Semi-Automated Extraction of Viral RNA using the MagMax Viral Pathogen (MVPII) 96 Kit for SARS-CoV-2 Detection v1 (protocols.io.brcvm2w6)

Dengue virus (DENV) is the most significant mosquito-borne viral pathogen in the world and is the cause of dengue fever. The DENV RNA-dependent RNA polymerase (RdRp) is conserved among the four viral serotypes and is an attractive target for antiviral drug development. During initiation of viral RNA synthesis, the polymerase switches from a “closed” to “open” conformation to accommodate the viral RNA template. Inhibitors that lock the “closed” or block the “open” conformation would prevent viral RNA synthesis. Herein, we describe a screening campaign that employed two biochemical assays to identify inhibitors of RdRp initiation and elongation. Using a DENV subgenomic RNA template that promotes RdRp de novo initiation, the first assay measures cytosine nucleotide analogue (Atto-CTP) incorporation. Liberated Atto fluorophore allows for quantification of RdRp activity via fluorescence. The second assay uses the same RNA template but is label free and directly detects RdRp-mediated liberation of pyrophosphates of native ribonucleotides via liquid chromatography–mass spectrometry. The ability of inhibitors to bind and stabilize a “closed” conformation of the DENV RdRp was further assessed in a differential scanning fluorimetry assay. Last, active compounds were evaluated in a renilla luciferase–based DENV replicon cell-based assay to monitor cellular efficacy. All assays described herein are medium to high throughput, are robust and reproducible, and allow identification of inhibitors of the open and closed forms of DENV RNA polymerase.

Download Full-text

Automated Extraction of Viral-Pathogen RNA and DNA for High-Throughput Quantitative Real-Time PCR

Journal of Clinical Microbiology ◽

10.1128/jcm.43.11.5541-5546.2005 ◽

2005 ◽

Vol 43 (11) ◽

pp. 5541-5546 ◽

Cited By ~ 53

Author(s):

K. Beuselinck ◽

M. van Ranst ◽

J. van Eldere

Keyword(s):

Real Time ◽

High Throughput ◽

Real Time Pcr ◽

Automated Extraction ◽

Quantitative Real Time Pcr ◽

Viral Pathogen ◽

Rna And Dna

Download Full-text

Identification of a Natural Viral RNA Motif That Optimizes Sensing of Viral RNA by RIG-I

mBio ◽

10.1128/mbio.01265-15 ◽

2015 ◽

Vol 6 (5) ◽

Cited By ~ 24

Author(s):

Jie Xu ◽

Xiomara Mercado-López ◽

Jennifer T. Grier ◽

Won-keun Kim ◽

Lauren F. Chun ◽

...

Keyword(s):

Rna Viruses ◽

Antiviral Immunity ◽

Antiviral Response ◽

Viral Rna ◽

Immune Recognition ◽

Immunostimulatory Activity ◽

Viral Pathogen ◽

Double Stranded Rna ◽

Rna Motif ◽

Viral Recognition

ABSTRACTStimulation of the antiviral response depends on the sensing of viral pathogen-associated molecular patterns (PAMPs) by specialized cellular proteins. During infection with RNA viruses, 5′-di- or -triphosphates accompanying specific single or double-stranded RNA motifs trigger signaling of intracellular RIG-I-like receptors (RLRs) and initiate the antiviral response. Although these molecular signatures are present during the replication of many viruses, it is unknown whether they are sufficient for strong activation of RLRs during infection. Immunostimulatory defective viral genomes (iDVGs) from Sendai virus (SeV) are among the most potent natural viral triggers of antiviral immunity. Here we describe an RNA motif (DVG70-114) that is essential for the potent immunostimulatory activity of 5′-triphosphate-containing SeV iDVGs. DVG70-114enhances viral sensing by the host cell independently of the long stretches of complementary RNA flanking the iDVGs, and it retains its stimulatory potential when transferred to otherwise inert viral RNA.In vitroanalysis showed that DVG70-114augments the binding of RIG-I to viral RNA and promotes enhanced RIG-I polymerization, thereby facilitating the onset of the antiviral response. Together, our results define a new natural viral PAMP enhancer motif that promotes viral recognition by RLRs and confers potent immunostimulatory activity to viral RNA.IMPORTANCEA discrete group of molecular motifs, including 5′-triphosphates associated with double-stranded RNA, have been identified as essential for the triggering of antiviral immunity. Most RNA viruses expose these motifs during their replication; however, successful viruses normally evade immune recognition and replicate to high levels before detection, indicating that unknown factors drive antiviral immunity. DVGs from SeV are among the most potent natural viral stimuli of the antiviral response known to date. These studies define a new natural viral motif present in DVGs that maximizes viral recognition by the intracellular sensor RIG-I, allowing fast and strong antiviral responses even in the presence of viral-encoded immune antagonists. This motif can be harnessed to increase the immunostimulatory potential of otherwise inert viral RNAs and represents a novel immunostimulatory enhancer that could be used in the development of vaccine adjuvants and antivirals.

Download Full-text

A streamlined approach to rapidly detect SARS-CoV-2 infection, avoiding RNA purification

10.1101/2020.04.06.20054114 ◽

2020 ◽

Cited By ~ 11

Author(s):

Stefania Marzinotto ◽

Catia Mio ◽

Adriana Cifù ◽

Roberto Verardo ◽

Corrado Pipan ◽

...

Keyword(s):

Nucleic Acid ◽

Molecular Analysis ◽

Rna Extraction ◽

Extraction Methods ◽

Viral Rna ◽

Proteinase K ◽

Automated Extraction ◽

Inexpensive Method ◽

Rna Purification ◽

Extraction Step

AbstractIn the current pandemic, the presence of SARS-CoV-2 RNA in samples by nucleic acid (NA) molecular analysis is the only method available to diagnose COVID-19 disease and to assess patients’ infectiveness. Recently, the demand for laboratory reagents has greatly increased; in particular, there is a worldwide shortage of RNA extraction kits. Here, we describe a fast, simple and inexpensive method for the detection of SARS CoV-2 RNA, which includes a pretreatment with Proteinase K and a heating-cooling cycle before the amplification. This method bypasses the RNA extraction step; it leads to a higher amount of available viral RNA compared to the automated extraction methods, and generates the same profile in the subsequent amplification phase.

Download Full-text

Standardized preservation, extraction and quantification techniques for detection of fecal SARS-CoV-2 RNA

Nature Communications ◽

10.1038/s41467-021-25576-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Aravind Natarajan ◽

Alvin Han ◽

Soumaya Zlitni ◽

Erin F. Brooks ◽

Summer E. Vance ◽

...

Keyword(s):

Respiratory Infection ◽

Viral Rna ◽

Viral Pathogen ◽

Patient Health ◽

Reliable Quantification

AbstractPatients with COVID-19 shed SARS-CoV-2 RNA in stool, sometimes well after their respiratory infection has cleared. This may be significant for patient health, epidemiology, and diagnosis. However, methods to preserve stool, and to extract and quantify viral RNA are not standardized. We test the performance of three preservative approaches at yielding detectable SARS-CoV-2 RNA: the OMNIgene-GUT kit, Zymo DNA/RNA shield kit, and the most commonly applied, storage without preservative. We test these in combination with three extraction kits: QIAamp Viral RNA Mini Kit, Zymo Quick-RNA Viral Kit, and MagMAX Viral/Pathogen Kit. We also test the utility of ddPCR and RT-qPCR for the reliable quantification of SARS-CoV-2 RNA from stool. We identify that the Zymo DNA/RNA preservative and the QiaAMP extraction kit yield more detectable RNA than the others, using both ddPCR and RT-qPCR. Taken together, we recommend a comprehensive methodology for preservation, extraction and detection of RNA from SARS-CoV-2 and other coronaviruses in stool.

Download Full-text

Automated Extraction of Plant Viral RNA Using the Nucleic Acid Instrument Based on Magnetic Nanobeads

Applied Mechanics and Materials ◽

10.4028/www.scientific.net/amm.303-306.646 ◽

2013 ◽

Vol 303-306 ◽

pp. 646-649

Author(s):

Yong Jie Dou ◽

Xiao Li Zhao ◽

Jian Sheng Xu ◽

Cong Liang Deng ◽

Leng Nie ◽

...

Keyword(s):

Nucleic Acid ◽

Plant Virus ◽

Rna Extraction ◽

Viral Rna ◽

Acid Extraction ◽

Automated Extraction ◽

Nucleic Acid Extraction ◽

Pcr Assay ◽

Initial Concentration ◽

Magnetic Nanobeads

In this paper, an automated nucleic acid extraction instrument based on magnetic nanobeads is described. Also, a prototype is designed and constructed. Some plant viral RNA extraction experiments were done using the platform. And the experimental results demonstrated that the instrument could be successfully used for extraction of plant viral RNA，and the extracted RNA was successfully used in an automated PCR assay for the detection of plant virus. The Ct values of CGMMV RNA, LSV RNA and ArMV RNA at the initial concentration were 12.51, 17.81 and 23.48.

Download Full-text

Standardized and optimized preservation, extraction and quantification techniques for detection of fecal SARS-CoV-2 RNA

10.1101/2021.04.10.21255250 ◽

2021 ◽

Author(s):

Aravind Natarajan ◽

Alvin Han ◽

Soumaya Zlitni ◽

Erin F Brooks ◽

Summer E Vance ◽

...

Keyword(s):

Clinical Trial ◽

Viral Rna ◽

Detection Methods ◽

Common Condition ◽

Viral Pathogen ◽

Standardized Protocol ◽

Patient Health ◽

Stool Samples ◽

Key Features ◽

Rna Concentration

COVID-19 patients shed SARS-CoV-2 viral RNA in their stool, sometimes well after they have cleared their respiratory infection. This feature of the disease may be significant for patient health, epidemiology, and diagnosis. However, to date, methods to preserve stool samples from COVID patients, and to extract and quantify viral RNA concentration have yet to be optimized. We sought to meet this urgent need by developing and benchmarking a standardized protocol for the fecal detection of SARS-CoV-2 RNA. We test three preservative conditions for their ability to yield detectable SARS-CoV-2 RNA: OMNIgene-GUT, Zymo DNA/RNA shield kit, and the most common condition, storage without any preservative. We test these in combination with three extraction kits: the QIAamp Viral RNA Mini Kit, Zymo Quick-RNA Viral Kit, and MagMAX Viral/Pathogen Kit. Finally, we also test the utility of two detection methods, ddPCR and RT-qPCR, for the robust quantification of SARS-CoV-2 viral RNA from stool. We identify that the Zymo DNA/RNA shield collection kit and the QiaAMP viral RNA mini kit yield more detectable RNA than the others, using both ddPCR and RT-qPCR assays. We also demonstrate key features of experimental design including the incorporation of appropriate controls and data analysis, and apply these techniques to effectively extract viral RNA from fecal samples acquired from COVID-19 outpatients enrolled in a clinical trial. Finally, we recommend a comprehensive methodology for future preservation, extraction and detection of RNA from SARS-CoV-2 and other coronaviruses in stool.

Download Full-text