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

Schulz, Martin; Calabrese, Silvia; Hausladen, Florian; Wurm, Holger; Drossart, Dominik; Stock, Karl; Sobieraj, Anna M.; Eichenseher, Fritz; Loessner, Martin J.; Schmelcher, Mathias; Gerhardts, Anja; Goetz, Ulrike; Handel, Marina; Serr, Annerose; Haecker, Georg; Li, Jia; Specht, Mara; Koch, P.; Meyer, M Cobas; Tepper, Philipp; Rother, Raimund; Jehle, Michael; Wadle, Simon; Zengerle, Roland; Stetten, Felix von; Paust, Nils; Borst, Nadine

doi:10.1039/d0lc00294a

Cited by 46 publications

(41 citation statements)

References 57 publications

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“…We present a centrifugal, microfluidic, single-use cartridge (LabDisk) with 12 identical ddPCR units. In contrast to the state of the art, where until now only one-plex ddPCR assays have been automated by centrifugal microfluidics [9,[17][18][19], we present an increased multiplexing degree of four. This elevated degree of multiplexing is particularly relevant for cancer diagnostics, where a comprehensive overview of the mutation status of a patient must be generated from a limited sample amount [3].…”

Section: Introductionmentioning

confidence: 80%

“…In the recent past, centrifugal microfluidics developed into a mature technology which enabled the efficient miniaturization, parallelization and integration of assays for both sample preparation and analysis [13][14][15]. In the past, several groups have presented centrifugal, microfluidic cartridges that were able to emulsify a reaction mix [6,9,[16][17][18][19][20]. We present a centrifugal, microfluidic, single-use cartridge (LabDisk) with 12 identical ddPCR units.…”

Section: Introductionmentioning

confidence: 99%

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Centrifugal Microfluidic Integration of 4-Plex ddPCR Demonstrated by the Quantification of Cancer-Associated Point Mutations

et al. 2021

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We present the centrifugal microfluidic implementation of a four-plex digital droplet polymerase chain reaction (ddPCR). The platform features 12 identical ddPCR units on a LabDisk cartridge, each capable of generating droplets with a diameter of 82.7 ± 9 µm. By investigating different oil–surfactant concentrations, we identified a robust process for droplet generation and stabilization. We observed high droplet stability during thermocycling and endpoint fluorescence imaging, as is required for ddPCRs. Furthermore, we introduce an automated process for four-color fluorescence imaging using a commercial cell analysis microscope, including a customized software pipeline for ddPCR image evaluation. The applicability of ddPCRs is demonstrated by the quantification of three cancer-associated KRAS point mutations (G12D, G12V and G12A) in a diagnostically relevant wild type DNA background. The four-plex assay showed high sensitivity (3.5–35 mutant DNA copies in 15,000 wild type DNA copies) and linear performance (R² = 0.99) across all targets in the LabDisk.

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Section: Introductionmentioning

confidence: 80%

Section: Introductionmentioning

confidence: 99%

Centrifugal Microfluidic Integration of 4-Plex ddPCR Demonstrated by the Quantification of Cancer-Associated Point Mutations

et al. 2021

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“…A centrifugal microfluidic LabDisk device has recently been introduced for the on‐site and rapid detection of MRSA, which improves the disease screening and timely decision‐making process. [ 122 ] The proposed design also increases the antibiotic therapy efficiency via clear differentiation of methicillin‐sensitive and resistive bacteria.…”

Section: Potential Of Non‐invasive Sampling In Integrated Devicesmentioning

confidence: 99%

Integrated Devices for Non‐Invasive Diagnostics

Ates

Brunauer

Stetten

et al. 2021

Adv Funct Materials

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“Sample‐in‐answer‐out” type integrated diagnostic devices have been widely recognized as the ultimate solution to simplify testing across healthcare systems. Such systems are equipped with advanced fluidic, mechanical, chemical, biological, and electronic components to handle patient samples without any manual steps therefore have the potential to accelerate intervention and improve patient outcomes. In this regard, the combination of integrated devices and non‐invasive sampling has gained a substantial interest to further improve the comfort and safety of patients. In this Review, the pioneering developments in integrated diagnostics are covered and their potential in non‐invasive sampling is discussed. The key properties of possible sample types are highlighted by addressing their relevance for the clinical practice. Last, the factors affecting the transition of integrated devices from academia to the market are identified by analyzing the technology readiness levels of selected examples and alternative remedies are explored to increase the rate of survival during this transition.

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“…For the excitation, oligonucleotides specific for HTLV-1 were labelled with the green fluorescent dye, FAM, and excited with an LED at 465 nm. Further technical information is provided in [18].…”

Section: Functional Model Of the Poct Instrumentmentioning

confidence: 99%

“…In this work, we present a novel centrifugal POCT test for the automated, nucleic acid-based quantification of HTLV-1 directly from whole blood. Originally, this digital POC sample-to-answer platform was introduced for the rapid detection of species and resistance markers in methicillin-resistant Staphylococcus aureus (MRSA) on a single-cell level [18]. By adding additional sample pre-processing workflow steps, such as a beadbased extraction module, even more challenging laboratory protocols can be automated on the same digital POC sample-to-answer platform.…”

Section: Introductionmentioning

confidence: 99%

Point-of-Care System for HTLV-1 Proviral Load Quantification by Digital Mediator Displacement LAMP

Becherer

Hess

Frischmann³

et al. 2021

Micromachines

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This paper presents a universal point-of-care system for fully automated quantification of human T-cell lymphotropic virus type 1 (HTLV-1) proviral load, including genomic RNA, based on digital reverse RNA transcription and c-DNA amplification by MD LAMP (mediator displacement loop-mediated isothermal amplification). A disposable microfluidic LabDisk with pre-stored reagents performs automated nucleic acid extraction, reaction setup, emulsification, reverse transcription, digital DNA amplification, and quantitative fluorogenic endpoint detection with universal reporter molecules. Automated nucleic acid extraction from a suspension of HTLV-1-infected CD4+ T-lymphocytes (MT-2 cells) yielded 8 ± 7 viral nucleic acid copies per MT-2 cell, very similar to the manual reference extraction (7 ± 2 nucleic acid copies). Fully automated sample processing from whole blood spiked with MT-2 cells showed a comparable result of 7 ± 3 copies per MT-2 cell after a run time of two hours and 10 min.

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

Abstract: Automated point-of-care testing system for rapid single cell detection of MRSA directly from nasal swab samples.

Cited by 46 publications

References 57 publications

Centrifugal Microfluidic Integration of 4-Plex ddPCR Demonstrated by the Quantification of Cancer-Associated Point Mutations

Centrifugal Microfluidic Integration of 4-Plex ddPCR Demonstrated by the Quantification of Cancer-Associated Point Mutations

Integrated Devices for Non‐Invasive Diagnostics

Point-of-Care System for HTLV-1 Proviral Load Quantification by Digital Mediator Displacement LAMP

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