Ce travail a été soutenu par le National Institutes of Health (NIH) sous subvention RC3 AI089106. Acides nucléiques de MDR-TB ont été fournis par le Fonds / développement des Nations Unies Programme / Banque mondiale / Organisation mondiale de la Santé Programme spécial des Nations Unies pour l&#39;enfance pour la recherche et la formation concernant les maladies tropicales (TDR), Genève, Suisse. Nous remercions le Dr Tom Shinnick des US Centers for Disease Control and Prevention des indications sur les gènes et les mutations spécifiques à inclure dans le test de prototype.

Pour les laboratoires qui suivent les consignes de PCR universelle, il est opérationnel plus efficace d&#39;inclure plusieurs puces d&#39;amplification et joints par substrat et laver tous les puces d&#39;amplification simultanément dans un conteneur, comme décrit ici. Formats consommables sont disponibles pour effectuer des post-amplification microarray étapes de lavage dans un test d&#39;amplicon fermée entièrement scellé, comme rapporté ailleurs 30,33. 1. Configuration <ol><li> Extraire et de purifier des acides nucléiques à partir de l&#39;échantillon dans des conditions de sécurité biologique appropriées avec une méthode de choix. L&#39;exigence est que les acides nucléiques être d&#39;une pureté suffisante pour asymétrique amplification, PCR multiplex. </li><li> Préparer l&#39;espace de travail en essuyant les surfaces et l&#39;équipement avec des solutions de décontamination. </li><li> Placer les réactifs d&#39;amplification sur la glace ou bloc froid. </li><li> Etiqueter un tube de 1,5 ml centrifuger stérile pour le mélange maître d&#39;amplification, et étiqueter un stérile de 0,2 ml centrifuger tusoit pour chaque échantillon. </li><li> Après réactifs sont décongelées, vortex brièvement et de recueillir le contenu au fond du tube avec une impulsion de spin dans une mini-centrifugeuse. Ne pas vortex la Taq polymérase. Tapotez doucement le tube pour mélanger et de suivre avec une impulsion de spin dans la mini-centrifugeuse. </li><li> Préparer un mélange maître d&#39;amplification en utilisant les volumes par-échantillon réactionnelles indiquées dans le tableau 1. Afin de tenir compte des imprécisions possibles de pipetage, préparer au moins un volume de réaction de plus que le nombre total d&#39;échantillons en cours de traitement. Notez que le mélange maître contient plus Taq polymérase, au-delà de ce qui est prévu avec le tampon muliplex PCR. Seulement ajouter le contrôle d&#39;amplification / d&#39;inhibition au mélange maître après tous les autres réactifs sont combinés, et les tubes stock réactifs renvoyée au stockage. </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Réactif </td><td> Volume par-échantillon (pl) </td><td> &lt;strong&gt; Finale [] </td></tr><tr><td> PCR Buffer Mulitplex avec HotStar Taq plus </td><td> 25 </td><td> 1x </td></tr><tr><td> albumine de sérum bovin (BSA) </td><td> 0,55 </td><td> 0,6 mg / ml </td></tr><tr><td> Formamide </td><td> 3.8 </td><td> 7,6% </td></tr><tr><td> Taq polymérase supplémentaires </td><td> 0,8 </td><td> unités / pl (4 unités au total) </td></tr><tr><td> MDR-TB mélange d&#39;amorces </td><td> 15.75 </td><td> - </td></tr><tr><td> RNase-H 2 O </td><td> 2.1 </td><td> - </td></tr><tr><td> Amplification / Contrôle inhibition </td><td> 1 </td><td> 5.0 fg / pl </td></tr><tr><td> Total </td><td> 49 </td><td></td></tr></tbody></table> La composition du mélange tableau 1. MDR-TB amplification microarray maître. <ol start="7"><li> Vortex le mélange maître et de recueillir le contenu au fond du tube avec une impulsion de spin dans une mini-centrifugeuse. </li><li> Mélanger 49 pi de mélange maître et 1 pl de M. ADN de la tuberculose à chacun des tubes échantillons issus de l&#39;étape 1.4 ci-dessus. Pour une externe, pas de contrôle de modèle (NTC), utiliser une pi d&#39;eau moléculaire de qualité de biologie à la place de la M. tuberculose échantillon d&#39;ADN. Vortex et impulsion de spin tube (s) lorsque vous avez terminé. </li></ol> 2. Chargez amplification puces à ADN <ol><li> Ajouter 48 ul de chaque Master Mix / échantillon sur le centre de leurs puces respectives, en faisant attention de ne pas toucher la puce elle-même. </li><li> Placez une lamelle sur le dessus de la garniture de puces à ADN et le joint, en faisant attention à ne pas coincer les bulles d&#39;air dans la chambre de puces à ADN. Des bulles d&#39;air peuvent affecter négativement l&#39;efficacité d&#39;amplificationet peut créer des artefacts ou de bruit dans l&#39;image de puces à ADN ultérieure. </li></ol> 3. Cycle thermique <ol><li> Une fois toutes les puces sont chargés de l&#39;échantillon, le lieu substrats sur le cycleur thermique de bloc plat. Assurez-vous que l&#39;option chauffée de couvercle est en position &quot;Off&quot;. Équipement obtenu de Akonni Biosystems sera déjà préqualifiés pour utilisation. Sinon, il est très important de vérifier que le cycleur thermique de bloc plat (s) donne (nt) uniforme, chauffage uniforme dans tous les supports de puces à ADN. </li><li> Ouvrez le logiciel de cycleur thermique, sélectionnez le programme approprié, entrez &quot;50 pl&quot; pour le volume de réaction, et de lancer la course. Le protocole de cyclage thermique décrit ici se compose de: <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Thermiques étapes à vélo </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> Répétez les étapes (2-4) pendant 50 cycles </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 heures </td></tr></tbody></table></li><li> Le nombre total de cycles thermiques et 55 ° C l&#39;après-amplification temps d&#39;attente sont des variables indépendantes que l&#39;utilisateur peut modifier, si nécessaire ou approprié pour l&#39;expérience spécifique. Parce que le mélange maître TB-MR crée asymétriques amplicons (principalement simple brin), il est généralement utile d&#39;inclure des cycles thermiques plus que, autrement, pourraient être utilisés pour un protocole d&#39;amplification exponentielle classique, PCR. </li><li> Lorsque le programme de cyclisme et l&#39;hybridation thermique est terminée, la fin du programme, retirez les puces d&#39;amplification, fermez le logiciel, et éteignez le cycleur (s) thermique.</li></ol> 4. Laver et sécher <ol><li> Pour le lavage jusqu&#39;à 24 substrats simultanément, préparer au moins 250 ml 1x SSPE - 0,1% de Triton X-100 du tampon de lavage, et deux conteneurs avec au moins 250 ml d&#39;eau désionisée ou de qualité Milli-Q chacun. Le tampon de lavage peut être préparé à l&#39;avance. </li><li> Retirez délicatement la lamelle et le joint de chaque chambre de microréseau d&#39;amplification en utilisant une pince plate-end. Cette étape est le moment où il est le plus grand risque de causer de blessures réseaux d&#39;éléments de gel. Utiliser les contrôles de contamination PCR appropriées pour prévenir la propagation des acides nucléiques amplifiés à l&#39;intérieur de l&#39;environnement de travail. </li><li> Placez substrats de puces à ADN dans un support histologie de diapositives, et placer toutes les diapositives dans le bac de lavage contenant le tampon de lavage. Couvrir le récipient avec un couvercle. </li><li> Laver microarrays pendant 10 min à température ambiante avec agitation douce. </li><li> Tremper les puces trois fois dans chacune des deux lavages successifs de déminéralisée ouEau de qualité Milli-Q, et l&#39;air sec. </li></ol> 5. Imaging Le mélange d&#39;amorces asymétrique MDR-TB génère amplicons Cy3 marqués. puces d&#39;éléments de gel peuvent être visualisés avec n&#39;importe quel imageur microarray norme capable de l&#39;imagerie Cy3. La procédure d&#39;imagerie suivante est spécifique pour le mélange maître TB-MR, domaine Dx2100 imageur portable et un logiciel d&#39;analyse automatisée fournie par Akonni. <ol><li> Assurez-vous que le câble Ethernet de l&#39;imageur est connecté à l&#39;ordinateur. </li><li> Tournez sur l&#39;imageur. L&#39;interrupteur d&#39;alimentation est situé sur le panneau arrière de l&#39;instrument. La LED bleue sur le devant de la caméra s&#39;allume lorsque imageur est sous tension. </li><li> Allumez l&#39;ordinateur. </li><li> Sur le bureau de l&#39;ordinateur, double-cliquez sur l&#39;icône du logiciel pour lancer le logiciel d&#39;analyse d&#39;image automatisée. </li><li> Attendez que le logiciel pour établir une connexion avec l&#39;appareil photo de l&#39;imageur. Une fois la connexion établie, le bouton Analyser devient disponible, et lamessage «Connexion réussie» apparaît dans le coin inférieur gauche de l&#39;écran. </li><li> Insérer la puce d&#39;amplification séché avec le microréseau opposé à l&#39;utilisateur, et en direction de la lentille d&#39;objectif. Si la puce n&#39;est pas correctement orientée par rapport à la lentille et de la caméra, le logiciel d&#39;analyse automatisé échoue à placer une grille sur l&#39;image de fluorescence. </li><li> Fermez la porte d&#39;accès. Un aperçu sera disponible sur l&#39;écran d&#39;ordinateur. </li><li> Sélectionnez le script d&#39;analyse de TB-MR dans le menu déroulant et entrez le temps de l&#39;exposition souhaitée (en millisecondes). Un point de départ typique pour élément de gel puces d&#39;amplification est 100-500 ms. </li><li> Pixels saturés seront affichés sur l&#39;image de prévisualisation en rouge. Ajuster le temps d&#39;exposition afin de minimiser le nombre de pixels à l&#39;intérieur de spots saturés individuels. </li><li> Cliquer Analyser à acquérir et à analyser l&#39;image de fluorescence. Le logiciel placera automatiquement uneMDR-TB réseau spécifique au test sur l&#39;image, extraire intensité intégrée et des valeurs de fond, et de faire rapport résistance aux médicaments et de génotypage des résultats. L&#39;analyse automatique prend généralement quelques secondes. Pour les images difficiles qui contiennent des rayures, de la poussière, des objets fluorescents ou des dommages physiques aux caractéristiques de puces à ADN, le logiciel peut nécessiter jusqu&#39;à 1 min pour compléter l&#39;analyse. </li><li> Une fois l&#39;analyse terminée, cliquez sur Enregistrer, sélectionnez le dossier de destination, tapez un nom de fichier, et cliquez sur OK. Données de biopuces sous-jacents sont enregistrées dans un fichier xls. Et peuvent être exportés pour des analyses hors ligne, si désiré. </li><li> Une fois l&#39;analyse terminée, retirez la puce d&#39;amplification de l&#39;imageur, et cliquez sur Nouveau pour lancer l&#39;analyse suivante. </li><li> Lorsque vous avez terminé la collecte de données, fermez le logiciel, éteignez l&#39;ordinateur, et d&#39;éteindre la caméra. </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>

Les auteurs déclarent aucun intérêt financier concurrents.

L&#39;étendue de multiplexage avec un microréseau d&#39;amplification est finalement dictée par l&#39;efficacité de la PCR multiplex asymétrique, et non le microréseau. Dans notre expérience, 10-12 paires d&#39;amorces uniques peuvent être facilement multiplexés dans un format amplification de puces à ADN. Critères d&#39;amorces et de conception de la sonde classiques s&#39;appliquent donc à de nouveaux essais, à l&#39;exception que l&#39;on doit aussi tenir compte des interactions potentielles entre les ac...

La détection précoce des cas et un traitement rapide sont considérés comme des stratégies de lutte les plus efficaces pour réduire Mycobacterium tuberculosis (MTB) transmission 1, et il est maintenant un large consensus dans la communauté de la tuberculose que d&#39;un point de service (PDS) ou près de test de POC pour diagnostiquer simultanément la tuberculose et la résistance aux médicaments (DR) est nécessaire. Les technologies telles que GeneXpert de Cepheid et autres tests d&#39;amplification des acides nucléiques de réduire le temps de diagnostic pour de nombreux patients atteints de tuberculose, et fournissent une lecture rapide indique la résistance à la rifampicine ou mutations sélectionnées conférant une résistance à d&#39;autres médicaments de première ou deuxième ligne 2. Bien que les tests d&#39;amplification en temps réel et de l&#39;acide nucléique isotherme sont conçus pour identifier les mutations de résistance aux médicaments qui mènent à la TB-MR, le spectre des mutations qu&#39;ils détectent est souvent insuffisante pour concevoir un schéma thérapeutique individualisé correspondant au profil de résistance aux médicaments du patient, et les contraintes techniques liéesà la diaphonie optique ou de la complexité de l&#39;amplification et de reporting chimiques 3 au 7 mai limiter le nombre de loci ou des mutations qui sont détectés. Ainsi, les technologies de détection avec une capacité de multiplexage élevé sont nécessaires pour combler les lacunes connues dans le diagnostic de TB-MR au PS. Les puces à ADN et les Hain essais de sonde de la ligne approuvée par l&#39;OMS peuvent répondre à la «gène multiple, multiples mutations&quot; défi de diagnostiquer la TB-MR 8-29. Malheureusement, ces plates-formes, de détection multiplex à base d&#39;hybridation utilisent plusieurs étapes, compliqué, et les protocoles ouverts amplicon qui exigent une formation importante et la maîtrise des techniques moléculaires. L&#39;amplification microréseau 30 a été conçu pour répondre à certaines de ces puces à ADN flux de travail et des problèmes opérationnels. Les principes fluidiques sont simplificatrices pour amplifier, s&#39;hybrident, et détecter des cibles d&#39;acides nucléiques dans une seule chambre microfluidique. L&#39;utilisateur introduit les acides nucléiques et d&#39;amplification master mélanger dans une chambre fluidique avec une pipette et commence le protocole de cycle thermique. Pour la méthode de traitement par lots représenté ici, les microréseaux sont ensuite lavées dans une solution en vrac, séchées et imagés. Cette étude démontre la fonctionnalité d&#39;un microréseau d&#39;amplification à l&#39;aide d&#39;un test de TB-MR microarray pour rpoB (30 mutations), katG (2 mutations), inhA (4 mutations), rpsL (2 mutations), embB (1 mutation), IS1245, IS6110 , et une amplification interne et de contrôle de l&#39;hybridation. Au moins une paire appariée de microréseau de sondes (type sauvage (WT) et mutantes de nucléotide unique (MU)) est inclus pour chaque mutation d&#39;intérêt. Acides nucléiques purifiés à partir de multi-drogue M. résistant la tuberculose sont de la souche TDR tuberculose 31 Banque. puces d&#39;éléments de gel sont fabriqués sur des substrats de verre par copolymérisation essentiellement comme décrit par ailleurs 32, sauf que nous utilisons 4% de monomère et 0,05 mM chaque sonde dans le polymérisablesmélange d&#39;ation. Les tableaux sont entourés d&#39;un joint de 50 ml avant l&#39;utilisation. Après cyclage thermique, hybridation et lavage étapes, les puces d&#39;amplification sont visualisés sur un analyseur portable Akonni. Fond corrigée, les intensités de signaux intégrés sont obtenus à partir de la première. Images tif en utilisant un algorithme de cercle fixe. Bruit pour chaque élément en gel est égale à trois fois l&#39;écart type des milieux de tache locales. cibles de gènes sont généralement considérés comme détectable pour le rapport signal sur bruit (SNR) des valeurs ≥ 3. Afin de déterminer la présence ou l&#39;absence d&#39;une mutation spécifique dans chaque gène ou codon, un rapport discriminant est calculée à partir des valeurs de SNR que (WT-MU) / (WT + MU). Ratios discriminants &lt;0 sont indicatifs d&#39;une mutation de résistance aux médicaments au niveau du locus, tandis que les ratios&gt; 0 sont indicatifs de la séquence de type sauvage.

<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

Simplifier microarray flux de travail est une première étape nécessaire pour la création de MDR-TB diagnostic des biopuces qui peuvent être utilisées en routine dans des environnements à faibles ressources. Un microréseau d&#39;amplification combine amplification PCR asymétrique, la sélection de la taille de la cible, l&#39;étiquetage cible, et microarray hybridation dans une solution unique et dans une seule chambre microfluidique. Une méthode de traitement par lots est démontrée avec un mélange maître asymétrique 9 complexes et à faible densité élément de gel pour le génotypage microréseau multi-drogue Mycobacterium tuberculosis résistantes (MDR-TB). Le protocole décrit ici peut être complété en 6 heures et fournir correct génotypage d&#39;au moins 1 000 équivalents cellulaires d&#39;ADN génomique. L&#39;intégration sur puce des étapes de lavage est possible, qui se traduira par une méthode d&#39;amplicon entièrement clos et système. L&#39;étendue de multiplexage avec un microréseau d&#39;amplification est finalement limitée par le nombre de paires d&#39;amorces qui peuvent être combinés en un seul maître mix et encore obtenir une sensibilité désirée et des mesures de performance de spécificité, plutôt que le nombre de sondes qui sont immobilisées sur la matrice. De même, le temps total d&#39;analyse peut être raccourcie ou allongée en fonction de l&#39;utilisation spécifique prévue, question de recherche, et les limites souhaitées de détection. Néanmoins, l&#39;approche générale simplifie considérablement microarray flux de travail pour l&#39;utilisateur final en réduisant le nombre d&#39;étapes manuellement intensives et longues traitement, et fournit une voie biochimique et microfluidique simplifiée pour traduire diagnostic des biopuces dans la pratique clinique de routine.

Analyse d&#39;image qualitative peut donner un aperçu des sources de bruit ou de la variabilité expérimentale qui sont difficile à identifier dans les tableaux de données générés par un logiciel automatisé d&#39;analyse d&#39;image. Ainsi, il peut être utile de visuellement s&#39;assurer que 1) tous les éléments de gel sont intacts et en bon état, 2) le contexte mondial est sans artefacts fluorescents qui pourraient affecter signal individuel de bruit SNR) de valeurs (3) il n&#39;existe aucune preuve pour l...

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 microréseau d&#39;amplification asymétrique combine l&#39;amplification par PCR et l&#39;hybridation microarray dans une seule chambre, qui simplifie considérablement microarray flux de travail pour l&#39;utilisateur final. Simplifier microarray flux de travail est une première étape nécessaire pour la création de diagnostics des biopuces qui peuvent être utilisées en routine dans des environnements à faibles ressources.

Faisant preuve d&#39;une multi-résistante<em&gt; Mycobacterium tuberculosis</em&gt; Amplification de puces à ADN

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 vues.  Akonni Biosystems, Inc..  Un microréseau d'amplification asymétrique combine l'amplification par PCR et l'hybridation microarray dans une seule chambre, qui simplifie considérablement microarray flux de travail pour l'utilisateur final. Simplifier microarray flux de travail est une première étape nécessaire pour la création de diagnostics des biopuces qui peuvent être utilisées en routine dans des environnements à faibles ressources. 

Vidéo: 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 de sensibilité aux antimicrobiens du complexe Mycobacterium tuberculosis des drogues de première et deuxième par dilution en bouillon dans un format microplaque

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

Recherche

Immunologie et infection

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.

Préparation de l&#39;échantillon de<em&gt; Mycobacterium tuberculosis</em&gt; Extraits pour Magnétique Nucléaire études métabolomiques résonance

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.

La croissance de la Mycobacterium tuberculosis Les biofilms

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.

Protein Misfolding Cyclic Amplification des prions

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 modèle expérimental pour étudier la tuberculose-paludisme co-infection à transmission naturelle de Mycobacterium tuberculosis Et 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.

Une nouvelle approche microdissection de la récupération<em&gt; Mycobacterium tuberculosis</em&gt; Transcriptions spécifiques de formol paraffine fixe embarqué poumon granulomes

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 modèle de tissu 3D Poumon humain pour les études fonctionnelles sur<em&gt; Mycobacterium tuberculosis</em&gt; 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 ...

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.

Système d&#39;efficacité et de dépistage des inhibiteurs Cytotoxicité ciblage intracellulaire<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 haut-débit Assay Compatible pour évaluer l&#39;efficacité des médicaments contre Macrophage repiquées<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 de 18FDG TEP/CT d’imagerie comme un outil pour étudier Mycobacterium tuberculosis Infection et traitement chez les Primates Non humains

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