Biosensors and Diagnostics for Pandemic Response
Abstract not provided.
Publications
Exhaled breath biomarkers of influenza infection and influenza vaccination
Journal of Breath Research
Respiratory viral infections are considered a major public health threat, and breath metabolomics can provide new ways to detect and understand how specific viruses affect the human pulmonary system. In this pilot study, we characterized the metabolic composition of human breath for an early diagnosis and differentiation of influenza viral infection, as well as other types of upper respiratory viral infections. We first studied the non-specific effects of planned seasonal influenza vaccines on breath metabolites in healthy subjects after receiving the immunization. We then investigated changes in breath content from hospitalized patients with flu-like symptoms and confirmed upper respiratory viral infection. The exhaled breath was sampled using a custom-made breath condenser, and exhaled breath condensate (EBC) samples were analysed using liquid chromatography coupled to quadruplole-time-of-flight mass spectrometer (LC-qTOF). All metabolomic data was analysed using both targeted and untargeted approaches to detect specific known biomarkers from inflammatory and oxidative stress biomarkers, as well as new molecules associated with specific infections. We were able to find clear differences between breath samples collected before and after flu vaccine administration, together with potential biomarkers that are related to inflammatory processes and oxidative stress. Moreover, we were also able to discriminate samples from patients with flu-related symptoms that were diagnosed with confirmatory respiratory viral panels (RVPs). RVP positive and negative differences were identified, as well as differences between specific viruses defined. These results provide very promising information for the further study of the effect of influenza A and other viruses in human systems by using a simple and non-invasive specimen like breath.
Evaluation of Lyophilized Amplification Reagents for Detection of SARS-CoV-2
Abstract not provided.
Biosensors and Diagnostics for Pandemic Response
Abstract not provided.
Thermodynamic Predictions of Primer Interactions and Impact on LAMP Assay Performance
Abstract not provided.
Smart LAMP for 'no resource' infectious disease detection
Abstract not provided.
SpinDx - Platform for Point-of-Care Medical Diagnostics
Abstract not provided.
Engineering Isothermal Molecular Diagnostic Assays for Better Performance with Fewer Resources
Abstract not provided.
Dual gene expression analysis identifies factors associated with Staphylococcus aureus virulence in diabetic mice
Infection and Immunity
Staphylococcus aureus is a major human pathogen of the skin. The global burden of diabetes is high, with S. aureus being a major complication of diabetic wound infections. We investigated how the diabetic environment influences S. aureus skin infection and observed an increased susceptibility to infection in mouse models of both type I and type II diabetes. A dual gene expression approach was taken to investigate transcriptional alterations in both the host and bacterium after infection. While analysis of the host response revealed only minor changes between infected control and diabetic mice, we observed that S. aureus isolated from diabetic mice had significant increases in the levels of genes associated with translation and posttranslational modification and chaperones and reductions in the levels of genes associated with amino acid transport and metabolism. One family of genes upregulated in S. aureus isolated from diabetic lesions encoded the Clp proteases, associated with the misfolded protein response. The Clp proteases were found to be partially glucose regulated as well as influencing the hemolytic activity of S. aureus. Strains lacking the Clp proteases ClpX, ClpC, and ClpP were significantly attenuated in our animal model of skin infection, with significant reductions observed in dermonecrosis and bacterial burden. In particular, mutations in clpP and clpX were significantly attenuated and remained attenuated in both normal and diabetic mice. Our data suggest that the diabetic environment also causes changes to occur in invading pathogens, and one of these virulence determinants is the Clp protease system.
Ultrasensitive multi-species detection of CRISPR-Cas9 by a portable centrifugal microfluidic platform
Analytical Methods
The discovery of the RNA-guided DNA nuclease CRISPR-Cas9 has enabled the targeted editing of genomes from diverse organisms, but the permanent and inheritable nature of genome modification also poses immense risks. The potential for accidental exposure, malicious use, or undesirable persistence of Cas9 therapeutics and off-target genome effects highlight the need for detection assays. Here we report a centrifugal microfluidic platform for the measurement of both Cas9 protein levels and nuclease activity. Because Cas9 from many bacterial species have been adapted for biotechnology applications, we developed the capability to detect Cas9 from the widely-used S. pyogenes, as well as S. aureus, N. meningitidis, and S. thermophilus using commercially-available antibodies. Further, we show that the phage-derived anti-CRISPR protein AcrIIC1, which binds to Cas9 from several species, can be used as a capture reagent to broaden the species range of detection. As genome modification generally requires Cas9 nuclease activity, a fluorescence-based sedimentation nuclease assay was also incorporated to allow the sensitive and simultaneous measurement of both Cas9 protein and activity in a single biological sample.
More with Less: Novel Approaches to LAMP Assay Design for Better Performance with Fewer Resources
Abstract not provided.
Multi-Species Detection of Cas9 Presence and Activity on a Portable Centrifugal Microfluidic Platform
Abstract not provided.
Development of Rapid Diagnostic Tools for Detection and Quantification of Cas9 Presence and Activity
Abstract not provided.
Colorimetric-Luminance Readout for Quantitative Analysis of Fluorescence Signals with a Smartphone CMOS Sensor
Analytical Chemistry
Smartphones have shown promise as an enabling technology for portable and distributed point-of-care diagnostic tests. The CMOS camera sensor can be used for detecting optical signals, including fluorescence for applications such as isothermal nucleic acid amplification tests. However, such analysis is typically limited mostly to end point detection of single targets. Here we present a smartphone-based image analysis pipeline that utilizes the CIE xyY (chromaticity-luminance) color space to measure the luminance (in lieu of RGB intensities) of fluorescent signals arising from nucleic acid amplification targets, with a discrimination sensitivity (ratio between the positive to negative signals), which is an order of magnitude more than traditional RGB intensity based analysis. Furthermore, the chromaticity part of the analysis enables reliable multiplexed detection of different targets labeled with spectrally separated fluorophores. We apply this chromaticity-luminance formulation to simultaneously detect Zika and chikungunya viral RNA via end point RT-LAMP (Reverse transcription Loop-Mediated isothermal amplification). We also show real time LAMP detection of Neisseria gonorrhoeae samples down to a copy number of 3.5 copies per 10 μL of reaction volume in our smartphone-operated portable LAMP box. Our chromaticity-luminance analysis is readily adaptable to other types of multiplexed fluorescence measurements using a smartphone camera.
Molecular detection of emerging pathogens in low-resource settings
Abstract not provided.
Smartphone Assays for Detecting Emerging Pathogens in Low Resource Settings
Abstract not provided.
Impact of primer dimers and self-amplifying hairpins on reverse transcription loop-mediated isothermal amplification detection of viral RNA
Analyst
Loop-mediated isothermal amplification (LAMP), coupled with reverse transcription (RT), has become a popular technique for detection of viral RNA due to several desirable characteristics for use in point-of-care or low-resource settings. The large number of primers in LAMP (six per target) leads to an increased likelihood of primer dimer interactions, and the inner primers in particular are prone to formation of stable hairpin structures due to their length (typically 40-45 bases). Although primer dimers and hairpin structures are known features to avoid in nucleic acid amplification techniques, there is little quantitative information in literature regarding the impact of these structures on LAMP or RT-LAMP assays. In this study, we examine the impact of primer dimers and hairpins on previously published primer sets for dengue virus and yellow fever virus. We demonstrate that minor changes to the primers to eliminate amplifiable primer dimers and hairpins improves the performance of the assays when monitored in real time with intercalating dyes, and when monitoring a fluorescent endpoint using the QUASR technique. We also discuss the thermodynamic implications of these minor changes on the overall stability of amplifiable secondary structures, and we present a single thermodynamic parameter that can be correlated to the probability of non-specific amplification associated with LAMP primers.
Molecular detection of emerging pathogens in low-resource settings
Abstract not provided.
Smart Phone Diagnostic Platform For Detection of Emerging Viral Pathogens
Smart trap for autonomous monitoring of mosquito-borne viruses
Abstract not provided.
A smartphone-based diagnostic platform for rapid detection of Zika chikungunya and dengue viruses
Abstract not provided.
Single-Step One-Pot DNA Amplification & Electrochemical Detection via Loop-Mediated Isothermal Amplification (LAMP)
Abstract not provided.
Single-Step One-Pot DNA Amplification & Electrochemical Detection via Loop-Mediated Isothermal Amplification (LAMP)
Abstract not provided.
A smartphone-based diagnostic platform for rapid detection of Zika, chikungunya, and dengue viruses
Scientific Reports
Current multiplexed diagnostics for Zika, dengue, and chikungunya viruses are situated outside the intersection of affordability, high performance, and suitability for use at the point-of-care in resource-limited settings. Consequently, insufficient diagnostic capabilities are a key limitation facing current Zika outbreak management strategies. Here we demonstrate highly sensitive and specific detection of Zika, chikungunya, and dengue viruses by coupling reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with our recently developed quenching of unincorporated amplification signal reporters (QUASR) technique. We conduct reactions in a simple, inexpensive and portable "LAMP box" supplemented with a consumer class smartphone. The entire assembly can be powered by a 5 V USB source such as a USB power bank or solar panel. Our smartphone employs a novel algorithm utilizing chromaticity to analyze fluorescence signals, which improves the discrimination of positive/negative signals by 5-fold when compared to detection with traditional RGB intensity sensors or the naked eye. The ability to detect ZIKV directly from crude human sample matrices (blood, urine, and saliva) demonstrates our device's utility for widespread clinical deployment. Together, these advances enable our system to host the key components necessary to expand the use of nucleic acid amplification-based detection assays towards point-of-care settings where they are needed most.
Quenching of Unincorporated Amplification Signal Reporters (QUASR) for Robust Monitoring of Isothermal DNA and RNA Amplification Assays
Abstract not provided.
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