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Search results for: DirectPCR Lysis Reagent (cell)

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#32568322   2020/06/22 To Up

Point-of-care testing system for digital single cell detection of MRSA directly from nasal swabs.

We present an automated point-of-care testing (POCT) system for rapid detection of species- and resistance markers in methicillin-resistant Staphylococcus aureus (MRSA) at the level of single cells, directly from nasal swab samples. Our novel system allows clear differentiation between MRSA, methicillin-sensitive S. aureus (MSSA) and methicillin-resistant coagulase-negative staphylococci (MR-CoNS), which is not the case for currently used real-time quantitative PCR based systems. On top, the novel approach outcompetes the culture-based methods in terms of its short time-to-result (1 h vs. up to 60 h) and reduces manual labor. The walk-away test is fully automated on the centrifugal microfluidic LabDisk platform. The LabDisk cartridge comprises the unit operations swab-uptake, reagent pre-storage, distribution of the sample into 20 000 droplets, specific enzymatic lysis of Staphylococcus spp. and recombinase polymerase amplification (RPA) of species (vicK) - and resistance (mecA) -markers. LabDisk actuation, incubation and multi-channel fluorescence detection is demonstrated with a clinical isolate and spiked nasal swab samples down to a limit of detection (LOD) of 3 ± 0.3 CFU μl-1 for MRSA. The novel approach of the digital single cell detection is suggested to improve hospital admission screening, timely decision making, and goal-oriented antibiotic therapy. The implementation of a higher degree of multiplexing is required to translate the results into clinical practice.
Martin Schulz, Silvia Calabrese, Florian Hausladen, Holger Wurm, Dominik Drossart, Karl Stock, Anna M Sobieraj, Fritz Eichenseher, Martin J Loessner, Mathias Schmelcher, Anja Gerhardts, Ulrike Goetz, Marina Handel, Annerose Serr, Georg Haecker, Jia Li, Mara Specht, Philip Koch, Martin Meyer, Philipp Tepper, Raimund Rother, Michael Jehle, Simon Wadle, Roland Zengerle, Felix von Stetten, Nils Paust, Nadine Borst

2669 related Products with: Point-of-care testing system for digital single cell detection of MRSA directly from nasal swabs.

50 ml1kit10 ml1 kit50 ml1 kit10 ml 5 lt

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#32376283   2020/05/04 To Up

Comparative evaluation of nucleic acid stabilizing reagents for RNA- and DNA-based Leishmania detection in blood as proxy for visceral burdens.

Molecular detection techniques using peripheral blood are preferred over invasive tissue aspiration for the diagnosis and post-treatment follow-up of visceral leishmaniasis (VL) patients. This study aims to identify suitable stabilizing reagents to prevent DNA and RNA degradation during storage and transport to specialized laboratories where molecular diagnosis is performed.
Eline Eberhardt, Rik Hendrickx, Magali Van den Kerkhof, Severine Monnerat, Fabiana Alves, Sarah Hendrickx, Louis Maes, Guy Caljon

1993 related Products with: Comparative evaluation of nucleic acid stabilizing reagents for RNA- and DNA-based Leishmania detection in blood as proxy for visceral burdens.

100Tests 100 G100 assays 1 G100 tests1kg100tests 500 G 1 G

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#32255836   // To Up

Electrochemical cell lysis of gram-positive and gram-negative bacteria: DNA extraction from environmental water samples.

Cell lysis is an essential step for the nucleic acid-based surveillance of bacteriological water quality. Recently, electrochemical cell lysis (ECL), which is based on the local generation of hydroxide at a cathode surface, has been reported to be a rapid and reagent-free method for cell lysis. Herein, we describe the development of a milliliter-output ECL device and its performance characterization with respect to the DNA extraction efficiency for gram-negative bacteria ( and Typhi) and gram-positive bacteria ( and ). Both gram-negative and gram-positive bacteria were successfully lysed within a short but optimal duration of 1 min at a low voltage of ∼5 V. The ECL method described herein, is demonstrated to be applicable to various environmental water sample types, including pond water, treated wastewater, and untreated wastewater with DNA extraction efficiencies similar to a commercial DNA extraction kit. The ECL system outperformed homogeneous chemical lysis in terms of reaction times and DNA extraction efficiencies, due in part to the high pH generated at the cathode surface, which was predicted by simulations of the hydroxide transport in the cathodic chamber. Our work indicates that the ECL method for DNA extraction is rapid, simplified and low-cost with no need for complex instrumentation. It has demonstrable potential as a prelude to PCR analyses of waterborne bacteria in the field, especially for the gram-negative ones.
Siwen Wang, Yanzhe Zhu, Yang Yang, Jing Li, Michael R Hoffmann

2721 related Products with: Electrochemical cell lysis of gram-positive and gram-negative bacteria: DNA extraction from environmental water samples.

1 mL100 100 samples1 mg0.25 ml1 mg50 samples100 extractions1 mL1 mg

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#32084228   2020/02/21 To Up

Comparison of procedures for RNA-extraction from peripheral blood mononuclear cells.

RNA quality and quantity are important factors for ensuring the accuracy of gene expression analysis and other RNA-based downstream applications. Thus far, only a limited number of methodological studies have compared sample storage and RNA extraction procedures for human cells. We compared three commercially available RNA extraction kits, i.e., (NucliSENS) easyMAG, RNeasy (Mini Kit) and RiboPure (RNA Purification Kit-blood). In addition, additional conditions, such as storage medium and storage temperature of human peripheral blood mononuclear cells were evaluated, i.e., 4 °C for RNAlater or -80 °C for QIAzol and for the respective cognate lysis buffers; easyMAG, RNeasy or RiboPure. RNA was extracted from aliquots that had been stored for one day (Run 1) or 83 days (Run 2). After DNase treatment, quantity and quality of RNA were assessed by means of a NanoDrop spectrophotometer, 2100 Bioanalyzer and RT-qPCR for the ACTB reference gene. We observed that high-quality RNA can be obtained using RNeasy and RiboPure, regardless of the storage medium, whereas samples stored in RNAlater resulted in the least amount of RNA extracted. In addition, RiboPure combined with storage of samples in its cognate lysis buffer yielded twice as much RNA as all other procedures. These results were supported by RT-qPCR and by the reproducibility observed for two independent extraction runs.
Antonio Rodríguez, Hans Duyvejonck, Jonas D Van Belleghem, Tessa Gryp, Leen Van Simaey, Stefan Vermeulen, Els Van Mechelen, Mario Vaneechoutte

2428 related Products with: Comparison of procedures for RNA-extraction from peripheral blood mononuclear cells.

100 extractions100 extractions100ml30ml100ml10ml1 Unit10ml30ml30ml30ml

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#31906315   2020/01/01 To Up

Digital Microfluidic Platform to Maximize Diagnostic Tests with Low Sample Volumes from Newborns and Pediatric Patients.

"Children are not tiny adults" is an adage commonly used in pediatrics to emphasize the fact that children often have different physiological responses to sickness and trauma compared to adults. However, despite widespread acceptance of this concept, diagnostic blood testing is an excellent example of clinical care that is not yet customized to the needs of children, especially newborns. Cumulative blood loss resulting from clinical testing does not typically impact critically ill adult patients, but can quickly escalate in children, leading to iatrogenic anemia and related comorbidities. Moreover, the tests prioritized for rapid, near-patient testing in adults are not always the most clinically relevant tests for children or newborns. This report describes the development of a digital microfluidic testing platform and associated clinical assays purposely curated to address current shortcomings in pediatric laboratory testing by using microliter volumes (<50 µL) of samples. The automated platform consists of a small instrument and single-use cartridges, which contain all reagents necessary to prepare the sample and perform the assay. Electrowetting technology is used to precisely manipulate nanoliter-sized droplets of samples and reagents inside the cartridge. To date, we have automated three disparate types of assays (biochemical assays, immunoassays, and molecular assays) on the platform and have developed over two dozen unique tests, each with important clinical application to newborns and pediatric patients. Cell lysis, plasma preparation, magnetic bead washing, thermocycling, incubation, and many other essential functions were all performed on the cartridge without any user intervention. The resulting assays demonstrate performance comparable to standard clinical laboratory assays and are economical due to the reduced hands-on effort required for each assay and lower overall reagent consumption. These capabilities allow a wide range of assays to be run simultaneously on the same cartridge using significantly reduced sample volumes with results in minutes.
Rama S Sista, Rainer Ng, Miriam Nuffer, Michael Basmajian, Jacob Coyne, Jennifer Elderbroom, Daniel Hull, Kathryn Kay, Maithri Krishnamurthy, Christopher Roberts, Daniel Wu, Adam D Kennedy, Rajendra Singh, Vijay Srinivasan, Vamsee K Pamula

2093 related Products with: Digital Microfluidic Platform to Maximize Diagnostic Tests with Low Sample Volumes from Newborns and Pediatric Patients.

1,000 tests96 tests96 Tests n1000 TESTS/0.65ml1000 tests430 Tests / Kit600 Tests / Kit96 Tests n1 kit75 tests

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#31901461   2019/12/18 To Up

Application of hematoxylin reagent for sperm cell separation in sexual crime evidence.

Seminal evidence obtained from a sexual crime scene provides clues for solving a case through forensic analysis. However, most evidence collected from sexual crime scenes is a mixture of sperm cells and vaginal discharge. Therefore, separating the sperm cells from the seminal evidence is very important. In this study, we developed a separation method for effectively separating sperm cells using differential extraction with commercially available sperm staining reagents such as hematoxylin and nigrosin. Hematoxylin (0.03 % v/v) effectively stained the sperm cells in ATL and TNE lysis buffer, while nigrosin did not. The loss of sperm cells during washing of the specimen was minimized using the differential extraction method. Subsequently, genomic DNA was extracted from the hematoxylin-stained sperm cells and subjected to short tandem repeat genotyping. We observed no interference from hematoxylin. These results indicate that hematoxylin can be used to stain sperm cells and thus facilitate subsequent genetic identification.
Joo-Young Kim, Man Il Kim, Hye Hyeon Lee, Hye Lim Kim, Eun-Jung Lee, Yang-Han Lee, In-Kwan Hwang, Byung-Won Chun, Pil-Won Kang

1025 related Products with: Application of hematoxylin reagent for sperm cell separation in sexual crime evidence.

250 ml1 LITRE100 ml100ug Lyophilized100ug Lyophilized500 gm.100ug Lyophilized2 ml 100ul4 X 250 ml.

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#31896611   2020/01/02 To Up

Purification of Total RNA from Mammalian Cells and Tissues.

This protocol describes the use of a monophasic lysis reagent to isolate total RNA from mammalian cells (grown as either a monolayer or in suspension) or tissues. An alternative protocol for the isolation of RNA from a small quantity of cells (10-10) or tissue (1-10 mg) is also included. In the latter case, glycogen is added to the monophasic lysis reagent and is used as a carrier to increase the recovery of RNA.
Michael R Green, Joseph Sambrook

2413 related Products with: Purification of Total RNA from Mammalian Cells and Tissues.

100 extractions100 extractions 1 kit(s)

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#31836712   2019/12/13 To Up

Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying.

Sensitive simultaneous assessment of multiple signaling pathways within the same cells requires orthogonal reporters that can assay over large dynamic ranges. Luciferases are such genetically encoded candidates due to their sensitivity, versatility, and cost-effectiveness. We expand luciferase multiplexing in post-lysis endpoint luciferase assays from two to six. Light emissions are distinguished by a combination of distinct substrates and emission spectra deconvolution. All six luciferase reporter units are stitched together into one plasmid facilitating delivery of all reporter units through a process we termed solotransfection, minimizing experimental errors. We engineer a multiplex hextuple luciferase assay to probe pathway fluxes through five transcriptional response elements against a control constitutive promoter. We can monitor effects of siRNA, ligand, and chemical compound treatments on their target pathways along with the four other probed cellular pathways. We demonstrate the effectiveness and adaptiveness of multiplex luciferase assaying, and its broad application across different research fields.
Alejandro Sarrion-Perdigones, Lyra Chang, Yezabel Gonzalez, Tatiana Gallego-Flores, Damian W Young, Koen J T Venken

2472 related Products with: Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying.

50 ul20 ul50 ul20 ul16 Arrays/Slide16 Arrays/Slide16 Arrays/Slide16 Arrays/Slide16 Arrays/Slide

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#31821925   2019/11/22 To Up

Sodium azide inhibition of microbial activities and impact on sludge floc destabilization.

Absence of sludge deflocculation under prolonged (24 h or longer) conditions with dissolved oxygen (DO) less than 0.5 mg L⁻ was recently reported. The reduced aerobic microbial activity, was speculated, had been compensated by the activity of other bacterial (i.e. facultative) communities. To assess such a compensation mechanism and to better evaluate impact of overall microbial activity on the flocculation process, SBR sludge samples were inhibited by using sodium azide under various DO conditions. Sludge deflocculated only in the presence of sodium azide, regardless of DO conditions. This was linked to sodium azide's inhibitory effects on the microbes as indicated by the reduced ammonium and DOC removals. Extracellular potassium level in the mixed liquor of azide spiked samples also indicated simultaneous cell lysis. Fluorescence excitation emission matrix (FEEM) analysis of the extracted bound EPS and fluorescence quenching based interaction studies indicated sodium azide had interacted with the EPS components, and especially with the bound EPS proteins. The impact of such interactions on reduced floc stability needs consideration. This study confirmed the importance of overall microbial activity in the biological flocculation process and the role of bacterial communities, other than the aerobes, in mitigating deflocculation under low DO conditions.
Akshaykumar Suresh, Chaozhi Pan, Wun Jern Ng

2436 related Products with: Sodium azide inhibition of microbial activities and impact on sludge floc destabilization.

100 g 500 G1 mg 25 MG10 mg2 g 100 G96 assays 100 g 100 G100 MG

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#31816151   // To Up

Isolation of High-Quality RNA from Pichia pastoris.

Analysis of RNA structuromes provides new insights into cellular processes, enabling systems biology and biotechnology researchers to calculate promoter and terminator strengths and to directly observe how differing circuit states impact host gene expression and the burdens imposed by the circuits. Such analysis, however, is crucially dependent on the availability of highly pure, intact RNA isolated from fresh or frozen cell cultures. RNA extraction from the yeast Pichia pastoris requires specific pretreatment steps to ensure the reproducibility of downstream applications, but current methods and extraction kits are generally adapted for the conventional yeast Saccharomyces cerevisiae, which has a different cell wall composition. We therefore set out to compare the efficacy of two different RNA isolation methods when applied to P. pastoris: (i) phenol/chloroform extraction and (ii) silica spin-column absorption. We compared the yield, integrity, and purity of the resulting isolated RNA from the two methods (using two different types of commercial columns for silica spin-column absorption) and further optimized them through variations in the pretreatment steps. We also assessed two different methods of cell lysis: enzyme catalytic disruption using lyticase and mechanical disruption using acid-washed glass-beads in a TissueLyser. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: RNA isolation with phenol/chloroform extraction: monophasic lysis reagent Alternate Protocol 1: RNA isolation with silica-spin column absorption: High Pure RNA Isolation Kit (Roche Life Science) Alternate Protocol 2: RNA isolation with silica-spin column absorption: RNeasy Mini Kit (Qiagen).
Sibel Öztürk, İrem Demir, Pınar Çalık

2644 related Products with: Isolation of High-Quality RNA from Pichia pastoris.

1 mg1 mL100 µgUp to 200 ml cultures10x96, 2.0ml cultures10, 10ml whole blood 6 ml Ready-to-use 5 X 1000 U10 nmol200ul1

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