Graphene Oxide Conjugated Magnetic Beads for RNA Extraction

Abstract Genetic variants of the COVID-19 causative virus have been arising and circulating globally. In many countries especially in developing ones with a huge population, vaccination has become one of the major challenges. SARS-CoV-2 variants’ fast transmission rate has upsurge the COVID cases, leading to more stress on health systems. In the current COVID-19 scenario, there is the requirement of more adequate diagnostic approaches to check the COVID-19 spread. Out of many diagnostic approaches, a magnetic nanoparticle-based reverse transcription-polymerase chain reaction could be nontrivial. The use of magnetic nanoparticles to separate nucleic acid of SARS-CoV-2 from the patient samples and applied for detection is an easy and more effective way for COVID-19 patient detection. Herein, the magnetic nanoparticles are synthesized using the sol-gel autocombustion methods and then, successfully coated with biopolymer (chitosan) using ultra-sonication. Chitosan-coated nanoparticles are successfully integrated into the graphene oxide sheets to introduce carboxyl groups. Crystallite size calculation, morphological and magnetic studies of synthesized magnetic nanoparticles, and multifunctional magnetic nanoparticles are done using XRD, SEM, TEM, and VSM respectively. Besides the potentiality of the fabricated nanocomposites in RNA extraction protocol is also discussed with schematic representation.

Download Full-text

Detection of Tumor Marker Using ZnO@Reduced Graphene Oxide Decorated with Alkaline Phosphatase-Labeled Magnetic Beads

ACS Applied Nano Materials ◽

10.1021/acsanm.9b01797 ◽

2019 ◽

Vol 2 (12) ◽

pp. 7747-7754 ◽

Cited By ~ 2

Author(s):

Junxing Hao ◽

Caoling Li ◽

Kangbing Wu ◽

Chengguo Hu ◽

Nianjun Yang

Keyword(s):

Alkaline Phosphatase ◽

Graphene Oxide ◽

Tumor Marker ◽

Reduced Graphene Oxide ◽

Magnetic Beads ◽

Reduced Graphene

Download Full-text

Detection of both Hepatitis A Virus and Norwalk-Like Virus in Imported Clams Associated with Food-Borne Illness

Applied and Environmental Microbiology ◽

10.1128/aem.68.8.3914-3918.2002 ◽

2002 ◽

Vol 68 (8) ◽

pp. 3914-3918 ◽

Cited By ~ 77

Author(s):

David H. Kingsley ◽

Gloria K. Meade ◽

Gary P. Richards

Keyword(s):

Hepatitis A ◽

Magnetic Beads ◽

Hepatitis A Virus ◽

New York State ◽

Rna Extraction ◽

The United States ◽

Fecal Coliforms ◽

Most Probable Number ◽

Genotype I ◽

Probable Number

ABSTRACT Hepatitis A virus (HAV) and Norwalk-like virus (NLV) were detected by reverse transcription-PCR in clams imported into the United States from China. An epidemiological investigation showed that these clams were associated with five cases of Norwalk-like gastroenteritis in New York State in August 2000 (Food and Drug Administration Import Alert 16-50). They were labeled “cooked” but appeared raw. Viral RNA extraction was performed by using dissected digestive tissues rather than whole shellfish meats; this was followed by glycine buffer elution, polyethylene glycol precipitation, Tri-Reagent treatment, and purification of poly(A) RNA with magnetic beads coupled to poly(dT) oligonucleotides. We identified HAV and NLV as genotype I and genogroup II strains, respectively. Both viruses have high levels of homology to Asian strains. An analysis of fecal coliforms revealed a most-probable number of 93,000/100 g of clam meat, which is approximately 300-fold higher than the hygienic standard for shellfish meats.

Download Full-text

Development of a rapid pre-concentration protocol and a magnetic beads-based RNA extraction method for SARS-CoV-2 detection in raw municipal wastewater

Environmental Science Water Research & Technology ◽

10.1039/d1ew00539a ◽

2021 ◽

Author(s):

A. L. Parra-Guardado ◽

C. L. Sweeney ◽

E. K. Hayes ◽

B. F. Trueman ◽

Y. Huang ◽

...

Keyword(s):

Extraction Method ◽

Municipal Wastewater ◽

Magnetic Beads ◽

Rna Extraction ◽

Magnetic Bead ◽

Sample Treatment ◽

Raw Wastewater

We demonstrate the application of a rapid pre-concentration protocol and a magnetic bead-based RNA extraction method for the detection of SARS-CoV-2 RNA from raw wastewater without the need for extensive sample treatment.

Download Full-text

Rare SARS-CoV-2 antibody development in cancer patients

10.21203/rs.3.rs-71560/v1 ◽

2020 ◽

Author(s):

louisa hempel ◽

Jakob Molnar ◽

Sebastian Robert ◽

julia veloso ◽

Zeljka Trepotec ◽

...

Keyword(s):

Cancer Patients ◽

Multiple Testing ◽

Magnetic Beads ◽

Vaccine Development ◽

Rna Extraction ◽

Humoral Response ◽

Oncology Patients ◽

Previous Hypothesis ◽

Cast Doubt ◽

History Of

Abstract SARS-CoV-2 antibody development and immunity will be crucial for the further course of the pandemic. Until now, it has been assumed that patients who were infected with SARS-CoV-2 develop antibodies as it is the case with other coronaviruses, like MERS-CoV and SARS-CoV. In the present study, we analyzed the antibody development of 77 oncology patients 26 days after positive RT-qPCR testing for SARS-CoV-2. RT-qPCR and anti-SARS-CoV2-antibody methods from BGI (MGIEasy Magnetic Beads Virus DNA/RNA Extraction Kit) and Roche (Elecsys Anti-SARS-CoV-2 immunoassay) were used, respectively, according to the manufacturers’ specifications.Surprisingly, in only 6 of 77 individuals with a confirmed history of COVID-19 antibody development was detected. Despite of multiple testing, the remaining patients did not show measurable antibody concentrations in subsequent tests. These results undermine the previous hypothesis that SARS-CoV-2 infections are regularly associated with antibody development and cast doubt on the provided immunity to COVID-19. Understanding the adaptive and humoral response to SARS-CoV-2 will play a key-roll in vaccine development and gaining further knowledge on the pathogenesis.

Download Full-text

SARS-CoV-2 RNA extraction using magnetic beads for rapid large-scale testing by RT-qPCR and RT-LAMP

10.1101/2020.07.08.20147561 ◽

2020 ◽

Cited By ~ 1

Author(s):

Steffen Klein ◽

Thorsten G. Mueller ◽

Dina Khalid ◽

Vera Sonntag-Buck ◽

Anke-Mareil Heuser ◽

...

Keyword(s):

Large Scale ◽

Magnetic Beads ◽

Rna Extraction ◽

Magnetic Bead ◽

Viral Rna ◽

Detection Sensitivity ◽

Detection Methods ◽

Extraction Protocol ◽

Large Scale Testing ◽

Commercial Kits

Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and alternative, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric RT-LAMP using N primers, as well as RT-qPCR using E gene primers showing that the here presented RNA extraction protocol can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.

Download Full-text

Electroporation and lysis of marine microalga Karenia brevis for RNA extraction and amplification

Journal of The Royal Society Interface ◽

10.1098/rsif.2010.0445 ◽

2010 ◽

Vol 8 (57) ◽

pp. 601-608 ◽

Cited By ~ 18

Author(s):

M. M. Bahi ◽

M.-N. Tsaloglou ◽

M. Mowlem ◽

H. Morgan

Keyword(s):

Magnetic Beads ◽

Point Of Care ◽

Harmful Algal Bloom ◽

Rna Extraction ◽

Microfluidic System ◽

Karenia Brevis ◽

Integrated System ◽

Marine Microalga ◽

Point Of Care Diagnostics ◽

Motile Cells

We describe here a simple device for dielectrophoretic concentration of marine microalga Karenia brevis non-motile cells, followed by electric field-mediated lysis for RNA extraction. The lysate was purified using magnetic beads and pure RNA extracted. RNA quality was assessed off-chip by nucleic acid sequence-based amplification and the optimum conditions for lysis were determined. This procedure will form part of an integrated microfluidic system that is being developed with sub-systems for performing cell concentration and lysis, RNA extraction/purification and real-time quantitative RNA detection. The integrated system and its components could be used for a large range of applications including in situ harmful algal bloom detection, transcriptomics and point-of-care diagnostics.

Download Full-text

High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA v2

10.17504/protocols.io.b2mkqc4w ◽

2021 ◽

Cited By ~ 1

Author(s):

Aaron Topol ◽

marlene.wolfe not provided ◽

Krista Wigginton ◽

Bradley White ◽

Alexandria B Boehm

Keyword(s):

Nucleic Acids ◽

High Throughput ◽

Magnetic Beads ◽

Rna Extraction ◽

Poly Vinyl Alcohol ◽

Enzymatic Reactions ◽

Efficient Removal ◽

Dna And Rna ◽

Pcr Inhibitor ◽

Wastewater Samples

Please note that while this protocol is for TNA extraction using the Perkin Elmer Chemagic 360, RNA extraction with resuspended solids from this protocol has been verified to perform well using the Kingfisher MagMax kit as another high throughput, automated option and two manual Qiagen kits - the All Prep Powerviral DNA/RNA Kit and the Qiamp Viral RNA Mini Kit. This process instruction describes the steps for purification of nucleic acids from wastewater solids and preparation for downstream quantitative analysis with Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR). Due to the large quantities of substances that have inhibitory effects on PCR in wastewater samples, a subsequent PCR inhibitor removal step is required after nucleic acid purification. Both steps of the process are carried out in a 96-well plate format. This method uses the resuspended solids generated using this protocol: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. RNA purification is carried out using a kit optimized for the purification of viral on for the Perkin Elmer Chemagic 360. Although only RNA is used in the downstream applications from this protocol, DNA is also eluted in this process. A crucial component of the purification kit are the magnetic particles coated with poly vinyl alcohol (M-PVA Magnetic Beads) which have a hydrophilic surface giving them an affinity for nucleic acids but not many other biological molecules. The workflow involves binding nucleic acids in a sample to the beads which are then transferred through a series of wash buffers to remove debris with a robotic head with magnetic rods. The OneStep PCR Inhibitor Removal Kits are PCR inhibitor clean up kits that contain all the components needed for efficient removal of contaminants that can inhibit downstream enzymatic reactions (e.g. PCR and RT) from DNA and RNA preparations. The column matrices in these PCR inhibitor clean up kits have been specifically designed for the efficient removal of polyphenolic compounds, humic/fulvic acids, tannins, melanin, etc. from the most impure DNA and RNA preparations. This process instruction applies to extraction of RNA from wastewater samples using the Chemagic™ Viral DNA/RNA 300 Kit H96 for the Perkin Elmer Chemagic 360 followed by PCR Inhibitor Removal with the Zymo OneStep-96 PCR Inhibitor Removal Kit.

Download Full-text