A Rapid and Automated Device for Purifying Nucleic Acids

Uehara, Masayuki; Hanamura, Masato; Yamada, Kiyohito; Yamaguchi, Akemi; Murayama, Tetsuo; Saito, Yuji; Idegami, Kotaro; Honda, Takayuki

doi:10.2116/analsci.32.371

Cited by 9 publications

(8 citation statements)

References 12 publications

(13 reference statements)

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“…Over the last ten years, with the advent of isothermal amplification technologies (2), a new area of research has opened, raising hope to perform NAATs at the point of care, with portable devices much cheaper than extraction/PCR platforms and showing comparable performances. A new generation of NAATs has grown along the years, in laboratories, often coupling isothermal amplification to paper microfluidics, a field pioneered by G.Whitesides (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16).…”

Section: Introductionmentioning

confidence: 99%

Highly performing point-of-care molecular testing for SARS-CoV-2 with RNA extraction and isothermal amplification

Garneret

Coz

Martin

et al. 2020

Preprint

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In order to respond to the urgent request of massive testing, developed countries perform nucleic acid amplification tests (NAAT) of SARS-CoV-2 in centralized laboratories. Real-time RT - PCR (Reverse transcription - Polymerase Chain Reaction) is used to amplify the viral RNA and enable its detection. Although PCR is 37 years old, it is still considered, without dispute, as the gold standard. PCR is an efficient process, but the complex engineering required for automated RNA extraction and temperature cycling makes it incompatible for use in point of care settings. In the present work, by harnessing progress made in the past two decades in DNA amplification, microfluidics and membrane technologies, we succeeded to create a portable test, in which SARS-CoV-2 RNA is extracted, amplified isothermally by RT - LAMP (Loop-mediated Isothermal Amplification), and detected using intercalating dyes or highly fluorescent probes. Depending on the viral load, the detection takes between twenty minutes and one hour. Using pools of naso-pharyngal clinical samples, we estimated a sensitivity comparable to RT-qPCR (up to a Cycle threshold of 39, equivalent to <0.1 TCID50 per mL) and a 100% specificity, for other human coronaviruses and eight respiratory viruses currently circulating in Europe. We designed and fabricated an easy-to-use portable device called COVIDISC to carry out the test at the point of care. The low cost of the materials along with the absence of complex equipment paves the way towards a large dissemination of this device. The perspective of a reliable SARS-CoV-2 point of care detection, highly performing, that would deliver on-site results in less than one hour opens up a new efficient approach to manage the pandemics.

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Section: Introductionmentioning

confidence: 99%

Highly performing point-of-care molecular testing for SARS-CoV-2 with RNA extraction and isothermal amplification

Garneret

Coz

Martin

et al. 2020

Preprint

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“…2c). 9 The pushing system controls the cartridge plunger and pushes the reaction solution plug and oil plug into the PCR tube (Figs. 3a, 3b, and 3c).…”

Section: Device Constructionmentioning

confidence: 99%

“…We used a transport speed of 2.0 mm s -1 and a vibration frequency of 4.0 Hz. 9 Nucleic acids bound to the magnetic beads are thus washed into the washing solution and eluted in the reaction solution (Fig. 2b).…”

Section: Operationmentioning

confidence: 99%

“…Although other methods are available for extracting DNA or RNA, we used a combination of superparamagnetic beads and magnetic force. 9 Several bioassays have also already demonstrated the utility of magnetic beads in microfluidics. [10][11][12][13] Connection of such a cartridge extraction and purification system 9 to our novel droplet-real-time-PCR system 14 allows PCR tests to be performed simply in high-throughput assays by simple pouring of the sample into the cartridge.…”

Section: Introductionmentioning

confidence: 99%

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Development of a Novel and Rapid Fully Automated Genetic Testing System

Uehara

2016

ANAL. SCI.

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We have developed a rapid genetic testing system integrating nucleic acid extraction, purification, amplification, and detection in a single cartridge. The system performs real-time polymerase chain reaction (PCR) after nucleic acid purification in a fully automated manner. RNase P, a housekeeping gene, was purified from human nasal epithelial cells using silica-coated magnetic beads and subjected to real-time PCR using a novel droplet-real-time-PCR machine. The process was completed within 13 min. This system will be widely applicable for research and diagnostic uses.

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“…Recently, our lab [20, 21] and others [22–27] have shown that instead of pipetting liquids in and out of a tube that contains the PMPs, there is an advantage to using stationary microfluidics whereby different buffers needed for extraction are located in fixed positions, and an external magnet transfers the PMPs between the fluids through an immiscible phase filter (IPF) made up of a layer of oil or liquid wax that is immiscible with both aqueous solutions. In addition to eliminating the need for fluid pumping or pipetting, moving PMPs instead of fluids simplifies the instrumentation and consumable test cartridge required for nucleic acid extraction.…”

Section: Introductionmentioning

confidence: 99%

Immiscible phase filter extraction and equivalent amplification of genotypes 1–6 of hepatitis C RNA: The building blocks for point-of-care diagnosis

Neto

Butzler

Reed

et al. 2017

Journal of Virological Methods

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The lack of hepatitis C virus (HCV) diagnostic tests designed for use in decentralized settings is a major obstacle for providing access to treatment and prevention services particularly in low and middle income countries. Here we describe the development and validation of two building blocks of the HCV Quant Assay, a test in development for point-of-care use: 1) an RT-qPCR assay with noncompetitive internal control that equivalently detects the 6 major HCV genotypes and 2) an automated sample prep method using immiscible phase filter technology. This novel assay has wide dynamic range of HCV quantification and a limit of detection of 30 IU/ml with 200 μl specimen volume. In a preliminary study of 61 clinical specimens, the HCV Quant Assay demonstrated 100% sensitivity and specificity and gave comparable viral load results across 4 logs of IU/ml when compared to the Abbott RealTime HCV Assay.

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A Rapid and Automated Device for Purifying Nucleic Acids

Cited by 9 publications

References 12 publications

Highly performing point-of-care molecular testing for SARS-CoV-2 with RNA extraction and isothermal amplification

Highly performing point-of-care molecular testing for SARS-CoV-2 with RNA extraction and isothermal amplification

Development of a Novel and Rapid Fully Automated Genetic Testing System

Immiscible phase filter extraction and equivalent amplification of genotypes 1–6 of hepatitis C RNA: The building blocks for point-of-care diagnosis

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