scholarly journals Sandwich-Type DNA Micro-Optode Based on Gold–Latex Spheres Label for Reflectance Dengue Virus Detection

Sensors ◽  
2020 ◽  
Vol 20 (7) ◽  
pp. 1820
Author(s):  
Jeningsih ◽  
Ling Ling Tan ◽  
Alizar Ulianas ◽  
Lee Yook Heng ◽  
Nur-Fadhilah Mazlan ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Limit Of Detection ◽  
Virus Detection ◽  
Average Diameter ◽  
Optical Biosensor ◽  
Clinical Samples ◽  
Sandwich Hybridization ◽  
Latex Spheres ◽  
Sandwich Type ◽  
Thiol Binding

A DNA micro-optode for dengue virus detection was developed based on the sandwich hybridization strategy of DNAs on succinimide-functionalized poly(n-butyl acrylate) (poly(nBA-NAS)) microspheres. Gold nanoparticles (AuNPs) with an average diameter of ~20 nm were synthesized using a centrifugation-based method and adsorbed on the submicrometer-sized polyelectrolyte-coated poly(styrene-co-acrylic acid) (PSA) latex particles via an electrostatic method. The AuNP–latex spheres were attached to the thiolated reporter probe (rDNA) by Au–thiol binding to functionalize as an optical gold–latex–rDNA label. The one-step sandwich hybridization recognition involved a pair of a DNA probe, i.e., capture probe (pDNA), and AuNP–PSA reporter label that flanked the target DNA (complementary DNA (cDNA)). The concentration of dengue virus cDNA was optically transduced by immobilized AuNP–PSA–rDNA conjugates as the DNA micro-optode exhibited a violet hue upon the DNA sandwich hybridization reaction, which could be monitored by a fiber-optic reflectance spectrophotometer at 637 nm. The optical genosensor showed a linear reflectance response over a wide cDNA concentration range from 1.0 × 10−21 M to 1.0 × 10−12 M cDNA (R2 = 0.9807) with a limit of detection (LOD) of 1 × 10−29 M. The DNA biosensor was reusable for three consecutive applications after regeneration with mild sodium hydroxide. The sandwich-type optical biosensor was well validated with a molecular reverse transcription polymerase chain reaction (RT-PCR) technique for screening of dengue virus in clinical samples, e.g., serum, urine, and saliva from dengue virus-infected patients under informed consent.

scholarly journals Defining the analytical and clinical sensitivity of the ARTIC method for the detection of SARS-CoV-2

2021 ◽  
Author(s):  
Nabil-Fareed Alikhan ◽  
Joshua Quick ◽  
Alexander J. Trotter ◽  
Samuel C. Robson ◽  
Matthew Bashton ◽  
...  
Keyword(s):  
Performance Metrics ◽  
Limit Of Detection ◽  
Diagnostic Testing ◽  
Virus Detection ◽  
Detection Methods ◽  
Clinical Samples ◽  
Sequencing Data ◽  
Clinical Sensitivity ◽  
Diagnostic Assays ◽  
Sequencing Method

The SARS-CoV-2 ARTIC amplicon protocol is the most widely used genome sequencing method for SARS-CoV-2, accounting for over 43% of publicly-available genome sequences. The protocol utilises 98 primers to amplify ~400bp fragments of the SARS-CoV-2 genome covering all 30,000 bases. Understanding the analytical performance metrics of this protocol will improve how the data is used and interpreted. Different concentrations of SARS-CoV-2 control material were used to establish the limit of detection (LoD) of the ARTIC protocol. Results demonstrated the LoD was a minimum of 25-50 virus particles per mL. The sensitivity of ARTIC was comparable to the published sensitivities of commercial diagnostics assays and could therefore be used to confirm diagnostic testing results. A set of over 3,600 clinical samples from three UK regions were then evaluated to compare the protocols performance to clinical diagnostic assays (Roche Lightcycler 480 II, AusDiagnostics, Roche Cobas, Hologic Panther, Corman RdRp, Roche Flow, ABI QuantStudio 5, Seegene Nimbus, Qiagen Rotorgene, Abbott M2000, Thermo TaqPath, Xpert). We developed a Python tool, RonaLDO, to perform this validation (available under the GNU GPL3 open-source licence from https://github.com/quadram-institute-bioscience/ronaldo). Positives detected by diagnostic platforms were generally supported by sequencing data; platforms that used RT-qPCR were the best predictors of whether the sample would subsequently sequence successfully. To maximise success of sample sequencing for phylogenetic analysis, samples with Ct <31 should be chosen. For diagnostic tests that do not provide a quantifiable Ct value, adding a quantification step is recommended. The ARTIC SARS-CoV-2 sequencing protocol is highly sensitive, capable of detecting SARS-CoV-2 in samples with Cts in the high 30s. However, to routinely obtain whole genome coverage, samples with Ct <31 are recommended. Comparing different virus detection methods close to their LoD was challenging and significant discordance was observed.

scholarly journals Application of One-Step Reverse Transcription Droplet Digital PCR for Dengue Virus Detection and Quantification in Clinical Specimens

Diagnostics ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 639
Author(s):  
Dumrong Mairiang ◽  
Adisak Songjaeng ◽  
Prachya Hansuealueang ◽  
Yuwares Malila ◽  
Paphavee Lertsethtakarn ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Lower Limit ◽  
Reverse Transcription ◽  
Limit Of Detection ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Clinical Samples ◽  
Qrt Pcr ◽  
One Step ◽  
Detection And Quantification

Detection and quantification of viruses in laboratory and clinical samples are standard assays in dengue virus (DENV) studies. The quantitative reverse transcription polymerase chain reaction (qRT-PCR) is considered to be the standard for DENV detection and quantification due to its high sensitivity. However, qRT-PCR offers only quantification relative to a standard curve and consists of several “in-house” components resulting in interlaboratory variations. We developed and optimized a protocol for applying one-step RT-droplet digital PCR (RT-ddPCR) for DENV detection and quantification. The lower limit of detection (LLOD95) and the lower limit of quantification (LLOQ) for RT-ddPCR were estimated to be 1.851 log10-copies/reaction and 2.337 log10-copies/reaction, respectively. The sensitivity of RT-ddPCR was found to be superior to qRT-PCR (94.87% vs. 90.38%, p = 0.039) while no false positives were detected. Quantification of DENV in clinical samples was independently performed in three laboratories showing interlaboratory variations with biases <0.5 log10-copies/mL. The RT-ddPCR protocol presented here could help harmonize DENV quantification results and improve findings in the field such as identifying a DENV titer threshold correlating with disease severity.

scholarly journals Rapid and sensitive detection of SARS-CoV-2 infection using quantitative peptide enrichment LC-MS analysis

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Andreas Hober ◽  
Khue Hua Tran-Minh ◽  
Dominic Foley ◽  
Thomas McDonald ◽  
Johannes PC Vissers ◽  
...  
Keyword(s):  
Large Scale ◽  
Limit Of Detection ◽  
Virus Detection ◽  
Viral Proteins ◽  
Clinical Samples ◽  
Molecular Testing ◽  
Rt Pcr ◽  
Percent Agreement ◽  
Peptide Enrichment ◽  
Asymptomatic Individuals

Reliable, robust, large-scale molecular testing for SARS-CoV-2 is essential for monitoring the ongoing Covid-19 pandemic. We have developed a scalable analytical approach to detect viral proteins based on peptide immunoaffinity enrichment combined with liquid chromatography - mass spectrometry (LC-MS). This is a multiplexed strategy, based on targeted proteomics analysis and read-out by LC-MS, capable of precisely quantifying and confirming the presence of SARS-CoV-2 in PBS swab media from combined throat/nasopharynx/saliva samples.<br />The results reveal that the levels of SARS-CoV-2 measured by LC-MS correlate well with their corresponding RT-PCR readout (r=0.79). The analytical workflow shows similar turnaround times as regular RT-PCR instrumentation with a quantitative readout of viral proteins corresponding to cycle thresholds (Ct) equivalents ranging from 21 to 34. Using RT-PCR as a reference, we demonstrate that the LC-MS-based method has 100% negative percent agreement (estimated specificity) and 95% positive percent agreement (estimated sensitivity) when analyzing clinical samples collected from asymptomatic individuals with a Ct within the limit of detection of the mass spectrometer (Ct ≤30). These results suggest that a scalable analytical method based on LC-MS has a place in future pandemic preparedness centers to complement current virus detection technologies.

scholarly journals Femtosecond-Pulsed Laser Written and Etched Fiber Bragg Gratings for Fiber-Optical Biosensing

Sensors ◽  
2018 ◽  
Vol 18 (9) ◽  
pp. 2844 ◽  
Author(s):  
Sven Schulze ◽  
Michel Wehrhold ◽  
Carsten Hille
Keyword(s):  
Limit Of Detection ◽  
Fiber Bragg Gratings ◽  
Pulsed Laser ◽  
Bragg Gratings ◽  
Optical Biosensor ◽  
Clinical Samples ◽  
Bragg Wavelength ◽  
Optical Biosensing ◽  
Fiber Optical ◽  
Femtosecond Pulsed Laser

We present the development of a label-free, highly sensitive fiber-optical biosensor for online detection and quantification of biomolecules. Here, the advantages of etched fiber Bragg gratings (eFBG) were used, since they induce a narrowband Bragg wavelength peak in the reflection operation mode. The gratings were fabricated point-by-point via a nonlinear absorption process of a highly focused femtosecond-pulsed laser, without the need of prior coating removal or specific fiber doping. The sensitivity of the Bragg wavelength peak to the surrounding refractive index (SRI), as needed for biochemical sensing, was realized by fiber cladding removal using hydrofluoric acid etching. For evaluation of biosensing capabilities, eFBG fibers were biofunctionalized with a single-stranded DNA aptamer specific for binding the C-reactive protein (CRP). Thus, the CRP-sensitive eFBG fiber-optical biosensor showed a very low limit of detection of 0.82 pg/L, with a dynamic range of CRP detection from approximately 0.8 pg/L to 1.2 µg/L. The biosensor showed a high specificity to CRP even in the presence of interfering substances. These results suggest that the proposed biosensor is capable for quantification of CRP from trace amounts of clinical samples. In addition, the adaption of this eFBG fiber-optical biosensor for detection of other relevant analytes can be easily realized.

Detection of viruses by electron microscopy: Increased sensitivity by direct micro-ultracentrifugation of samples

1987 ◽  
Vol 45 ◽  
pp. 908-909
Author(s):  
Alain R. Trudel ◽  
M. Trudel
Keyword(s):  
Electron Microscopy ◽  
Fold Increase ◽  
Observation Time ◽  
Clinical Samples ◽  
Maximum Speed ◽  
Viral Particles ◽  
Structural Details ◽  
Latex Spheres ◽  
Increased Sensitivity ◽  
Size Of Particles

AirfugeR (Beckman) direct ultracentrifugation of viral samples on electron microscopy grids offers a rapid way to concentrate viral particles or subunits and facilitate their detection and study. Using the A-100 fixed angle rotor (30°) with a K factor of 19 at maximum speed (95 000 rpm), samples up to 240 μl can be prepared for electron microscopy observation in a few minutes: observation time is decreased and structural details are highlighted. Using latex spheres to calculate the increase in sensitivity compared to the inverted drop procedure, we obtained a 10 to 40 fold increase in sensitivity depending on the size of particles. This technique also permits quantification of viral particles in samples if an aliquot is mixed with latex spheres of known concentration.Direct ultracentrifugation for electron microscopy can be performed on laboratory samples such as gradient or column fractions, infected cell supernatant, or on clinical samples such as urine, tears, cephalo-rachidian liquid, etc..

Enhanced electrochemiluminescence of CdS quantum dots capped with mercaptopropionic acid activated by EDC for Zika virus detection

The Analyst ◽  
2021 ◽  
Author(s):  
Hui-Jun Zhang ◽  
Jin Zhu ◽  
Ning Bao ◽  
Shou-Nian Ding
Keyword(s):  
Quantum Dots ◽  
Zika Virus ◽  
Virus Detection ◽  
Cds Quantum Dots ◽  
Mercaptopropionic Acid ◽  
Sandwich Type ◽  
Cds Qds

The mechanism of enhanced ECL of MPA@CdS QDs by EDC activation was investigated, and a sandwich-type ECL immunosensor has been designed for Zika virus detection.

scholarly journals Development and validation of a multiplex qPCR assay for detection and relative quantification of HPV16 and HPV18 E6 and E7 oncogenes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexia Bordigoni ◽  
Anne Motte ◽  
Hervé Tissot-Dupont ◽  
Philippe Colson ◽  
Christelle Desnues
Keyword(s):  
Limit Of Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Human Papillomaviruses ◽  
Molecular Techniques ◽  
Clinical Samples ◽  
Gene Encoding ◽  
Hpv Dna ◽  
E6 And E7 ◽  
High Risk Hpv ◽  
Development And Validation

AbstractHuman papillomaviruses (HPV) play a key role in promoting human anogenital cancers. Current high-risk HPV screening or diagnosis tests involve cytological or molecular techniques mostly based on qualitative HPV DNA detection. Here, we describe the development of a rapid quantitative polymerase chain reaction (qPCR) detection test of HPV16 and HPV18 oncogenes (E6 and E7) normalized on human gene encoding GAPDH. Optimized qPCR parameters were defined, and analytical specificities were validated. The limit of detection was 101 for all genes tested. Assay performances were evaluated on clinical samples (n = 96). Concordance between the Xpert HPV assay and the triplex assay developed here was 93.44% for HPV16 and 73.58% for HPV18. HPV co-infections were detected in 15 samples. The systems developed in the present study can be used in complement to traditional HPV tests for specifically validating the presence of HPV16 and/or HPV18. It can also be used for the follow-up of patients with confirmed infection and at risk of developing lesions, through the quantification of E6 and E7 oncogene expression (mRNA) normalized on the GAPDH expression levels.

scholarly journals 423. SARS-CoV-2 NGS Assay Powered by Biotia COVID-DX Software

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S278-S279
Author(s):  
Dorottya Nagy-Szakal ◽  
Mara Couto-Rodriguez ◽  
Joseph Barrows ◽  
Heather L Wells ◽  
Marilyne Debieu ◽  
...  
Keyword(s):  
Genetic Variants ◽  
Viral Genome ◽  
Board Member ◽  
Limit Of Detection ◽  
Clinical Samples ◽  
Analysis Tool ◽  
Viral Titer ◽  
Target Enrichment ◽  
Library Preparation ◽  
Software Analysis

Abstract Background COVID-19 had spread quickly, causing an international public health emergency with an alarming global shortage of COVID-19 diagnostic tests. We developed and clinically validated a next-generation sequencing (NGS)-based target enrichment assay with the COVID-DX Software tailored for the detection, characterization, and surveillance of the SARS-CoV-2 viral genome. Methods The SARS-CoV-2 NGS assay consists of components including library preparation, target enrichment, sequencing, and a COVID-DX Software analysis tool. The NGS library preparation starts with extracted RNA from nasopharyngeal (NP) swabs followed by cDNA synthesis and conversion to Illumina TruSeq-compatible libraries using the Twist Library Preparation Kit via Enzymatic Fragmentation and Unique Dual Indices (UDI). The library is then enriched for SARS-CoV-2 sequences using a panel of dsDNA biotin-labeled probes, specifically designed to target the SARS-CoV-2 genome, then sequenced on an Illumina NextSeq 550 platform. The COVID-DX Software analyzes sequence results and provides a clinically oriented report, including the presence/absence of SARS-CoV-2 for diagnostic use. An additional research use only report describes the assay performance, estimated viral titer, coverage across the viral genome, genetic variants, and phylogenetic analysis. Results The SARS-CoV-2 NGS Assay was validated on 30 positive and 30 negative clinical samples. To measure the sensitivity and specificity of the assay, the positive and negative percent agreement (PPA, NPA) was defined in comparison to an orthogonal EUA RT-PCR assay (PPA [95% CI]: 96.77% [90.56%-100%] and NPA [95% CI]: 100% [100%-100%]). Data reported using our assay defined the limit of detection to be 40 copies/ml using heat-inactivated SARS-CoV-2 viral genome in clinical matrices. In-silico analysis provided &gt;99.9% coverage across the SARS-CoV-2 viral genome and no cross-reactivity with evolutionarily similar respiratory pathogens. Conclusion The SARS-CoV-2 NGS Assay powered by the COVID-DX Software can be used to detect the SARS-CoV-2 virus and provide additional insight into viral titer and genetic variants to track transmission, stratify risk, predict outcome and therapeutic response, and control the spread of infectious disease. Disclosures Dorottya Nagy-Szakal, MD PhD, Biotia (Employee) Mara Couto-Rodriguez, MS, Biotia (Employee) Joseph Barrows, MS, Biotia, Inc. (Employee, Shareholder) Heather L. Wells, MPH, Biotia (Consultant) Marilyne Debieu, PhD, Biotia (Employee) Courteny Hager, BS, Biotia (Employee) Kristin Butcher, MS, Twist Bioscience (Employee) Siyuan Chen, PhD, Twist Bioscience (Employee) Christopher Mason, PhD, Biotia (Board Member, Employee, Shareholder) Niamh B. O’Hara, PhD, Biotia (Board Member, Employee, Shareholder)Twist (Other Financial or Material Support, I am CEO of Biotia and Biotia has business partnership with Twist)

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