ibridazione microarray in una sola camera, che semplifica significativamente workflow microarray per l&#39;utente finale. La semplificazione del flusso di lavoro microarray è un primo passo necessario per la creazione di sistemi diagnostici basati su microarray, che possono essere utilizzati di routine in ambienti inferiore di risorse.

Dimostrazione di un multi-resistente ai farmaci<em&gt; Mycobacterium tuberculosis</em&gt; Amplificazione 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

12.5K Views.  Akonni Biosystems, Inc..  Un microarray amplificazione combina asimmetrica amplificazione PCR e ibridazione microarray in una sola camera, che semplifica significativamente workflow microarray per l'utente finale. La semplificazione del flusso di lavoro microarray è un primo passo necessario per la creazione di sistemi diagnostici basati su microarray, che possono essere utilizzati di routine in ambienti inferiore di risorse. 

Video: 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.

Test di suscettibilità antimicrobica del complesso Mycobacterium tuberculosis delle droghe prima e seconda linea per diluizione brodo in un formato micropiastra

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

Ricerca

Immunologia e infezioni

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.

Preparazione del campione di<em&gt; Mycobacterium tuberculosis</em&gt; Estratti di studi di risonanza magnetica nucleare metabolomica

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.

Crescita Mycobacterium tuberculosis I biofilm

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

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrPC) and the infectious protein (PrPSc), an abnormally-folded isoform of PrPC 8.
One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation13. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrPSc are poorly characterized due to their unusual conformation and aggregation states.
PrPSc can seed the conversion of PrPC to PrPSc in vitro14. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrPSc, at an accelerated rate, from a system containing excess amounts of PrPC and minute amounts of the PrPSc seed19. This technique has proven to effectively recapitulate the species and strain specificity of PrPSc conversion from PrPC, to emulate prion strain interference, and to amplify very low levels of PrPSc from infected tissues, fluids, and environmental samples6,7,16,23 .
This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.

Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.

Proteine ​​ciclica Amplificazione misfolding di prioni

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

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.

Un modello sperimentale per studiare la tubercolosi, la malaria coinfezione sulla trasmissione naturale di Mycobacterium tuberculosis E 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.

Un nuovo approccio Microdissezione per recuperare<em&gt; Mycobacterium tuberculosis</em&gt; Trascrizioni specifici da formalina paraffina fisso incorporato Lung granulomi

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.

Un 3D Polmone umano tessuto modello per studi funzionali su<em&gt; Mycobacterium tuberculosis</em&gt; Infezione

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.

Sistema per l&#39;efficacia e la citotossicità Proiezione di inibitori di targeting intracellulare<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.

Un test compatibile high-throughput per valutare Drug efficacia contro macrofagi diversi passaggi<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.

Analisi di 18FDG PET/CT Imaging come strumento per studiare l'infezione di tubercolosi del micobatterio e trattamento in primati Non umani

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