An Automated and Miniaturized Rotating-Disk Device for Rapid Nucleic Acid Extraction

The result of molecular diagnostic and detection greatly dependent on the quality and integrity of the isolated nucleic acid. In this work, we developed an automated miniaturized nucleic acid extraction device based on magnetic beads method, consisting of four components including a sample processing disc and its associated rotary power output mechanism, a pipetting module, a magnet module and an external central controller to enable a customizable and automated robust nucleic acid sample preparation. The extracted nucleic acid using 293T cells were verified using real-time polymerase chain reaction (PCR) and the data implies a comparable efficiency to a manual process, with the advantages of performing a flexible, time-saving (~10 min), and simple nucleic acid sample preparation.

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Effects of diverse materials-based methods on DNA extraction for Clostridium difficle from stool samples

Materials Express ◽

10.1166/mex.2019.1520 ◽

2019 ◽

Vol 9 (5) ◽

pp. 509-516 ◽

Cited By ~ 3

Author(s):

Ziqi Xiao ◽

Gaojian Yang ◽

Deng Yan ◽

Song Li ◽

Zhu Chen ◽

...

Keyword(s):

Nucleic Acid ◽

Dna Extraction ◽

Magnetic Beads ◽

Diagnostic Technique ◽

Proteinase K ◽

Molecular Diagnostic ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Spin Column ◽

Stool Samples

Nosocomial infections, including Clostridium difficile infection (CDI), and their fatality rates have increased in the past few decades. Despite emerging molecular diagnostic technologies with rapid, accurate outcomes, nucleic acid extraction from stool samples remains the first limiting step before downstream applications. Commercial nucleic acid extraction kits greatly decrease labor and time requirements, and also provide nucleic acid preparations with higher quality and purity for enzyme digestion analysis or genotyping. The magnetic bead based technique is a novel method compared with the conventional spin-column method, and currently has widespread use in nucleic acid extraction. We evaluated five DNA extraction kits with magnetic beads using materials with various properties (particle size, concentration of magnetic beads, grinding beads) and reagents (proteinase K, lysozyme, isopropanol, and absolute ethanol) to determine the cost, hands-on time, number of essential operations, and quality and purity of the DNA preparations, compared with those obtained using the QIAamp Fast DNA Stool Mini Kit. The six DNA extraction kits yielded A260/280 ratios ranging from 0.85 to 1.9 (average 1.57), and concentrations from 3.70 to 108.09 ng/μL (average 34.64 ng/μL). All the DNA samples had acceptable downstream application effects, except for those obtained using the TIANGEN Magnetic Soil and Stool DNA Kit. However, gel electrophoresis analysis of the DNA samples resulted in a light strip on the gel, indicating that the proteinaceous contaminant may not have been removed completely. A rapid and accurate molecular diagnostic technique could allow for more suitable treatment and prognosis outcomes for inpatients, depending, in large part, on the quality and purity of DNA preparations, which are frequently neglected. Our study focused on the quality of commercial kits with a primary focus on the treatment of stool samples and molecular diagnostic applications.

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Design and Application of a High-Throughput Sample Processing Module Based on Magnetic Beads

Nanoscience and Nanotechnology Letters ◽

10.1166/nnl.2018.2628 ◽

2018 ◽

Vol 10 (3) ◽

pp. 320-328 ◽

Cited By ~ 4

Author(s):

Yile Fang ◽

Yanqi Wu ◽

Pei Liao ◽

Zhu Chen ◽

Hui Chen ◽

...

Keyword(s):

Nucleic Acid ◽

Sample Preparation ◽

Magnetic Separation ◽

High Throughput ◽

Magnetic Beads ◽

Performance Evaluations ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Dc Power ◽

Processing Module

One of key challenges in diagnosis of infectious diseases is collecting a large quantity of purified nucleic acids from pathogens immediately. To address this challenge, a high-throughput magnetic separation module and a heating and vibrating module were developed to optimize the operation of biological sample preparation (like nucleic acid extraction) on our home-built liquid handing system. Both of the two modules have 4 working locations for Biomolecular Screening (BS) standard labware, and three functions (magnetic separation, heating and vibrating) were elaborately integrated into the two modules. Each module has its own core control circuit and a unique interface port containing a 24 V DC power supply connector and a CAN bus communication connector. When equipping with the two modules, the home-built liquid handing system transformed into an integrated automatic sample preparation workstation. Performance evaluations were carried out on the two modules and finally a nucleic acid extraction of human's whole blood was carried out on the workstation. Results showed that the two modules could cooperate well with the liquid handing system, and the workstation exhibited its ability for high-throughput sample preparation.

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Functional Magnetite Nanoparticle: A Review on the Particles Lysis and Nucleic Acid Separation

Journal of Metastable and Nanocrystalline Materials ◽

10.4028/www.scientific.net/jmnm.33.13 ◽

2021 ◽

Vol 33 ◽

pp. 13-27

Author(s):

Puspita Nurlilasari ◽

Camellia Panatarani ◽

Mia Miranti ◽

Savira Ekawardhani ◽

Ferry Faizal ◽

...

Keyword(s):

Nucleic Acid ◽

Magnetite Nanoparticles ◽

Magnetic Beads ◽

Functional Materials ◽

Cell Lysis ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Coating Materials ◽

Magnetite Nanoparticle ◽

Separation Cell

The functional magnetite nanoparticles are one of the most important functional materials for nucleic acid separation. Cell lysis and magnetic separation are two essential steps involve in optimizing nucleic acid extraction using the magnetic beads method. Many coating materials, coupling agents, chemical cell lysis, and several methods have been proposed to produce the specific desired properties for nucleic acid extraction. The important properties, such as biocompatibility, stability, linking ability, hydrophobicity, and biodegradable, were considered. The appropriate coating material of magnetite core and coupling agent are necessary to give biomolecules a possibility to link with each other through chemical conjugation. In this review, progress in functional magnetite nanoparticles to optimize the high binding performance in nucleic acid extraction is discussed.

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Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation

PLoS Biology ◽

10.1371/journal.pbio.3000107 ◽

2019 ◽

Vol 17 (1) ◽

pp. e3000107 ◽

Cited By ~ 36

Author(s):

Phil Oberacker ◽

Peter Stepper ◽

Donna M. Bond ◽

Sven Höhn ◽

Jule Focken ◽

...

Keyword(s):

Nucleic Acid ◽

High Throughput ◽

Magnetic Beads ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Open Platform

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Rapid and automated sample preparation for nucleic acid extraction on a microfluidic CD (compact disk)

10.1117/12.646932 ◽

2006 ◽

Cited By ~ 3

Author(s):

Jitae Kim ◽

Horacio Kido ◽

Jim V. Zoval ◽

Dominic Gagné ◽

Régis Peytavi ◽

...

Keyword(s):

Nucleic Acid ◽

Sample Preparation ◽

Acid Extraction ◽

Compact Disk ◽

Nucleic Acid Extraction ◽

Automated Sample Preparation

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Gargle-Direct: Extraction-Free Detection of SARS-CoV-2 using Real-time PCR (RT-qPCR) of Saline Gargle Rinse Samples

10.1101/2020.10.09.20203430 ◽

2020 ◽

Author(s):

Vijay J. Gadkar ◽

David M. Goldfarb ◽

Virginia Young ◽

Nicole Watson ◽

Linda Hoang ◽

...

Keyword(s):

Nucleic Acid ◽

Standard Method ◽

Molecular Diagnostic ◽

Heat Inactivation ◽

Acid Extraction ◽

Analytical Sensitivity ◽

Sample Type ◽

Mouth Rinse ◽

Nucleic Acid Extraction ◽

Single Tube

ABSTRACTBackgroundSaline mouth rinse/gargle samples have recently been shown to be a suitable option for swab-independent self-collection for SARS-CoV-2 diagnosis. We sought to evaluate a simplified process for direct reverse transcriptase PCR (RT-qPCR) testing of this novel sample type and to compare performance with routine RT-qPCR using automated nucleic acid extraction.MethodsClinical saline mouth rinse/gargle samples were subjected to automated nucleic acid extraction (“standard method”), followed by RT-qPCR using three assays including the FDA authorized US-CDC’s N1/N2 assay, which was the reference standard for determining sensitivity/specificity. For extraction-free workflow, an aliquot of each gargle sample underwent viral heat inactivation at 65 °C for 20 minutes followed by RT-qPCR testing, without an intermediate extraction step. An in-house validated RT-qPCR lab developed test (LDT), targeting the SARS-CoV-2’s S/ORF8 genes (SORP triplex assay) and the N1/N2 US-CDC assay was used to evaluate the extraction-free protocol. To improve the analytical sensitivity, we developed a single-tube hemi-nested (STHN) version of the SORP triplex assay.ResultsA total of 38 SARS-CoV-2 positive and 75 negative saline mouth rinse/gargle samples were included in this evaluation. A 100% concordance in detection rate was obtained between the standard method and the extraction-free approach for the SORP assay. An average increase of +2.63 to +5.74 of the cycle threshold (CT) values was observed for both the SORP and N1/N2 assay when extraction-free was compared between the standard method. The average ΔCT [ΔCT=CT(Direct PCR)-CT(Extracted RNA)], for each of the gene targets were: S (ΔCT= +4.24), ORF8 (ΔCT=+2.63), N1 (ΔCT=+2.74) and N2 (ΔCT=+5.74). The ΔCT for the STHN SORP assay was +1.51 and −2.05 for the S and ORF8 targets respectively, when extracted method was compared to the standard method.ConclusionOur Gargle-Direct SARS-CoV-2 method is operationally simple, minimizes pre-analytical sample processing and is potentially implementable by most molecular diagnostic laboratories. The empirical demonstration of single-tube hemi-nested RT-qPCR, to specifically address and alleviate the widely-acknowledged problem of reduced analytical sensitivity of detection of extraction-free templates, should help diagnostic laboratories in choosing Gargle-Direct protocol for high-throughput testing.

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Towards a “Sample-In, Answer-Out” Point-of-Care Platform for Nucleic Acid Extraction and Amplification: Using an HPV E6/E7 mRNA Model System

Journal of Oncology ◽

10.1155/2012/905024 ◽

2012 ◽

Vol 2012 ◽

pp. 1-12 ◽

Cited By ~ 16

Author(s):

Anja Gulliksen ◽

Helen Keegan ◽

Cara Martin ◽

John O'Leary ◽

Lars A. Solli ◽

...

Keyword(s):

Nucleic Acid ◽

Sample Preparation ◽

Clinical Sample ◽

Point Of Care ◽

Fluorescent Detection ◽

Clinical Samples ◽

Microfluidic Channels ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Hpv E6

The paper presents the development of a “proof-of-principle” hands-free and self-contained diagnostic platform for detection of human papillomavirus (HPV) E6/E7 mRNA in clinical specimens. The automated platform performs chip-based sample preconcentration, nucleic acid extraction, amplification, and real-time fluorescent detection with minimal user interfacing. It consists of two modular prototypes, one for sample preparation and one for amplification and detection; however, a common interface is available to facilitate later integration into one single module. Nucleic acid extracts (n=28) from cervical cytology specimens extracted on the sample preparation chip were tested using the PreTect HPV-Proofer and achieved an overall detection rate for HPV across all dilutions of 50%–85.7%. A subset of 6 clinical samples extracted on the sample preparation chip module was chosen for complete validation on the NASBA chip module. For 4 of the samples, a 100% amplification for HPV 16 or 33 was obtained at the 1 : 10 dilution for microfluidic channels that filled correctly. The modules of a “sample-in, answer-out” diagnostic platform have been demonstrated from clinical sample input through sample preparation, amplification and final detection.

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Multifunctional Device for Nucleic Acid Extraction Based on Magnetic Separation and Its Co-Working with Liquid Handling System for High Throughput Sample Preparation

Journal of Nanoscience and Nanotechnology ◽

10.1166/jnn.2016.12583 ◽

2016 ◽

Vol 16 (7) ◽

pp. 6919-6924 ◽

Cited By ~ 3

Author(s):

Yanqi Wu ◽

Hui Chen ◽

Zhu Chen ◽

Libo Nie ◽

Bin Liu ◽

...

Keyword(s):

Nucleic Acid ◽

Sample Preparation ◽

Magnetic Separation ◽

High Throughput ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Handling System ◽

Liquid Handling ◽

Multifunctional Device

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Parallel RNA extraction using magnetic beads and a droplet array

Lab on a Chip ◽

10.1039/c4lc01111b ◽

2015 ◽

Vol 15 (4) ◽

pp. 1059-1065 ◽

Cited By ~ 24

Author(s):

Xu Shi ◽

Chun-Hong Chen ◽

Weimin Gao ◽

Shih-hui Chao ◽

Deirdre R. Meldrum

Keyword(s):

Nucleic Acid ◽

Magnetic Beads ◽

Rna Extraction ◽

Lab On A Chip ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Droplet Array

Nucleic acid extraction is a necessary step for most genomic/transcriptomic analyses, but it often requires complicated mechanisms to be integrated into a lab-on-a-chip device.

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