Diese Forschung wurde von Naval Medical Research Center, Forschung Arbeitseinheit 6000.RAD1.J.A0310 unterstützt. Die in diesem Artikel sind die der Autoren und geben nicht unbedingt die offizielle Politik oder Position des Department of the Navy, Department of Defense, oder der US-Regierung widerspiegeln. Wei-Mei Ching ist ein Mitarbeiter der US-Regierung. Diese Arbeit wurde im Rahmen ihrer offiziellen Aufgaben vorbereitet. Titel 17 USC § 105 heißt es: &quot;Der Urheberrechtsschutz unter diesem Titel für jede Arbeit der Regierung der Vereinigten Staaten nicht zur Verfügung steht.&quot; Titel 17 USC §101 definiert eine US-Regierung Arbeit als Arbeit, die durch Militärdienst Mitglied oder Mitarbeiter der US-Regierung als Teil der Person durch Amtspflichten.

1. Bereiten Sie Plasmid-DNA Verdünnungen als Standard für den LAMP-Reaktion <ol><li> Verwenden eines Spektrophotometers die optische Dichte (OD) bei 260 nm zu messen , um das Plasmid (enthaltend Zielgen IS1111a von C. burnetii RSA 493) DNA - Konzentration zu bestimmen. Eine OD 260 von 1 entspricht 50 ug / ul DNA. </li><li> Konvertieren Sie die Menge von Plasmid-DNA in die Kopienzahl. Da die Größe des Plasmids 6330 bp ist, ist das Molekulargewicht dieses Plasmids 4,1 x 10 6 g (6.330 bp x 649 g / bp). Die DNA-Konzentration des Plasmids ist 34,2 ng / &amp; mgr; l (wie in Schritt bestimmt 1.1). 1 &amp; mgr; l dieser DNA - Probe enthält 34,2 ng Plasmid - DNA, zu gleich 5 x 10 9 Kopien (34,2 x 10 -9 g / 4,1 x 10 6 6,02 x 10 gx 23) von DNA. </li><li> Zugabe von 10 &amp; mgr; l Plasmid - DNA (5 x 10 9 Kopien / ul) und 90 ul Wasser für eine 10-fache Verdünnung einer DNA - Probe von 5 x 10 8 Kopien zu erstellen / ul. </li><li> Wiederholen Sie Schritt 1.3 zur Herstellung von DNA - Proben von 5 x 10 7, 5 x 10 6, 5 x 10 5, 5 x 10 4, 5 × 10 3, 5 x 10 2, 5 x 10 1 und 5 x 10 0 Kopien / ul. </li></ol> 2. Bereiten Sie DNA-Vorlage von Proben für die LAMP-Reaktion <ol><li> Führen DNA Extraktion aus Humanplasmaproben Kommerzielle DNA Mini Kit nach Herstellerprotokoll. Verwenden Sie eine Gesamtmenge von 200 ul Probe für die Extraktion und Elution extrahierten DNA in einem 20 &amp; mgr; l Volumen. </li></ol> 3. Bereiten Sie 2x LAMP-Reaktionspuffer <ol><li> Mischen Sie 200 ul 10x Pol Buffer, 320 ul 5 M Trimethylglycin, 12 ul 1 M MgSO 4, 280 &amp; mgr; l dNTP - Gemisch (jeweils 10 mM) und 188 ul Wasser zur Herstellung von 1 ml 2x LAMP - Puffer. Mix von Puls-Verwirbelung für 10 Sekunden. </li></ol> 4. Führen Sie Standard-LAMP-Reaktion <ol><li> Mix 12,5 ul 2x LAMP - Puffer (wie in Schritt 3 hergestellt; 40 mM Tris-HCl (pH 8,8), 20 mM KCl, 16 mM MgSO 4, 20 mM (NH 4) 2 SO 4, 0,2% Triton X-100, 1,6 M Trimethylglycin, 1.4 mM dNTP - Gemisch), 1,2 &amp; mgr; l Primer - Mix, 1 &amp; mgr; l Bst DNA - Polymerase (8 U / ul) und 5,3 ul Wasser Reaktionsmischung (20 ul) in einem 0,2 ml PCR - Röhrchen zu bilden. </li><li> Zugabe von 5 &amp; mgr; l DNA (aus Schritt 1 oder 2) in die 20 &amp; mgr; l Reaktionsgemisch und gut mischen. </li><li> Schließen Rohr und für 60 min in einem Wasserbad oder Heizblock bei 60 ° C inkubiert. Dies ist die optimale Reaktionstemperatur zuvor bestimmt. </li><li> In 5 ul 10x Gelladepuffer, um die Reaktion zu beenden. </li><li> Last 5 &amp; mgr; l Reaktionsprodukte zu einem 2% Agarosegel, gefärbt mit stain Nukleinsäure interkalierende. </li><li> Führen Gel bei 100 V für 35 min in 1x TBE-Puffer. </li><li> Visualisieren Sie die Ergebnisse durch UV-Licht. </li></ol> 5. Führen Sie LAMP Reaktion mit RekonstituiertesReagenzien <ol><li> Mischen Sie 125 ul von 10x Pol Puffer, 200 &amp; mgr; l von 5 M Trimethylglycin, 7,5 ul 1 M MgSO 4, 667,5 ul Wasser zur Herstellung von 1 ml Rekonstitution Puffer. Mix von Puls-Verwirbelung für 10 Sekunden. </li><li> Entfernen Sie Rohre, die die lyophilisierten Reagenzien aus dem versiegelten Aluminiumfolienbeutel enthalten. Sie nicht die Rohre verwenden, wenn die Aluminiumfolienbeutel nicht abgedichtet ist, oder wenn es beschädigt ist. </li><li> In 20 ul Rekonstitution Puffer in jedes Röhrchen lyophilisierten Reagenzien erneut zu suspendieren. </li><li> Mischen durch Pipettieren Puffer nach oben und unten 5-mal. Werfen Sie einen Blick um sicherzustellen, dass die lyophilisierten Reagenzien komplett neu aufgehängt sind. Das weiße Pulver sollte in der Röhre verschwinden. </li><li> In 5 ul DNA-Template (aus Schritt 1 oder 2) zu den rekonstituierten Reagenzien und gut mischen. </li><li> Schließen Rohr und für 60 min in einem Wasserbad oder Heizblock bei 60 ° C inkubiert. </li><li> In 5 ul 10x Gelladepuffer, um die Reaktion zu beenden. </li><li> Last 5 ul ReaktionsProdukte einem 2% Agarosegel, gefärbt mit Nukleinsäure interkalierende stain. </li><li> Führen Gel bei 100 V für 35 min in 1x TBE-Puffer. </li><li> Visualisieren Sie die Ergebnisse durch UV-Licht. </li></ol> 6. Führen Sie LAMP Reaktion mit Rekonstitution Puffer, der HNB oder Fluorescent interkalierende Farbstoff <ol><li> Mischungs 125 ul 10fach Pol Puffer, 200 &amp; mgr; l von 5 M Trimethylglycin, 7,5 ul 1 M MgSO 4, 7,5 ul 20 mM HNB (oder 12,5 &amp; mgr; l 100x fluoreszierenden interkalierenden Farbstoff) und 660 ul (oder 655 &amp; mgr; l) Wasser machen 1 ml Rekonstitution Puffer. Mix von Puls-Verwirbelung für 10 Sekunden. </li><li> Verwenden Sie das aufgelöste Puffer in Abschnitt 6 hergestellt, die Schritte 5.2 zu wiederholen - 5.6. </li><li> Visualisieren Sie die Ergebnisse mit dem bloßen Auge (Reaktion, die HNB) oder einem UV-Licht (Reaktion fluoreszierenden interkalierenden Farbstoff enthält). </li></ol> Führen 7. Echtzeit-LAMP-Reaktion mit Rohr Scanner <ol><li> In 5 ul DNA an die reconstituted Reagenzien fluoreszierenden interkalierenden Farbstoff, in der Nähe Rohr und legen Sie es auf das Rohr Scanner enthält. </li><li> Eingestellt Inkubationstemperatur auf 60 ° C. Messen der Fluoreszenz bei 520 nm für 60 min. </li></ol>

<ol>
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Diagn. 1 (112), (2012).</li><li>Pan, L., Zhang, L., Wang, G., Liu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+sensitive+detection+of+the+ompB+gene+of+spotted+fever+group+rickettsiae+by+loop-mediated+isothermal+amplification.">Rapid, sensitive detection of the ompB gene of spotted fever group rickettsiae by loop-mediated isothermal amplification.</a> BMC Infect. Dis. 12 (254), (2012).</li><li>Mori, Y., Nagamine, K., Tomita, N., Notomi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+loop-mediated+isothermal+amplification+reaction+by+turbidity+derived+from+magnesium+pyrophosphate+formation.">Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation.</a> Biochem. Biophys. Res. Commun. 289 (1), 150-154 (2001).</li><li>Qiao, Y. M., 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=Loop-mediated+isothermal+amplification+for+rapid+detection+of+Bacillus+anthracis.spores.">Loop-mediated isothermal amplification for rapid detection of Bacillus anthracis.spores.</a> Biotechnol. Lett. 29 (12), 1939-1946 (2007).</li><li>Tomita, N., Mori, Y., Kanda, H., Notomi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+(LAMP)+of+gene+sequences+and+simple+visual+detection+of+products.">Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products.</a> Nat. Protoc. 3 (5), 877-882 (2008).</li><li>Thekisoe, O. 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Microbiol Methods. 107, 176-181 (2014).</li><li>Wang, D., 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=A+Comparison+of+In-House+Real-Time+LAMP+Assays+with+a+Commercial+Assay+for+the+Detection+of+Pathogenic+Bacteria.">A Comparison of In-House Real-Time LAMP Assays with a Commercial Assay for the Detection of Pathogenic Bacteria.</a> Molecules. 20 (6), 9487-9495 (2015).</li></ol>

Die Autoren haben nichts zu offenbaren.

Bisher haben wir eine empfindliche und spezifische LAMP - Targeting - Assay das Einsatzelement IS1111a 14. Das IS1111 Element wurde ausgewählt , weil es stark unter den verschiedenen Stämmen von C. konserviert burnetii und die hohe Anzahl der Kopien (7-110) in den Bakterien (Klee 2006). Unsere Ergebnisse zeigten , dass LAMP können etwa 25 Kopien des IS1111 Element erkennen, die so wenig wie eine chromosomale Kopie von Coxiella DNA korrelieren. In dieser...

Die kleine Gram-negative Bakterium Coxiella burnetii ist der Erreger des Q - Fiebers, das ein weltweit Zoonose ist. Durch den weltweiten Vertrieb von Q - Fieber und die hohe Infektiosität von C. burnetii, US - Militär- und Zivilpersonal im Ausland im Einsatz sind in Gefahr, in den Endemiegebieten infiziert. Q-Fieber manifestiert sich in zwei Formen: akute und chronische Infektionen. Akute Q-Fieber präsentiert sich mit grippeähnlichen Symptomen, Hepatitis oder Pneumonie, und ist in der Regel eine selbstlimitierend Krankheit mit einer niedrigen Sterblichkeit. Chronische Q-Fieber, während weniger verbreitet, führt oft zu Endokarditis, die eine viel höhere Mortalitätsrate hat , wenn 1,2 unbehandelt. Daher ist eine frühzeitige Diagnose führen eine geeignete Behandlung für die Patientenversorgung kritisch ist. Polymerase - Kettenreaktion (PCR) und quantitative Echtzeit - PCR (qPCR) Assays wurden zum Nachweis von C. entwickelt burnetii DNA in Zellkulturen und klinische Proben 3-5 &lt;/ Sup&gt;. Beide PCR und qPCR sind teuer und oft nicht ohne weiteres verfügbar in ressourcenbeschränkte Bereiche für die Routinearbeit. Ursprünglich von Notomi 6 beschrieben, Loop-vermittelte isothermen Amplifikation (LAMP) bietet eine alternative DNA - Verstärkungsverfahren. LAMP verwendet Bst DNA - Polymerase zusammen mit speziell entwickelten Primer - Sets Verschiebung DNA Synthese Strang, der mindestens sechs unabhängigen Regionen des Zielgens zu erkennen. Der signifikanteste Vorteil ist, dass LAMP-Amplifikation unter isothermischen Bedingungen auftritt. Daher ist nur ein Wasserbad, Heizblock oder ein Inkubator ist nicht erforderlich. Dieses Verfahren wurde verwendet 7-9 verschiedene rickettsial Pathogene zu erkennen. Visualisierung von amplifizierte DNA-Produkte durch Gelelektrophorese ist die genaueste Methode, die wahren Positiven von Fehlalarme aufgrund unspezifischer Amplifikation unterscheiden kann. Jedoch sind die in der Gelelektrophorese angewandten Verfahren nicht praktikabel für begrenzten Ressource-areas. Es wurden verschiedene alternative Verfahren entwickelt, um die Reaktionsprodukte zu detektieren. Diese alternativen, wie Trübung , abgeleitet von Magnesiumpyrophosphat Formation 10 oder unter Verwendung eines fluoreszierenden interkalierenden Farbstoff unter UV - Licht visualisiert werden 11,12 sind günstiger als ein Gel läuft. Die LAMP - Reagenzien waren einen Monat lang stabil , wenn sie bei 25 ° C und 37 ° C gelagert, die sind Umgebungstemperaturen in tropischen und subtropischen Ländern , in denen Q - Fieber endemisch 13 ist. Ein LAMP - Assay wurde in unserem Labor entwickelt , um das Vorhandensein von C. zu erkennen burnetii in Plasmaproben 14. Hier beschreiben wir ein einfaches Protokoll für die Herstellung der LAMP Reaktionsmischung aus dem lyophilisierten Reagenzien. Die lyophilisierten Reagenzien sind stabil für einen Monat, wenn bei RT gelagert. Wenn sie mit einem einfachen Visualisierungsverfahren kombiniert werden , ist dies eine ideale Methode zum Nachweis C. verwenden burnetii in einer begrenzten Ressource-Einstellung.

<table><tbody><tr><td>LAMP primers</td><td>Eurofins MWG operon</td><td>10 nmol, salt free</td><td>Sequence designed by customer</td></tr><tr><td>Bst DNA polymerase</td><td>New England Biolabs</td><td>M0275L</td><td>8 units/ml</td></tr><tr><td>10x Thermo pol buffer</td><td>New England Biolabs</td><td>B9004S</td><td></td></tr><tr><td>dNTP mixture</td><td>New England Biolabs</td><td>N0447L</td><td>10 mM each</td></tr><tr><td>Betaine</td><td>Sigma-Aldrich</td><td>B0300</td><td>5 M, Trimethylglycine</td></tr><tr><td>Magnesium Sulfate</td><td>Sigma-Aldrich</td><td>M3409-1ML</td><td>1 M</td></tr><tr><td>10x Bluejuice</td><td>Invitrogen</td><td>10816-015</td><td>gel loading buffer</td></tr><tr><td>SYBR green</td><td>Invitrogen</td><td>S7585</td><td>10,000x, visualize products in tubes</td></tr><tr><td>GelRed</td><td>Phenix Research Products</td><td>RGB-4103</td><td>10,000x, visualize products in gels</td></tr><tr><td>Lyophilized reagents</td><td>Gene Reach</td><td></td><td></td></tr><tr><td>Hydroxynaphthol blue</td><td>Fluka</td><td>33936-10G</td><td></td></tr><tr><td>ESEQuant tube scanner</td><td>Qiagen</td><td></td><td>real-time detection</td></tr></tbody></table>

the development of lyophilized loop-mediated isothermal amplification reagents for the detection of coxiella burnetii

Coxiella burnetii, der Agent Q - Fieber verursacht, ist ein obligat intrazelluläres Bakterium. PCR - basierten diagnostischen Assays wurden zum Nachweis von C. entwickelt burnetii DNA in Zellkulturen und klinischen Proben. PCR erfordert spezielle Ausrüstung und umfassende Schulung von Endanwendern, und daher ist es für Routinearbeiten vor allem in einem ressourcenbeschränkte Bereich nicht geeignet. Wir haben eine loop-vermittelte isothermische Amplifikation (LAMP) Assay entwickelt , um die Anwesenheit von C. nachzuweisen burnetii in Patientenproben. Dieses Verfahren wird bei einer einzigen Temperatur etwa 60 ° C in einem Wasserbad oder Heizblock durchgeführt. Die Empfindlichkeit dieses LAMP-Test ist sehr ähnlich wie PCR mit einer Nachweisgrenze von etwa 25 Kopien pro Reaktion. Dieser Bericht beschreibt die Herstellung der Reaktion lyophilisierten Reagenzien und Visualisierung der Ergebnisse mit hydroxynaphthol blau (HNB) oder einer UV-Lampe mit fluoreszierenden interkalierenden Farbstoff in der Reaktion verwendet wird. Die LAMP-Reagenzien waren lyophilized und bei Raumtemperatur (RT) einen Monat lang ohne Verlust der Detektionsempfindlichkeit gespeichert. Diese LAMP-Test ist besonders robust, da die Reaktionsgemischaufbereitung nicht komplexe Schritte beinhaltet. Diese Methode ist ideal für den Einsatz in begrenzten Ressource-Einstellungen, wo Q-Fieber endemisch ist.

Die lyophilisierten LAMP Reagenzien innerhalb der 0,2 - ml - Röhrchen enthält Bst DNA - Polymerase, Primer und dNTPs. 20 ul Rekonstitution Puffer wird verwendet , die lyophilisierten Reagenzien erneut zu suspendieren. Abbildung 1 zeigt die LAMP Reaktion führt auf Agarosegelen mit frisch zubereiteten Reagenzien und gefriergetrocknet Reagenzien. Das gefriergetrocknete Reagenz verringert nicht seine Aktivität. LAMP Reaktionen von beiden Reagenzien hergestellt w...

Watch this Scientific Journal Video about The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii at JoVE.com

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Die Entwicklung der Lyophilisierte Loop-mediated Isothermal Amplification Reagenzien zum Nachweis von<em&gt; Coxiella burnetii</em

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

the-development-lyophilized-loop-mediated-isothermal-amplification

Research

JoVE Journal

Immunology and Infection

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

10.5K Aufrufe.  Naval Medical Research Center. We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

Serie: The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification

Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in vegetable production annually. Phytophthora capsici is soil-borne and a persistent problem in vegetable fields due to its long-lived survival structures (oospores and chlamydospores) that resist weathering and degradation. The main method of dispersal is through the production of zoospores, which are single-celled, flagellated spores that can swim through thin films of water present on surfaces or in water-filled soil pores and can accumulate in puddles and ponds. Therefore, irrigation ponds can be a source of the pathogen and initial points of disease outbreaks. Detection of P. capsici in irrigation water is difficult using traditional culture-based methods because other microorganisms present in the environment, such as Pythium spp., usually overgrow P. capsici making it undetectable. To determine the presence of P. capsici spores in water sources (irrigation water, runoff, etc.), we developed a hand pump-based filter paper (8-10 &#181;m) method that captures the pathogen&#8217;s spores (zoospores) and is later used to amplify the pathogen&#8217;s DNA through a novel loop-mediated isothermal amplification (LAMP) assay designed for the specific amplification of P. capsici. This method can amplify and detect DNA from a concentration as low as 1.2 x 102 zoospores/mL, which is 40 times more sensitive than conventional PCR. No cross-amplification was obtained when testing closely related species. LAMP was also performed using a colorimetric LAMP master mix dye, displaying results that could be read with the naked eye for on-site rapid detection. This protocol could be adapted to other pathogens that reside, accumulate, or are dispersed via contaminated irrigation systems.

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification

We developed a method to detect Phytophthora capsici zoospores in water sources using a filter paper DNA extraction method coupled with a loop-mediated isothermal amplification (LAMP) assay that can be analyzed in the field or in the lab.

Nachweis von Phytophthora capsici im Bewässerungswasser mit Loop-Mediad Isothermale Amplifikation

Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in ...

Forschung

Umwelt

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Generation und Multi-phänotypischen High-Content-Screening<em&gt; Coxiella burnetii</em&gt; Transposon-Mutanten

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Immunologie und Infektion

Ready-To-Use qPCR for Detection of DNA from Trypanosoma cruzi or Other Pathogenic Organisms

Real-time PCR (qPCR) is a remarkably sensitive and precise technique&#160;that allows for amplifying&#160;minute amounts of nucleic acid targets from a multitude of samples. It has been extensively used in many research areas and achieved industrial application in fields such as human diagnostics and trait selection in crops of genetically modified organisms (GMO) crops. However, qPCR is not an error-proof technique. Mixing all reagents into a single master mix subsequently distributed onto 96 wells of a regular qPCR plate might lead to operator mistakes such as incorrect mixing of reagents or inaccurate dispensing into the wells. Here, a technique called gelification is presented, whereby most of the water present in the master mix is substituted by reagents that form a sol-gel mixture when submitted to a vacuum. As a result, qPCR reagents are effectively preserved for a few weeks at room temperature or a few months at 2-8 oC. Details of preparing each solution are shown here along with the expected aspect of a gelified reaction designed to detect&#160;T. cruzi satellite DNA (satDNA). A similar procedure can be applied to detect other organisms. Starting a gelified qPCR run is as simple as removing the plate from the refrigerator, adding the samples to their respective wells, and starting the run, thus decreasing the setup time of a full-plate reaction to the time it takes to load the samples. Additionally, gelified PCR reactions can be produced and controlled for quality in batches, saving time and avoiding common operator mistakes while running routine PCR reactions.

Ready-To-Use qPCR for Detection of DNA from Trypanosoma cruzi or Other Pathogenic Organisms

The present work describes the steps for producing ready-to-use qPCR for T. cruzi DNA detection that can be pre-loaded on the reaction vessel and stored in the refrigerator for several months.

Gebrauchsfertige qPCR zum Nachweis von DNA aus Trypanosoma cruzi oder anderen pathogenen Organismen

Real-time PCR (qPCR) is a remarkably sensitive and precise technique&#160;that allows for amplifying&#160;minute amounts of nucleic acid targets from a ...

Biochemie

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

Anwenden von Fluorescence Resonance Energy Transfer (FRET) zu untersuchen Effektor Translokation Effizienz durch<em&gt; Coxiella burnetii</em&gt; Während siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

Visuelle Erkennung von mehreren Nukleinsäuren in einem Kapillar Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

Biotechnik

Open-Source Miniature Fluorimeter to Monitor Real-Time Isothermal Nucleic Acid Amplification Reactions in Resource-Limited Settings

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with integrated fluorescence detection of amplicons. Isothermal nucleic acid amplification technologies eliminate the need for thermal cycling; however, fluorescence-based detection of products is still required for real-time, quantitative results. Several portable isothermal heaters with integrated fluorescence detection are now commercially available; however, the cost of these devices remains a significant barrier to widespread adoption in resource-limited settings. Described here is a protocol for the design and assembly of a modular, low-cost fluorimeter constructed from off-the-shelf components. Enclosed in a compact 3D printed housing, the fluorimeter is designed to be placed atop a commercially available heat block holding a PCR tube. The fluorimeter described here was optimized to detect fluorescein isothiocyanate (FITC) dye, but the system can be modified for use with dyes commonly used as reporters in real-time nucleic acid amplification reactions. Clinical applicability of the system is demonstrated by performing real-time nucleic acid detection with two isothermal amplification technologies: recombinase polymerase amplification (RPA) for detection of positive control DNA provided in a commercial kit and reverse transcription loop-mediated isothermal amplification (RT-LAMP) for detection of clinically meaningful levels of SARS-CoV-2 RNA.

Detailed instructions are provided to build an open-source, modular fluorimeter that is compatible with many low-cost heaters to perform real-time, quantitative isothermal nucleic acid amplification.

Open-Source-Miniaturfluorimeter zur Überwachung isothermer Nukleinsäureverstärkungsreaktionen in Echtzeit in ressourcenbegrenten Umgebungen

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with ...

Diagnose von bakteriellen Atemwegsinfektionen beim Menschen mittels isothermer Amplifikation

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

Autor im Rampenlicht: Weiterentwicklung des schnellen Nachweises von Atemwegserregern mit mikrofluidischem Chip 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

Schneller Nachweis von bakteriellen Krankheitserregern, die Infektionen der unteren Atemwege verursachen, durch mikrofluidische Chip-basierte schleifenvermittelte isotherme Amplifikation

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, fungal, parasitic, and viral agents. Salmonella is a bacterial pathogen of worldwide food safety concern, including food for animals. Presented here is a multi-laboratory-validated Salmonella LAMP protocol that can be used to rapidly screen animal food for the presence of Salmonella contamination and can also be used to confirm presumptive Salmonella isolates recovered from all food categories. The LAMP assay specifically targets the Salmonella invasion gene (invA) and is rapid, sensitive, and highly specific. Template DNAs are prepared from enrichment broths of animal food or pure cultures of presumptive Salmonella isolates. The LAMP reagent mixture is prepared by combining an isothermal master mix, primers, DNA template, and water. The LAMP assay runs at a constant temperature of 65 &#176;C for 30 min. Positive results are monitored via real-time fluorescence and can be detected as early as 5 min. The LAMP assay exhibits high tolerance to inhibitors in animal food or culture medium, serving as a rapid, reliable, robust, cost-effective, and user-friendly method for screening and confirming Salmonella. The LAMP method has recently been incorporated into the U.S. Food and Drug Administration&#8217;s Bacteriological Analytical Manual (BAM) Chapter 5.

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification test (iNAAT) that has attracted broad interest in the pathogen detection field. Here, we present a multi-laboratory-validated Salmonella LAMP protocol as a rapid, reliable, and robust method for screening Salmonella in animal food and confirming presumptive Salmonella from culture isolation.

Loop-vermittelte isotherme Amplifikation zum Screening von Salmonellen in Tierfutter und Bestätigung von Salmonellen aus der Kulturisolierung

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, ...

Die Entwicklung der Lyophilisierte Loop-mediated Isothermal Amplification Reagenzien zum Nachweis von

Zusammenfassung

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Gebrauchsfertige qPCR zum Nachweis von DNA aus Trypanosoma cruzi oder anderen pathogenen Organismen

Anwenden von Fluorescence Resonance Energy Transfer (FRET) zu untersuchen Effektor Translokation Effizienz durch

Visuelle Erkennung von mehreren Nukleinsäuren in einem Kapillar Array

Open-Source-Miniaturfluorimeter zur Überwachung isothermer Nukleinsäureverstärkungsreaktionen in Echtzeit in ressourcenbegrenten Umgebungen

Schneller Nachweis von bakteriellen Krankheitserregern, die Infektionen der unteren Atemwege verursachen, durch mikrofluidische Chip-basierte schleifenvermittelte isotherme Amplifikation

Schneller Nachweis von bakteriellen Krankheitserregern, die Infektionen der unteren Atemwege verursachen, durch mikrofluidische Chip-basierte schleifenvermittelte isotherme Amplifikation

Schneller Nachweis von bakteriellen Krankheitserregern, die Infektionen der unteren Atemwege verursachen, durch mikrofluidische Chip-basierte schleifenvermittelte isotherme Amplifikation

Loop-vermittelte isotherme Amplifikation zum Screening von Salmonellen in Tierfutter und Bestätigung von Salmonellen aus der Kulturisolierung