scholarly journals Targeted proteomics as a tool to detect SARS-CoV-2 proteins in clinical specimens

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0259165
Author(s):  
Karel Bezstarosti ◽  
Mart M. Lamers ◽  
Wouter A. S. Doff ◽  
Peter C. Wever ◽  
Khoa T. D. Thai ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Limit Of Detection ◽  
High Sensitivity ◽  
Viral Proteins ◽  
Reaction Monitoring ◽  
Proof Of Concept ◽  
Specific Detection ◽  
Clinical Specimens ◽  
Additional Tool ◽  
Parallel Reaction Monitoring

The rapid, sensitive and specific detection of SARS-CoV-2 is critical in responding to the current COVID-19 outbreak. In this proof-of-concept study, we explored the potential of targeted mass spectrometry (MS) based proteomics for the detection of SARS-CoV-2 proteins in both research samples and clinical specimens. First, we assessed the limit of detection for several SARS-CoV-2 proteins by parallel reaction monitoring (PRM) MS in infected Vero E6 cells. For tryptic peptides of Nucleocapsid protein, the limit of detection was estimated to be in the mid-attomole range (9E-13 g). Next, this PRM methodology was applied to the detection of viral proteins in various COVID-19 patient clinical specimens, such as sputum and nasopharyngeal swabs. SARS-CoV-2 proteins were detected in these samples with high sensitivity in all specimens with PCR Ct values <24 and in several samples with higher CT values. A clear relationship was observed between summed MS peak intensities for SARS-CoV-2 proteins and Ct values reflecting the abundance of viral RNA. Taken together, these results suggest that targeted MS based proteomics may have the potential to be used as an additional tool in COVID-19 diagnostics.

scholarly journals Targeted proteomics as a tool to detect SARS-CoV-2 proteins in clinical specimens

2020 ◽  
Author(s):  
Karel Bezstarosti ◽  
Mart M. Lamers ◽  
Jeroen J. A. van Kampen ◽  
Bart L. Haagmans ◽  
Jeroen A. A. Demmers
Keyword(s):  
Mass Spectrometry ◽  
Potential Role ◽  
Limit Of Detection ◽  
Viral Proteins ◽  
Clinical Samples ◽  
Proof Of Concept ◽  
Specific Detection ◽  
Clinical Specimens ◽  
Parallel Reaction Monitoring

ABSTRACTThe rapid, sensitive and specific detection of SARS-CoV-2 is critical in responding to the current COVID-19 outbreak. Here, we explore the potential of targeted mass spectrometry based proteomics for the detection of SARS-CoV-2 proteins in both research and clinical samples. First, we assessed the limit of detection for several SARS-CoV-2 proteins by parallel reaction monitoring (PRM) mass spectrometry. For Nucleocapsid the limit of detection was found to be in the mid-attomole range (0.9 x 10−12 g). Next, we apply this PRM assay to the detection of viral proteins in in vitro mucus substitutes, as well as in various clinical specimens such as nasopharyngeal swabs and sputum. In this proof-of-concept study SARS-CoV-2 proteins could unambiguously be detected in clinical samples, suggesting that the sensitivity of this technology may be sufficiently high to further explore its potential role in diagnostics.

scholarly journals Detection of Toxoplasmic Encephalitis in HIV Positive Patients in Urine with Hydrogel Nanoparticles.

2020 ◽  
Author(s):  
Hannah Steinberg ◽  
Natalie M Bowman ◽  
Andrea Diestra ◽  
Cusi Ferradas ◽  
Paul Russo ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Toxoplasmic Encephalitis ◽  
Proof Of Concept ◽  
Resource Limited ◽  
Parallel Reaction Monitoring ◽  
Promising Target ◽  
Urine Diagnostics ◽  
Blotting Technique ◽  
Polymerase Chain

Diagnosis of toxoplasmic encephalitis (TE) is challenging under the best clinical circumstances. The poor sensitivity of quantitative polymerase chain reaction (qPCR) for Toxoplasma in blood and CSF and the limited availability of molecular diagnostics and imaging technology leaves clinicians in resource-limited settings with few options other than empiric treatment. Here we describe proof of concept for a novel urine diagnostics for TE using Poly-N-isoproplyacrylamide nanoparticles dyed with Reactive Blue-221 to concentrate antigens, substantially increasing the limit of detection. After nanoparticle-concentration, a standard western blotting technique with a monoclonal antibody was used for antigen detection. Limit of detection was 7.8pg/ml and 31.3pg/ml of T. gondii antigens GRA1 and SAG1, respectively. To characterize this diagnostic approach, 164 hospitalized HIV-infected patients with neurological symptoms compatible with TE were tested for 1) T. gondii serology (121/147, positive samples/total samples tested), 2) qPCR in cerebrospinal fluid (11/41), 3) qPCR in blood (10/112), and 4) urinary GRA1 (30/164) and SAG1 (12/164). GRA1 appears to be superior to SAG1 for detection of TE antigens in urine. Fifty-one HIV-infected, T. gondii seropositive but asymptomatic persons all tested negative by nanoparticle western blot and blood qPCR, suggesting the test has good specificity for TE for both GRA1 and SAG1. In a subgroup of 44 patients, urine samples were assayed with mass spectrometry parallel-reaction-monitoring (PRM) for the presence of T. gondii antigens. PRM identified antigens in 8 samples, 6 of which were concordant with the urine diagnostic. Our results demonstrate nanoparticle technology potential for a noninvasive diagnostic test for TE. Moving forward, GRA1 is a promising target for antigen based diagnostics for TE.

scholarly journals Giant gauge factor of Van der Waals material based strain sensors

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Wenjie Yan ◽  
Huei-Ru Fuh ◽  
Yanhui Lv ◽  
Ke-Qiu Chen ◽  
Tsung-Yin Tsai ◽  
...  
Keyword(s):  
Carrier Mobility ◽  
Large Range ◽  
Van Der Waals ◽  
High Sensitivity ◽  
Strain Sensors ◽  
Strain Gauges ◽  
Gauge Factor ◽  
Proof Of Concept ◽  
High Flexibility ◽  
Small Deformations

AbstractThere is an emergent demand for high-flexibility, high-sensitivity and low-power strain gauges capable of sensing small deformations and vibrations in extreme conditions. Enhancing the gauge factor remains one of the greatest challenges for strain sensors. This is typically limited to below 300 and set when the sensor is fabricated. We report a strategy to tune and enhance the gauge factor of strain sensors based on Van der Waals materials by tuning the carrier mobility and concentration through an interplay of piezoelectric and photoelectric effects. For a SnS2 sensor we report a gauge factor up to 3933, and the ability to tune it over a large range, from 23 to 3933. Results from SnS2, GaSe, GeSe, monolayer WSe2, and monolayer MoSe2 sensors suggest that this is a universal phenomenon for Van der Waals semiconductors. We also provide proof of concept demonstrations by detecting vibrations caused by sound and capturing body movements.

scholarly journals Validation of TaqMan-Based Assays for Specific Detection and Differentiation of Wild-Type and Neethling Vaccine Strains of LSDV

2021 ◽  
Vol 9 (6) ◽  
pp. 1234
Author(s):  
Dejan Vidanović ◽  
Bojana Tešović ◽  
Milanko Šekler ◽  
Zoran Debeljak ◽  
Nikola Vasković ◽  
...  
Keyword(s):  
Diagnostic Performance ◽  
High Sensitivity ◽  
Animal Disease ◽  
Wild Type ◽  
Notifiable Disease ◽  
Specific Detection ◽  
Lumpy Skin Disease ◽  
Systemic Reactions ◽  
Vaccination Campaigns ◽  
Significant Health

Lumpy skin disease (LSD) is an important animal disease with significant health and economic impacts. It is considered a notifiable disease by the OIE. Attenuated strains of LSDV have been successfully used as vaccines (LAV) but can also produce mild or systemic reactions. Vaccination campaigns using LAVs are therefore only viable if accompanying DIVA assays are available. Two DIVA qPCR assays able to distinguish Neethling-based LAVs and wild-type LSDV were developed. Upon validation, both assays were shown to have high sensitivity and specificity with a diagnostic performance comparable to other published DIVA assays. This confirmed their potential as reliable tools to confirm infection in animals during vaccination campaigns based on Neethling vaccine strains.

scholarly journals Ratiometric Fluorescent Biosensors for Glucose and Lactate Using an Oxygen-Sensing Membrane

Biosensors ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 208
Author(s):  
Hong Dinh Duong ◽  
Jong Il Rhee
Keyword(s):  
Limit Of Detection ◽  
Oxygen Sensing ◽  
High Sensitivity ◽  
Glucose Sensing ◽  
Oxidation Reactions ◽  
Lactate Oxidase ◽  
Lactate Oxidation ◽  
Detection Range ◽  
Quenching Effect ◽  
Sensing Membrane

In this study, ratiometric fluorescent glucose and lactate biosensors were developed using a ratiometric fluorescent oxygen-sensing membrane immobilized with glucose oxidase (GOD) or lactate oxidase (LOX). Herein, the ratiometric fluorescent oxygen-sensing membrane was fabricated with the ratio of two emission wavelengths of platinum meso-tetra (pentafluorophenyl) porphyrin (PtP) doped in polystyrene particles and coumarin 6 (C6) captured into silica particles. The operation mechanism of the sensing membranes was based on (i) the fluorescence quenching effect of the PtP dye by oxygen molecules, and (ii) the consumption of oxygen levels in the glucose or lactate oxidation reactions under the catalysis of GOD or LOX. The ratiometric fluorescent glucose-sensing membrane showed high sensitivity to glucose in the range of 0.1–2 mM, with a limit of detection (LOD) of 0.031 mM, whereas the ratiometric fluorescent lactate-sensing membrane showed the linear detection range of 0.1–0.8 mM, with an LOD of 0.06 mM. These sensing membranes also showed good selectivity, fast reversibility, and stability over long-term use. They were applied to detect glucose and lactate in artificial human serum, and they provided reliable measurement results.

scholarly journals Ag nanoparticles outperform Au nanoparticles for the use as label in electrochemical point-of-care sensors

2021 ◽  
Author(s):  
Franziska Beck ◽  
Carina Horn ◽  
Antje J. Baeumner
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Automatic Measurement ◽  
High Sensitivity ◽  
Differential Pulse ◽  
Small Sample ◽  
Blood Protein ◽  
User Friendliness ◽  
Response Curves ◽  
Assay Protocol

AbstractElectrochemical immunosensors enable rapid analyte quantification in small sample volumes, and have been demonstrated to provide high sensitivity and selectivity, simple miniaturization, and easy sensor production strategies. As a point-of-care (POC) format, user-friendliness is equally important and most often not combinable with high sensitivity. As such, we demonstrate here that a sequence of metal oxidation and reduction, followed by stripping via differential pulse voltammetry (DPV), provides lowest limits of detection within a 2-min automatic measurement. In exchanging gold nanoparticles (AuNPs), which dominate in the development of POC sensors, with silver nanoparticles (AgNPs), not only better sensitivity was obtained, but more importantly, the assay protocol could be simplified to match POC requirements. Specifically, we studied both nanoparticles as reporter labels in a sandwich immunoassay with the blood protein biomarker NT-proBNP. For both kinds of nanoparticles, the dose-response curves easily covered the ng∙mL−1 range. The mean standard deviation of all measurements of 17% (n ≥ 4) and a limit of detection of 26 ng∙mL−1 were achieved using AuNPs, but their detection requires addition of HCl, which is impossible in a POC format. In contrast, since AgNPs are electrochemically less stable, they enabled a simplified assay protocol and provided even lower LODs of 4.0 ng∙mL−1 in buffer and 4.7 ng∙mL−1 in human serum while maintaining the same or even better assay reliability, storage stability, and easy antibody immobilization protocols. Thus, in direct comparison, AgNPs clearly outperform AuNPs in desirable POC electrochemical assays and should gain much more attention in the future development of such biosensors.

Analytical assessment of ortho clinical diagnostics high-sensitivity cardiac troponin I assay

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Peter A. Kavsak ◽  
Tara Edge ◽  
Chantele Roy ◽  
Paul Malinowski ◽  
Karen Bamford ◽  
...  
Keyword(s):  
Cardiac Troponin ◽  
Troponin I ◽  
Limit Of Detection ◽  
Clinical Performance ◽  
Characteristic Curve ◽  
Area Under The Curve ◽  
High Sensitivity ◽  
Cardiac Troponin I ◽  
Clinical Diagnostics ◽  
Ortho Clinical Diagnostics

AbstractObjectivesTo analytically evaluate Ortho Clinical Diagnostics VITROS high-sensitivity cardiac troponin I (hs-cTnI) assay in specific matrices with comparison to other hs-cTn assays.MethodsThe limit of detection (LoD), imprecision, interference and stability testing for both serum and lithium heparin (Li-Hep) plasma for the VITROS hs-cTnI assay was determined. We performed Passing-Bablok regression analyses between sample types for the VITROS hs-cTnI assay and compared them to the Abbott ARCHITECT, Beckman Access and the Siemens ADVIA Centaur hs-cTnI assays. We also performed Receiver-operating characteristic curve analyses with the area under the curve (AUC) determined in an emergency department (ED)-study population (n=131) for myocardial infarction (MI).ResultsThe VITROS hs-cTnI LoD was 0.73 ng/L (serum) and 1.4 ng/L (Li-Hep). Stability up to five freeze-thaws was observed for the Ortho hs-cTnI assay, with the analyte stability at room temperature in serum superior to Li-Hep with gross hemolysis also affecting Li-Hep plasma hs-cTnI results. Comparison of Li-Hep to serum concentrations (n=202), yielded proportionally lower concentrations in plasma with the VITROS hs-cTnI assay (slope=0.85; 95% confidence interval [CI]:0.83–0.88). In serum, the VITROS hs-cTnI concentrations were proportionally lower compared to other hs-cTnI assays, with similar slopes observed between assays in samples frozen <−70 °C for 17 years (ED-study) or in 2020. In the ED-study, the VITROS hs-cTnI assay had an AUC of 0.974 (95%CI:0.929–0.994) for MI, similar to the AUCs of other hs-cTn assays.ConclusionsLack of standardization of hs-cTnI assays across manufacturers is evident. The VITROS hs-cTnI assay yields lower concentrations compared to other hs-cTnI assays. Important differences exist between Li-Hep plasma and serum, with evidence of stability and excellent clinical performance comparable to other hs-cTn assays.

scholarly journals Confirming putative variants at ≤ 5% allele frequency using allele enrichment and Sanger sequencing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yan Helen Yan ◽  
Sherry X. Chen ◽  
Lauren Y. Cheng ◽  
Alyssa Y. Rodriguez ◽  
Rui Tang ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Limit Of Detection ◽  
High Sensitivity ◽  
Sequencing Errors ◽  
Whole Exome ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Fresh Frozen ◽  
The Cost ◽  
Allelic Fraction

AbstractWhole exome sequencing (WES) is used to identify mutations in a patient’s tumor DNA that are predictive of tumor behavior, including the likelihood of response or resistance to cancer therapy. WES has a mutation limit of detection (LoD) at variant allele frequencies (VAF) of 5%. Putative mutations called at ≤ 5% VAF are frequently due to sequencing errors, therefore reporting these subclonal mutations incurs risk of significant false positives. Here we performed ~ 1000 × WES on fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue biopsy samples from a non-small cell lung cancer patient, and identified 226 putative mutations at between 0.5 and 5% VAF. Each variant was then tested using NuProbe NGSure, to confirm the original WES calls. NGSure utilizes Blocker Displacement Amplification to first enrich the allelic fraction of the mutation and then uses Sanger sequencing to determine mutation identity. Results showed that 52% of the 226 (117) putative variants were disconfirmed, among which 2% (5) putative variants were found to be misidentified in WES. In the 66 cancer-related variants, the disconfirmed rate was 82% (54/66). This data demonstrates Blocker Displacement Amplification allelic enrichment coupled with Sanger sequencing can be used to confirm putative mutations ≤ 5% VAF. By implementing this method, next-generation sequencing can reliably report low-level variants at a high sensitivity, without the cost of high sequencing depth.

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