scholarly journals Monitoring SARS-CoV-2 in sewage: toward sentinels with analytical accuracy

2021 ◽  
Author(s):  
David Calderón-Franco ◽  
Laura Orschler ◽  
Susanne Lackner ◽  
Shelesh Agrawal ◽  
David G. Weissbrodt
Keyword(s):  
Magnetic Beads ◽  
Rna Isolation ◽  
Viral Rna ◽  
Clinical Patient ◽  
Rna Purification ◽  
Viral Particles ◽  
Diethylaminoethyl Cellulose ◽  
Isolation Methods ◽  
Analytical Accuracy ◽  
Epidemiology Studies

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemia has been one of the most difficult challenges humankind has recently faced. Wastewater-based epidemiology has emerged as a tool for surveillance and mitigation of potential viral outbreaks, circumventing biases introduced by clinical patient testing. Due to the situation urgency, protocols followed for isolating viral RNA from sewage were not adapted for such sample matrices. In parallel to their implementation for fast collection of data to sustain surveillance and mitigation decisions, molecular protocols need to be harmonized to deliver accurate, reproducible, and comparable analytical outputs. Here we studied analytical variabilities linked to viral RNA isolation methods from sewage. Three different influent wastewater volumes were used to assess the effect of filtered volumes (50, 100 or 500 mL) for capturing viral particles. Three different concentration strategies were tested by electronegative membranes, polyethersulfone membranes, and anion-exchange diethylaminoethyl cellulose columns. To compare the number of viral particles, different RNA isolation methods (column-based vs. magnetic beads) were compared. The effect of extra RNA purification steps and different RT-qPCR strategies (one step vs. two-step) were also evaluated. Results showed that the combination of 500 mL filtration volume through electronegative membranes and without multiple RNA purification steps (using column-based RNA purification) using two-step RT-qPCR avoided false negatives when basal viral load in sewage are present and yielded more consistent results during the surveillance done during the second-wave in Delft (The Hague area, The Netherlands). By paving the way for standardization of methods for the sampling, concentration and molecular detection of SARS-CoV-2 viruses from sewage, these findings can help water and health surveillance authorities to use and trust results coming from wastewater based epidemiology studies in order to anticipate SARS-CoV-2 outbreaks.

scholarly journals Inactivation of Human Norovirus in Contaminated Oysters and Clams by High Hydrostatic Pressure

2014 ◽  
Vol 80 (7) ◽  
pp. 2248-2253 ◽  
Author(s):  
Mu Ye ◽  
Xinhui Li ◽  
David H. Kingsley ◽  
Xi Jiang ◽  
Haiqiang Chen
Keyword(s):  
Hydrostatic Pressure ◽  
High Hydrostatic Pressure ◽  
Magnetic Beads ◽  
Viral Rna ◽  
Pressure Treatment ◽  
Rt Pcr ◽  
Human Norovirus ◽  
Porcine Gastric Mucin ◽  
Food Borne ◽  
Viral Particles

ABSTRACTHuman norovirus (NoV) is the most frequent causative agent of food-borne disease associated with shellfish consumption. In this study, the effect of high hydrostatic pressure (HHP) on inactivation of NoV was determined. Genogroup I.1 (GI.1) or genogroup II.4 (GII.4) NoV was inoculated into oyster homogenates and treated at 300 to 600 MPa at 25, 6, and 1°C for 5 min. After HHP, samples were treated with RNase and viral particles were extracted with porcine gastric mucin (PGM)-conjugated magnetic beads (PGM-MBs). Viral RNA was then quantified by real-time reverse transcription (RT)-PCR. Since PGM contains histo-blood group-like antigens, which can act as receptors for NoV, deficiency for binding to PGM is an indication of loss of infectivity of NoV. After binding to PGM-MBs, RT-PCR-detectable NoV RNA in oysters was reduced by 0.4 to >4 log10by HHP at 300 to 600 MPa. The GI.1 NoV was more resistant to HHP than the GII.4 NoV (P< 0.05). HHP at lower temperatures significantly enhanced the inactivation of NoV in oysters (P< 0.05). Pressure treatment was also conducted for clam homogenates. Treatment at 450 MPa at 1°C achieved a >4 log10reduction of GI.1 NoV in both oyster and clam homogenates. It is therefore concluded that HHP could be applied as a potential intervention for inactivating NoV in raw shellfish. The method of pretreatment of samples with RNase, extraction of viral particles using PGM-MB binding, and quantification of viral RNA using RT-PCR can be explored as a practical means of distinguishing between infectious and noninfectious NoV.

Spermatozoa input concentrations and RNA isolation methods on RNA yield and quality in bull (Bos taurus)

2015 ◽  
Vol 482 ◽  
pp. 32-39 ◽  
Author(s):  
Sivashanmugam Parthipan ◽  
Sellappan Selvaraju ◽  
Lakshminarayana Somashekar ◽  
Atul P. Kolte ◽  
Arunachalam Arangasamy ◽  
...  
Keyword(s):  
Bos Taurus ◽  
Rna Isolation ◽  
Yield And Quality ◽  
Isolation Methods

The Need for Harmonization in Detection of Human Noroviruses in Food

2013 ◽  
Vol 96 (5) ◽  
pp. 998-1005 ◽  
Author(s):  
Ambroos Stals ◽  
Mieke Uyttendaele ◽  
Els Van Coillie
Keyword(s):  
Food Safety ◽  
Real Time ◽  
Point Of View ◽  
Rna Purification ◽  
Copy Numbers ◽  
Viral Particles ◽  
Extraction Step ◽  
Foodborne Outbreaks ◽  
Automated Methods ◽  
Selection Of

Abstract Noroviruses (NoV) have been recognized worldwide as a leading cause of foodborne gastroenteritis over the last decade. A broad range of foods—shellfish, fresh produce, and ready-to-eat/catered foods—has been implicated in NoV foodborne outbreaks. The recognition of NoV as an important food pathogen has been aided by the development of sensitive molecular methods for detection of the NoV genome. However, despite advances, NoV detection is still hampered by several limitations. First, NoV detection can often only be implemented by expert laboratories due to the complexity of the virus extraction step, which in most protocols is cumbersome and labor-intensive. Moreover, a very wide selection of automated methods for virus extraction from foods is available, so selection of an adequate method is not straightforward. On the other hand, automated systems have been made available or the RNA purification and real-time RT-PCR (RT-qPCR) is considered the gold standard for detection of NoV. Second, correct interpretation of real-time PCR results is often difficult. From a technical point of view, the interpretation of the often nonsigmoidal amplification curves remains difficult, even for experts. From a food safety perspective, interpretation of very high Cq (or Ct) values—and thus, of low viral genomic copy numbers—is not straightforward, as RT-(q)PCR merely detects the presence of viral genomic material that is not necessarily linked to the presence of infectious viral particles. Despite efforts, both limitations have not completely resolved thus far. Harmonization may be a first step to comprehend and deal with these limitations. The current review provides an overview of a number of validated methods that have been published by food safety and other authorities.

scholarly journals Evaluation of RNA isolation methods for microRNA quantification in a range of clinical biofluids.

2020 ◽  
Author(s):  
Henk Roest ◽  
Jan N.M. IJzermans ◽  
Luc. J.W. van der Laan
Keyword(s):  
Rna Isolation ◽  
Sample Type ◽  
Cdna Synthesis ◽  
Inhibitory Compounds ◽  
Isolation Methods ◽  
Synthetic Mirnas ◽  
Mirna Detection ◽  
Endogenous Mirnas ◽  
Heparinase I ◽  
Bile Samples

Abstract BACKGROUND Extracellular microRNAs (miRNAs), released from cells into biofluids, have emerged as promising biomarkers for diagnostic and prognostic purposes. For the analysis of these cell-free miRNAs in various biofluids by RT-qPCR, several RNA isolation methods are available. However, not all methods are equally suitable for different biofluids. The aim of this study is to evaluate the potential of different RNA isolation methods in a range of clinical biofluid samples.METHODS Total RNA was isolated from serum, urine, bile, and graft preservation fluid (perfusate) using four different protocols: phenol-chloroform extraction in combination with a precipitation carrier, and three different column-based isolation methods. Co-purification of heparin, a known RT-qPCR inhibitor, was assessed using heparinase I during cDNA synthesis. Synthetic miRNAs, spiked-in during RNA workup (cel-miR-39) or during cDNA synthesis (cel-miR-54), and endogenous miRNAs were quantified using RT-qPCR.RESULTS Recovery of cel-miR-39 significantly differed between methods with miRNeasy columns providing the best overall recovery in the four biofluids tested, as was also observed for endogenous miRNAs. Contamination of RNA with heparin differed between sample type and isolation method, and could be counteracted using heparinase I. Other co-isolated RT-qPCR inhibitors were not identified, except for biliverdin which co-isolated from some bile samples with one of the methods.CONCLUSIONS For reliable measurements of miRNA-based biomarkers in biofluids, optimization of RNA isolation procedures is recommended as methods can differ in miRNA detection and co-purification of RT-qPCR inhibitory compounds. Heparinase I treatment confirmed that heparin appeared to be the major RT-qPCR-inhibiting compound but also biliverdin, co-isolated from bile, could interfere with detection.

scholarly journals A Novel Method for the Capture-based Purification of Whole Viral Native RNA Genomes

2018 ◽  
Author(s):  
Cedric Chih Shen Tan ◽  
Sebastian Maurer-Stroh ◽  
Yue Wan ◽  
October Michael Sessions ◽  
Paola Florez de Sessions
Keyword(s):  
Rna Sequencing ◽  
Turnaround Time ◽  
Viral Rna ◽  
Nucleic Acid Hybridization ◽  
Proof Of Concept ◽  
Purification Method ◽  
Viral Genomes ◽  
Viral Particles ◽  
Sequencing Strategy ◽  
Enrichment Protocol

ABSTRACTCurrent technologies for targeted characterization and manipulation of viral RNA primarily involve amplification or ultracentrifugation with isopycnic gradients of viral particles to decrease host RNA background. The former strategy is non-compatible for characterizing properties innate to RNA strands such as secondary structure, RNA-RNA interactions, and also for nanopore direct RNA sequencing involving the sequencing of native RNA strands. The latter strategy, ultracentrifugation, causes loss in genomic information due to its inability to retrieve unassembled viral RNA. To address this, we developed a novel application of current nucleic acid hybridization technologies for direct characterization of RNA. In particular, we modified a current enrichment protocol to capture whole viral native RNA genomes for downstream RNA assays to circumvent the abovementioned problems. This technique involves hybridization of biotinylated baits at 500 nucleotides (nt) intervals, stringent washes and release of free native RNA strands using DNase I treatment, with a turnaround time of about 6 h 15 min. RT-qPCR was used as the primary proof of concept that capture-based purification indeed removes host background. Subsequently, capture-based purification was applied to direct RNA sequencing as proof of concept that capture-based purification can be coupled with downstream RNA assays. We report that this protocol was able to successfully purify viral RNA by 561-791 fold. We also report that application of this protocol to direct RNA sequencing yielded a reduction in human host RNA background by 1580 fold, a 99.91% recovery of viral genome with at least 15x coverage, and a mean coverage across the genome of 120x. This report is, to the best of our knowledge, the first description of a capture-based purification method for assays that involve direct manipulation or characterisation of native RNA. This report also describes a successful application of capture-based purification as a direct RNA sequencing strategy that addresses certain limitations of current strategies in sequencing RNA viral genomes.

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

2020 ◽  
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.

scholarly journals Early postmortem mapping of SARS-CoV-2 RNA in patients with COVID-19 and the correlation with tissue damage

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Stefanie Deinhardt-Emmer ◽  
Daniel Wittschieber ◽  
Juliane Sanft ◽  
Sandra Kleemann ◽  
Stefan Elschner ◽  
...  
Keyword(s):  
Tissue Damage ◽  
Systemic Disease ◽  
The Body ◽  
Viral Rna ◽  
Viral Loads ◽  
Clinical Observations ◽  
Transmission Electron ◽  
Viral Particles ◽  
Tissue Degradation ◽  
Early Postmortem

Clinical observations indicate that COVID-19 is a systemic disease. An investigation of the viral distribution within the human body and its correlation with tissue damage can aid in understanding the pathophysiology of SARS-CoV-2 infection. We present a detailed mapping of the viral RNA in 61 tissues and organs of 11 deceased patients with COVID-19. The autopsies were performed within the early postmortem interval (between 1.5 and 15 hr, mean: 5.6 hr) to minimize the bias due to viral RNA and tissue degradation. Very high viral loads (>104copies/ml) were detected in most patients' lungs, and the presence of intact viral particles in the lung tissue could be verified by transmission electron microscopy. Interestingly, viral RNA was detected throughout various extrapulmonary tissues and organs without visible tissue damage. The dissemination of SARS-CoV-2-RNA throughout the body supports the hypothesis that there is a maladaptive host response with viremia and multiorgan dysfunction.

scholarly journals The near-atomic cryoEM structure of a flexible filamentous plant virus shows homology of its coat protein with nucleoproteins of animal viruses

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Xabier Agirrezabala ◽  
Eduardo Méndez-López ◽  
Gorka Lasso ◽  
M Amelia Sánchez-Pina ◽  
Miguel Aranda ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Coat Protein ◽  
Mosaic Virus ◽  
Plant Virus ◽  
Plant Pathogens ◽  
Viral Rna ◽  
Electron Cryomicroscopy ◽  
Pepino Mosaic Virus ◽  
Viral Particles

Flexible filamentous viruses include economically important plant pathogens. Their viral particles contain several hundred copies of a helically arrayed coat protein (CP) protecting a (+)ssRNA. We describe here a structure at 3.9 Å resolution, from electron cryomicroscopy, of Pepino mosaic virus (PepMV), a representative of the genus Potexvirus (family Alphaflexiviridae). Our results allow modeling of the CP and its interactions with viral RNA. The overall fold of PepMV CP resembles that of nucleoproteins (NPs) from the genus Phlebovirus (family Bunyaviridae), a group of enveloped (-)ssRNA viruses. The main difference between potexvirus CP and phlebovirus NP is in their C-terminal extensions, which appear to determine the characteristics of the distinct multimeric assemblies – a flexuous, helical rod or a loose ribonucleoprotein. The homology suggests gene transfer between eukaryotic (+) and (-)ssRNA viruses.

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