High-throughput, Automated Extraction of DNA and RNA from Clinical Samples using TruTip Technology on Common Liquid Handling Robots

TruTip is a simple nucleic acid extraction  technology whereby a porous, monolithic binding matrix is inserted into a  pipette tip. The geometry of the ...

Demonstrating a Multi-drug Resistant <em>Mycobacterium tuberculosis</em> Amplification Microarray

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

Akonni Biosystems, Inc.

Clinical laboratories are recognizing the importance of implementing sensitive and specific molecular diagnostic tests. However, widespread adoption of these tests requires simplified workflows without requiring expensive supporting instrumentation. To enable microarray-based analysis to meet these requirements, we describe a valveless flow cell for disposable use that supports PCR coupled with microarray hybridization in the same chamber. The flow cell assembly consists simply of double-faced tape, a plastic microarray substrate, an absorbent, and a commercially-available hydrophilic thin film. The simple construction lends itself to low-cost and ease of manufacturing, yet several features reduce the complexity of the standard microarray workflow. First, there is no requirement for custom instrumentation. Second, the hydrophilic thin film allows uniform filling of a microfluidic chamber. Third, a geometric capillary stop design confines liquid to the microarray chamber during PCR, and thus eliminates the need for a valve or hydrophobic surface treatment. And fourth, imbibition drives the uniform removal of liquid reagents from the array chamber. Three hundred genomic copies of methicillin-resistant Staphylococcus aureus (MRSA) are detected in a flow cell with gel drop microarrays printed on an unmodified plastic substrate. This sensitivity is shown to be comparable to conventional methods (i.e., PCR in a tube, with separate hybridization in a microarray chamber, where amplicon is exposed to the workspace before and after hybridization). However, the flow cell combines these multiple steps into a simple, compact workflow without the need for complex valves or custom instrumentation and is less susceptible to contamination of the workspace than conventional methods because the amplicon is confined to the device.

A plastic, disposable microfluidic flow cell for coupled on-chip PCR and microarray detection of infectious agents.

Laboratoires cliniques reconnaissent l&#39;importance de la mise en &oelig;uvre de tests de diagnostic mol&eacute;culaires sensibles et sp&eacute;cifiques. Toutefois, l&#39;adoption g&eacute;n&eacute;ralis&eacute;e de ces tests n&eacute;cessite des flux de travail simplifi&eacute; sans n&eacute;cessiter d&#39;instrumentation soutien co&ucirc;teuse. Pour activer l&#39;analyse microarray r&eacute;pondre &agrave; ces exigences, nous d&eacute;crivons une cellule d&#39;&eacute;coulement sans soupapes usage jetable qui prend en charge PCR coupl&eacute; avec microarray hybridation dans la m&ecirc;me chambre. L&#39;ensemble de cellule de flux se compose simplement de ruban adh&eacute;sif double face, un substrat en plastique microarray, absorbante et une couche mince hydrophile disponible dans le commerce. La construction simple se pr&ecirc;te &agrave; faible co&ucirc;t et facilit&eacute; de fabrication, mais plusieurs fonctionnalit&eacute;s r&eacute;duisent la complexit&eacute; du flux de travail standard de microarray. Tout d&#39;abord, il n&#39;y a pas d&#39;exigence pour l&#39;instrumentation personnalis&eacute;e. Deuxi&egrave;mement, la couche mince hydrophile permet un remplissage uniform d&#39;une chambre de la microfluidique. Troisi&egrave;mement, un capillaire g&eacute;om&eacute;trique arr&ecirc;ter conception liquide de limites &agrave; la chambre de microarray pendant l&#39;ACP et &eacute;limine donc la n&eacute;cessit&eacute; d&#39;une vanne ou un traitement de surface hydrophobe. Et Quatri&egrave;mement, imbibition entra&icirc;ne la suppression uniforme des r&eacute;actifs liquides de la chambre de tableau. Trois cents copies g&eacute;nomiques de methicillin-resistant Staphylococcus aureus (MRSA) sont d&eacute;tect&eacute;s dans une cellule d&#39;&eacute;coulement avec gel goutte microarrays imprim&eacute;es sur un substrat en plastique non modifi&eacute;. Cette sensibilit&eacute; est av&eacute;r&eacute;e comparable aux m&eacute;thodes conventionnelles (c.-&agrave;-d., PCR dans un tube, avec l&#39;hybridation s&eacute;par&eacute;e dans une chambre de microarray, o&ugrave; amplicon est expos&eacute; &agrave; l&#39;espace de travail avant et apr&egrave;s l&#39;hybridation). Cependant, la cellule de flux combine ces &eacute;tapes multiples dans un workflow simple, compact, sans besoin de valves complexes ou instrumentation personnalis&eacute;e et est moins sensible &agrave; la contamination de l&#39;espace de travail que les m&eacute;thodes conventionnelles, car l&#39;amplicon se limite &agrave; l&#39;appareil.

Une cellule d&#39;&eacute;coulement plastique, jetable microfluidiques pour coupl&eacute; sur puce PCR et microarray la d&eacute;tection d&#39;agents infectieux.

Laboratori clinici stanno riconoscendo l&#39;importanza di attuare sensibili e specifici test diagnostici molecolari. Tuttavia, diffusa adozione di questi test richiede semplificate i flussi di lavoro senza la necessit&agrave; di costosi strumenti di supporto. Per abilitare l&#39;analisi basata su microarray soddisfare questi requisiti, descriviamo una cella di flusso senza valvola per utilizzo monouso che supporta PCR accoppiato con l&#39;ibridazione del microarray nella stessa camera. L&#39;Assemblea di cella di flusso &egrave; costituito semplicemente da un nastro, un substrato di plastica microarray, un assorbente e una sottile pellicola di idrofila disponibile in commercio. La costruzione semplice si presta a basso costo e facilit&agrave; di fabbricazione, ma diverse caratteristiche riducono la complessit&agrave; del flusso di lavoro standard microarray. In primo luogo, non non c&#39;&egrave; nessun requisito per strumentazione personalizzata. In secondo luogo, la sottile pellicola idrofila permette un riempimento uniforme di una camera microfluidica. In terzo luogo, un capillare geometrico smettere di progettazione confini liquido nella camera di microarray durante la PCR e quindi elimina la necessit&agrave; di una valvola o un trattamento di superficie idrofoba. E quarto, imbibizione spinge la rimozione uniforme di reagenti liquidi dalla camera di matrice. Trecento copie genomiche di Staphylococcus aureus meticillino-resistente (MRSA) sono rilevate in una cella di flusso con gel goccia microarrays stampato su un substrato di plastica non modificato. Questa sensibilit&agrave; &egrave; mostrata essere paragonabile ai metodi convenzionali (cio&egrave;, la PCR in un tubo, con l&#39;ibridazione separato in una camera di microarray, dove amplicon &egrave; esposto nell&#39;area di lavoro prima e dopo l&#39;ibridazione). Tuttavia, la cellula di flusso combina questi passaggi multipli in un flusso di lavoro semplice e compatto, senza la necessit&agrave; di complesse valvole o strumentazione personalizzata e meno sensibile alla contaminazione dell&#39;area di lavoro rispetto ai metodi convenzionali perch&eacute; l&#39;amplicon &egrave; confinata al dispositivo.

Una cella di flusso in plastica, monouso microfluidica per accoppiato on-chip PCR e microarray rilevamento di agenti infettivi.

This report describes the development and pre-clinical testing of a new, random-access RNA sample preparation system (TruTip) for nasopharyngeal samples. The system is based on a monolithic, porous nucleic acid binding matrix embedded within an aerosol-resistant pipette tip and can be operated with single or multi-channel pipettors. Equivalent extraction efficiencies were obtained between automated QIAcube and manual TruTip methods at 10(6) gene copies influenza A per mL nasopharyngeal aspirate. Influenza A and B amended into nasopharyngeal swabs (in viral transport medium) were detected by real-time RT-PCR at approximately 745 and 370 gene copies per extraction, respectively. RNA extraction efficiency in nasopharyngeal swabs was also comparable to that obtained on an automated QIAcube instrument over a range of input concentrations; the correlation between threshold cycles (or nucleic acid recovery) for TruTip and QIAcube-purified RNA was R(2)>0.99. Preclinical testing of TruTip on blinded nasopharyngeal swab samples resulted in 98% detection accuracy relative to a clinically validated easyMAG extraction method. The physical properties of the TruTip binding matrix and ability to customize its shape and dimensions likewise make it amenable to automation and/or fluidic integration.

Rapid, simple influenza RNA extraction from nasopharyngeal samples.

Ce rapport d&eacute;crit le d&eacute;veloppement des essais pr&eacute;cliniques d&#39;un nouveau vive RNA &eacute;chantillon pr&eacute;paration syst&egrave;me (TruTip) pour les &eacute;chantillons rhinopharyng&eacute;s. Le syst&egrave;me est bas&eacute; sur une matrice de monolithique, poreux nucleic acid binding int&eacute;gr&eacute;e dans l&#39;embout de la pipette a&eacute;rosol r&eacute;sistant et peut &ecirc;tre actionn&eacute; avec pipettes simples ou multicanal. Efficacit&eacute; d&#39;extraction &eacute;quivalentes ont &eacute;t&eacute; obtenues entre QIAcube automatis&eacute;e et m&eacute;thodes manuelles de TruTip &agrave; 10 (6) copies de g&egrave;ne grippe A par aspiration nasopharyng&eacute;e mL. Grippe A et B modifi&eacute; dans le rhinopharynx (en milieu de transport viral) ont &eacute;t&eacute; d&eacute;tect&eacute;s par RT-PCR en temps r&eacute;el &agrave; des copies du g&egrave;ne environ 745 et 370 par extraction, respectivement. L&#39;efficacit&eacute; de la RNA extraction dans le rhinopharynx &eacute;tait &eacute;galement comparable &agrave; celle obtenue sur un instrument automatis&eacute; de QIAcube sur une plage de concentrations d&#39;entr&eacute;e ; la corr&eacute;lation entre les cycles seuil (ou la r&eacute;cup&eacute;ration de l&#39;acide nucl&eacute;ique) pour TruTip et l&#39;ARN purifi&eacute; QIAcube &eacute;tait r &gt; 0,99. Essais pr&eacute;cliniques de TruTip sur des &eacute;chantillons d&#39;&eacute;couvillonnage du rhinopharynx aveugle a entra&icirc;n&eacute; dans la pr&eacute;cision de d&eacute;tection de 98 % par rapport &agrave; une m&eacute;thode d&#39;extraction easyMAG cliniquement valid&eacute;s. Les propri&eacute;t&eacute;s physiques de la matrice de liaison TruTip et de la possibilit&eacute; de personnaliser sa forme et ses dimensions le rendent m&ecirc;me se pr&ecirc;tent &agrave; l&#39;automation et/ou int&eacute;gration fluidique.

Extraction rapide et simple grippe RNA provenant d&#39;&eacute;chantillons rhinopharyng&eacute;s.

Questo rapporto descrive lo sviluppo e il test preclinici di un nuovo accesso casuale RNA campione preparazione sistema (TruTip) per campioni nasofaringei. Il sistema &egrave; basato su una matrice monolitica, porosa dell&#39;acido nucleico associazione incorporata all&#39;interno di un puntale anti-aerosol e pu&ograve; essere azionato con pipette mono o multicanale. Efficienza equivalente di estrazione sono stato ottenuto tra QIAcube automatici e metodi manuali TruTip a 10 copie di gene influenza A per aspirato nasofaringeo mL. Influenza A e B modificato in tamponi nasofaringei (in terreno di trasporto virale) sono state rilevate da RT-PCR in tempo reale a circa 745 e 370 gene copie per estrazione, rispettivamente. L&#39;efficienza di estrazione di RNA in tamponi nasofaringei anche era paragonabile a quella ottenuta su uno strumento automatizzato di QIAcube sopra una gamma di concentrazioni di ingresso; la correlazione tra soglia cicli (o recupero dell&#39;acido nucleico) per TruTip e QIAcube-purificato RNA era R(2) &amp;gt; 0,99. Test preclinici di TruTip su campioni da tampone nasofaringeo accecato ha portato nella precisione di rilevamento del 98% rispetto a un metodo di estrazione easyMAG clinicamente validato. Le propriet&agrave; fisiche della matrice associazione TruTip e possibilit&agrave; di personalizzare la forma e le dimensioni rendono similarmente suscettibili di automazione e/o integrazione fluidici.

Estrazione rapida, semplice influenza RNA da campioni nasofaringei.

Rapid, simple and efficient influenza RNA purification from clinical samples is essential for sensitive molecular detection of influenza infection. Automation of the TruTip extraction method can increase sample throughput while maintaining performance.

Automated, simple, and efficient influenza RNA extraction from clinical respiratory swabs using TruTip and epMotion.

Genetic polymorphisms in the CYP2C9 and VKORC1 genes have been linked to sensitivity of the anticoagulant drug warfarin. The aim of this study is to demonstrate a method for warfarin sensitivity genotyping using gel element microarray technology in a simplified workflow from sample collection to analysis and detection.

Christopher G. Cooney

Engineering

Akonni Biosystems, Inc.

High-throughput, Automated Extraction of DNA and RNA from Clinical Samples using TruTip Technology on Common Liquid Handling Robots

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Christopher G. Cooney

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Engineering

Akonni Biosystems, Inc.

High-throughput, Automated Extraction of DNA and RNA from Clinical Samples using TruTip Technology on Common Liquid Handling Robots

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Engineering