Highly multiplexed oligonucleotide probe-ligation testing enables efficient extraction-free SARS-CoV-2 detection and viral genotyping

AbstractThe emergence of SARS-CoV-2 has caused the current COVID-19 pandemic with catastrophic societal impact. Because many individuals shed virus for days before symptom onset, and many show mild or no symptoms, an emergent and unprecedented need exists for development and deployment of sensitive and high throughput molecular diagnostic tests. RNA-mediated oligonucleotide Annealing Selection and Ligation with next generation DNA sequencing (RASL-seq) is a highly multiplexed technology for targeted analysis of polyadenylated mRNA, which incorporates sample barcoding for massively parallel analyses. Here we present a more generalized method, capture RASL-seq (“cRASL-seq”), which enables analysis of any targeted pathogen-(and/or host-) associated RNA molecules. cRASL-seq enables highly sensitive (down to ∼1-100 pfu/ml or cfu/ml) and highly multiplexed (up to ∼10,000 target sequences) detection of pathogens. Importantly, cRASL-seq analysis of COVID-19 patient nasopharyngeal (NP) swab specimens does not involve nucleic acid extraction or reverse transcription, steps that have caused testing bottlenecks associated with other assays. Our simplified workflow additionally enables the direct and efficient genotyping of selected, informative SARS-CoV-2 polymorphisms across the entire genome, which can be used for enhanced characterization of transmission chains at population scale and detection of viral clades with higher or lower virulence. Given its extremely low per-sample cost, simple and automatable protocol and analytics, probe panel modularity, and massive scalability, we propose that cRASL-seq testing is a powerful new surveillance technology with the potential to help mitigate the current pandemic and prevent similar public health crises.

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Characterization of a monoclonal antibody to turnip mosaic virus and its use in immunodiagnosis of infection

Phytoprotection ◽

10.7202/706004ar ◽

2005 ◽

Vol 72 (2) ◽

pp. 61-68 ◽

Cited By ~ 2

Author(s):

P. Horsewood ◽

M.R. McDermott ◽

L.W. Stobbs ◽

P.L.J. Brais ◽

B.J. Underdown

Keyword(s):

Mosaic Virus ◽

Turnip Mosaic Virus ◽

Buffer System ◽

Sensitive Assay ◽

Virus Infections ◽

Extraction Buffer ◽

Highly Sensitive ◽

Efficient Extraction ◽

Sandwich Enzyme Immunoassay

Monoclonal antibodies specifie for turnip mosaic virus (TuMV) were produced and used in a double antibody sandwich enzyme immunoassay to detect virus in infected plants. One particular antibody from a hybridoma clone having desirable growth, specificity and antibody production properties was characterized in detail. This antibody was shown by immunocytochemical electron microscopy and immunoblotting to react with a virion coat protein. Conditions providing efficient extraction of virus from leaves were investigated by using the antibody in both capture and detection steps of a sandwich immunoassay. With an extraction buffer System containing multiple detergents, a highly sensitive assay was produced that reliably detected virus in infected plants. This assay is now in routine use for immunodiagnosis of turnip mosaic virus infections.

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Electron Microscope Study of RNA's of Paramyxoviruses

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100094231 ◽

1972 ◽

Vol 30 ◽

pp. 196-197

Author(s):

Ruchama Baum ◽

J.T. Seto

Keyword(s):

Electron Microscopy ◽

Electron Microscope ◽

Electron Microscope Study ◽

Sendai Virus ◽

Sodium Dodecyl ◽

Rna Molecules ◽

Virus Particles ◽

Molecular Weights ◽

Length Measurements

The ribonucleic acid (RNA) of paramyxoviruses has been characterized by biochemical and physiochemical methods. However, paramyxovirus RNA molecules have not been studied by electron microscopy. The molecular weights of these single-stranded viral RNA molecules are not known as yet. Since electron microscopy has been found to be useful for the characterization of single-stranded RNA, this investigation was initiated to examine the morphology and length measurements of paramyxovirus RNA's.Sendai virus Z strain and Newcastle disease virus (NDV), Milano strain, were used. For these studies it was necessary to develop a method of extracting RNA molecules from purified virus particles. Highly purified Sendai virus was treated with pronase (300 μg/ml) at 37°C for 30 minutes and the RNA extracted by the sodium dodecyl sulfate (SDS)-phenol procedure.

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The glycomes of Caenorhabditis elegans and other model organisms

Biochemical Society Symposium ◽

10.1042/bss0690117 ◽

2002 ◽

Vol 69 ◽

pp. 117-134 ◽

Cited By ~ 47

Author(s):

Stuart M. Haslam ◽

David Gems ◽

Howard R. Morris ◽

Anne Dell

Keyword(s):

Caenorhabditis Elegans ◽

Current Knowledge ◽

Structural Data ◽

Model Organisms ◽

Entire Genome ◽

C Elegans ◽

The Face ◽

Area Of Concern ◽

Nematode Worm

There is no doubt that the immense amount of information that is being generated by the initial sequencing and secondary interrogation of various genomes will change the face of glycobiological research. However, a major area of concern is that detailed structural knowledge of the ultimate products of genes that are identified as being involved in glycoconjugate biosynthesis is still limited. This is illustrated clearly by the nematode worm Caenorhabditis elegans, which was the first multicellular organism to have its entire genome sequenced. To date, only limited structural data on the glycosylated molecules of this organism have been reported. Our laboratory is addressing this problem by performing detailed MS structural characterization of the N-linked glycans of C. elegans; high-mannose structures dominate, with only minor amounts of complex-type structures. Novel, highly fucosylated truncated structures are also present which are difucosylated on the proximal N-acetylglucosamine of the chitobiose core as well as containing unusual Fucα1–2Gal1–2Man as peripheral structures. The implications of these results in terms of the identification of ligands for genomically predicted lectins and potential glycosyltransferases are discussed in this chapter. Current knowledge on the glycomes of other model organisms such as Dictyostelium discoideum, Saccharomyces cerevisiae and Drosophila melanogaster is also discussed briefly.

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Synthesis and Characterization of a 6,6'-((1E,1'E)-(1,2phenylenebis(azanylylidene))bis(methanylylidene))b is(2-methoxy-3-((6-methoxybenzo[d]thiazol-2yl)diazenyl)phenol): as a highly sensitive reagent for determination cadmium(II) ion in the real samples

International Journal of Pharmaceutical Research ◽

10.31838/ijpr/2018.10.04.080 ◽

2018 ◽

Vol 10 (4) ◽

Keyword(s):

Synthesis And Characterization ◽

Real Samples ◽

The Real ◽

Highly Sensitive

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Synthesis and 31 P NMR characterization of new low toxic highly sensitive pH probes designed for in vivo acidic pH studies

Bioorganic & Medicinal Chemistry ◽

10.1016/s0968-0896(01)00414-x ◽

2002 ◽

Vol 10 (5) ◽

pp. 1451-1458 ◽

Cited By ~ 16

Author(s):

Sophie Martel ◽

Jean-Louis Clément ◽

Agnès Muller ◽

Marcel Culcasi ◽

Sylvia Pietri

Keyword(s):

Acidic Ph ◽

Highly Sensitive ◽

Nmr Characterization ◽

Low Toxic ◽

Ph Probes

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Shiga Toxin (Stx)-Binding Glycosphingolipids of Primary Human Renal Cortical Epithelial Cells (pHRCEpiCs) and Stx-Mediated Cytotoxicity

Toxins ◽

10.3390/toxins13020139 ◽

2021 ◽

Vol 13 (2) ◽

pp. 139

Author(s):

Johanna Detzner ◽

Elisabeth Krojnewski ◽

Gottfried Pohlentz ◽

Daniel Steil ◽

Hans-Ulrich Humpf ◽

...

Keyword(s):

Fatty Acid ◽

Epithelial Cells ◽

Shiga Toxin ◽

Saturated Fatty Acids ◽

Human Kidney ◽

Enterohemorrhagic Escherichia Coli ◽

Highly Sensitive ◽

Liquid Ordered ◽

Uremic Syndrome

Human kidney epithelial cells are supposed to be directly involved in the pathogenesis of the hemolytic–uremic syndrome (HUS) caused by Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC). The characterization of the major and minor Stx-binding glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), respectively, of primary human renal cortical epithelial cells (pHRCEpiCs) revealed GSLs with Cer (d18:1, C16:0), Cer (d18:1, C22:0), and Cer (d18:1, C24:1/C24:0) as the dominant lipoforms. Using detergent-resistant membranes (DRMs) and non-DRMs, Gb3Cer and Gb4Cer prevailed in the DRM fractions, suggesting their association with microdomains in the liquid-ordered membrane phase. A preference of Gb3Cer and Gb4Cer endowed with C24:0 fatty acid accompanied by minor monounsaturated C24:1-harboring counterparts was observed in DRMs, whereas the C24:1 fatty acid increased in relation to the saturated equivalents in non-DRMs. A shift of the dominant phospholipid phosphatidylcholine with saturated fatty acids in the DRM to unsaturated species in the non-DRM fractions correlated with the GSL distribution. Cytotoxicity assays gave a moderate susceptibility of pHRCEpiCs to the Stx1a and Stx2a subtypes when compared to highly sensitive Vero-B4 cells. The results indicate that presence of Stx-binding GSLs per se and preferred occurrence in microdomains do not necessarily lead to a high cellular susceptibility towards Stx.

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Terahertz characterization of two-dimensional low-conductive layers enabled by metal gratings

Scientific Reports ◽

10.1038/s41598-021-82560-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Prashanth Gopalan ◽

Yunshan Wang ◽

Berardi Sensale-Rodriguez

Keyword(s):

Optical Transmission ◽

Terahertz Spectroscopy ◽

Design Guidelines ◽

Two Dimensional ◽

Highly Sensitive ◽

Transmission Measurements ◽

Metal Gratings ◽

Low Sensitivity ◽

Non Destructive

AbstractWhile terahertz spectroscopy can provide valuable information regarding the charge transport properties in semiconductors, its application for the characterization of low-conductive two-dimensional layers, i.e., σs < < 1 mS, remains elusive. This is primarily due to the low sensitivity of direct transmission measurements to such small sheet conductivity levels. In this work, we discuss harnessing the extraordinary optical transmission through gratings consisting of metallic stripes to characterize such low-conductive two-dimensional layers. We analyze the geometric tradeoffs in these structures and provide physical insights, ultimately leading to general design guidelines for experiments enabling non-contact, non-destructive, highly sensitive characterization of such layers.

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The Increasing Impact of Translational Research in the Molecular Diagnostics of Neuromuscular Diseases

International Journal of Molecular Sciences ◽

10.3390/ijms22084274 ◽

2021 ◽

Vol 22 (8) ◽

pp. 4274

Author(s):

Dèlia Yubero ◽

Daniel Natera-de Benito ◽

Jordi Pijuan ◽

Judith Armstrong ◽

Loreto Martorell ◽

...

Keyword(s):

Molecular Diagnosis ◽

Clinical Symptoms ◽

Neuromuscular Diseases ◽

Genomic Analysis ◽

Molecular Diagnostic ◽

Diagnostic Process ◽

Targeted Analysis ◽

Whole Exome ◽

Referral Hospitals ◽

Uncertain Significance

The diagnosis of neuromuscular diseases (NMDs) has been progressively evolving from the grouping of clinical symptoms and signs towards the molecular definition. Optimal clinical, biochemical, electrophysiological, electrophysiological, and histopathological characterization is very helpful to achieve molecular diagnosis, which is essential for establishing prognosis, treatment and genetic counselling. Currently, the genetic approach includes both the gene-targeted analysis in specific clinically recognizable diseases, as well as genomic analysis based on next-generation sequencing, analyzing either the clinical exome/genome or the whole exome or genome. However, as of today, there are still many patients in whom the causative genetic variant cannot be definitely established and variants of uncertain significance are often found. In this review, we address these drawbacks by incorporating two additional biological omics approaches into the molecular diagnostic process of NMDs. First, functional genomics by introducing experimental cell and molecular biology to analyze and validate the variant for its biological effect in an in-house translational diagnostic program, and second, incorporating a multi-omics approach including RNA-seq, metabolomics, and proteomics in the molecular diagnosis of neuromuscular disease. Both translational diagnostics programs and omics are being implemented as part of the diagnostic process in academic centers and referral hospitals and, therefore, an increase in the proportion of neuromuscular patients with a molecular diagnosis is expected. This improvement in the process and diagnostic performance of patients will allow solving aspects of their health problems in a precise way and will allow them and their families to take a step forward in their lives.

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A New Ultrasensitive Bioluminescence-Based Method for Assaying Monoacylglycerol Lipase

International Journal of Molecular Sciences ◽

10.3390/ijms22116148 ◽

2021 ◽

Vol 22 (11) ◽

pp. 6148

Author(s):

Matteo Miceli ◽

Silvana Casati ◽

Pietro Allevi ◽

Silvia Berra ◽

Roberta Ottria ◽

...

Keyword(s):

Arachidonic Acid ◽

Kinetic Constants ◽

New Method ◽

Monoacylglycerol Lipase ◽

Enzymatic Cleavage ◽

Highly Sensitive ◽

Bioluminescence Assay ◽

Identification And Characterization ◽

In Cells

A novel bioluminescent Monoacylglycerol lipase (MAGL) substrate 6-O-arachidonoylluciferin, a D-luciferin derivative, was synthesized, physico-chemically characterized, and used as highly sensitive substrate for MAGL in an assay developed for this purpose. We present here a new method based on the enzymatic cleavage of arachidonic acid with luciferin release using human Monoacylglycerol lipase (hMAGL) followed by its reaction with a chimeric luciferase, PLG2, to produce bioluminescence. Enzymatic cleavage of the new substrate by MAGL was demonstrated, and kinetic constants Km and Vmax were determined. 6-O-arachidonoylluciferin has proved to be a highly sensitive substrate for MAGL. The bioluminescence assay (LOD 90 pM, LOQ 300 pM) is much more sensitive and should suffer fewer biological interferences in cells lysate applications than typical fluorometric methods. The assay was validated for the identification and characterization of MAGL modulators using the well-known MAGL inhibitor JZL184. The use of PLG2 displaying distinct bioluminescence color and kinetics may offer a highly desirable opportunity to extend the range of applications to cell-based assays.

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