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1.
Anal Chem ; 93(9): 4160-4165, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33631932

RESUMO

The rapid onset of the global COVID-19 pandemic has led to challenges for accurately diagnosing the disease, including supply shortages for sample collection, preservation, and purification. Currently, most diagnostic tests require RNA extraction and detection by RT-PCR; however, extraction is expensive and time-consuming and requires technical expertise. With these challenges in mind, we report extraction-free, multiplexed amplification of SARS-CoV-2 RNA from 246 clinical samples, resulting in 86% sensitivity and 100% specificity. The multiplex RT-PCR uses the CDC singleplex targets and has an LoD of 2 c/µL. We also report on amplification using a range of master mixes in different transport media. This work can help guide which combinations of reagents will enable accurate results when availability of supplies changes throughout the pandemic. Implementing these methods can reduce complexity and cost, minimize reagent usage, expedite time to results, and increase testing capacity.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , COVID-19/virologia , Reação em Cadeia da Polimerase Multiplex/métodos , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Humanos , RNA Viral/genética , Sensibilidade e Especificidade
2.
Anal Chem ; 92(5): 3535-3543, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-31999432

RESUMO

Immunoassays are important for the detection of proteins to enable disease identification and monitor treatment, but many immunoassays suffer from sensitivity limitations. The development of digital assays has enabled highly sensitive biomarker detection and quantification, but the necessary devices typically require precisely controlled volumes to reduce biases in concentration estimates from compartment size variation. These constraints have led to systems that are often expensive, cumbersome, and challenging to operate, confining many digital assays to centralized laboratories. To overcome these limitations, we have developed a simplified digital immunoassay performed in polydisperse droplets that are prepared without any specialized equipment. This polydisperse digital droplet immunoassay (ddIA) uses proximity ligation to remove the need for wash steps and simplifies the system to a single reagent addition step. Using interleukin-8 (IL-8) as an example analyte, we demonstrated the concept with samples in buffer and diluted whole blood with limits of detection of 0.793 pM and 1.54 pM, respectively. The development of a one-pot, washless assay greatly improves usability compared to traditional immunoassays or digital-based systems that rely heavily on wash steps and can be run with common and readily available laboratory equipment such as a heater and simple fluorescent microscope. We also developed a stochastic model with physically meaningful parameters that can be utilized to optimize the assay and enable quantification without standard curves, after initial characterization of the parameters. Our polydisperse ddIA assay serves as an example of sensitive, lower-cost, and simpler immunoassays suitable for both laboratory and point-of-care applications.


Assuntos
Imunoensaio/instrumentação , Dispositivos Lab-On-A-Chip , Interleucina-8/análise , Limite de Detecção
3.
Analyst ; 144(24): 7209-7219, 2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31663521

RESUMO

In digital assays, devices are typically considered to require precisely controlled volumes since variation in compartment volumes causes biases in concentration estimates. To enable more possibilities in device design, we derived two methods to accurately calculate target concentrations from raw results when the compartment volume may vary and may not follow known parametrically described distributions. The Digital Variable Volume (dvv) method uses volumes of ON compartments (those with positive signals) and the total sample volume, while the Digital Variable Volume Approximation (dvva) method uses the number of ON compartments, the total number of compartments, and a set of separately measured volumes. We verified the trueness of the dvv and dvva methods using simulated assays where volumes followed an empirical distribution (based on measured droplet volumes) and well known distributions with a wide range of standard deviations. We applied both methods to digital PCR experiments with polydisperse volumes, and also derived equations to estimate standard errors and limits of detection. The dvv method allows the compartment volume to follow any distribution in each assay run, the dvva method allows for quantification without in-assay volume measurements, and both methods potentially enable new designs of digital assays.

4.
Anal Chem ; 90(15): 9374-9380, 2018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-29985594

RESUMO

Nucleic acid amplification technology, such as polymerase chain reaction (PCR), has enabled highly sensitive and specific disease detection and quantification, leading to more accurate diagnosis and treatment regimens. Lab-on-a-chip applications have developed methods to partition single biomolecules, such as DNA and RNA, into picoliter-sized droplets. These individual reaction vessels lead to digitization of PCR enabling improved time to detection and direct quantification of nucleic acids without a standard curve, therefore simplifying assay analysis. Though impactful, these improvements have generally been restricted to centralized laboratories with trained personnel and expensive equipment. To address these limitations and make this technology more applicable for a variety of settings, we have developed a statistical framework to apply to droplet PCR performed in polydisperse droplets prepared without any specialized equipment. The polydisperse droplet system allows for accurate quantification of droplet digital PCR (ddPCR) and reverse transcriptase droplet digital PCR (RT-ddPCR) that is comparable to commercially available systems such as BioRad's ddPCR. Additionally, this approach is compatible with a range of input sample volumes, extending the assay dynamic range beyond that of commercial ddPCR systems. In this work, we show that these ddPCR assays can reduce overall assay time while still providing quantitative results. We also report a multiplexed ddPCR assay and demonstrate proof-of-concept methods for rapid droplet preparation in multiple samples simultaneously. Our simple polydisperse droplet preparation and statistical framework can be extended to a variety of settings for the quantification of nucleic acids in complex samples.


Assuntos
Dispositivos Lab-On-A-Chip , Reação em Cadeia da Polimerase/métodos , DNA/análise , Emulsões , RNA/análise
5.
Analyst ; 143(12): 2828-2836, 2018 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-29781480

RESUMO

Microbiological culture remains the most sensitive method for detecting viable and infectious bacteria, but these methods often require at least 24 hours to visibly identify bacterial growth. Lab-on-a-chip applications have utilized methods to isolate bacteria in picoliter-sized reaction vessels, resulting in digitized signals that offer improved time-to-detection and improved quantification. Although a great improvement, these approaches typically require expensive and specialized equipment, trained laboratory personnel, and maximum addressable volumes that can be orders of magnitude less than needed for clinically relevant limits of detection. To address these limitations, we have developed a simple method for preparing and semi-quantitatively analyzing small-volume droplets for performing digital culture, allowing for the detection of bacteria. This work includes a description of the method, characterization of resulting droplet sizes, comparison to traditional culture, and a statistical framework to quantify results. Though polydisperse, the droplet size distribution was consistent over different experiments, and there was a correlation between the observed number of positive droplets and the bulk concentration that can serve as a calibration curve for samples with unknown droplet size distributions. This statistical framework enables the simplification of droplet preparation and allows for accurate quantification even with polydisperse droplet sizes. The application of this method can also be extended to a variety of settings for the detection or quantification of bacteria in complex samples.


Assuntos
Bactérias/isolamento & purificação , Técnicas Bacteriológicas/métodos , Dispositivos Lab-On-A-Chip , Bioensaio , Emulsões
6.
ACS Omega ; 6(39): 25116-25123, 2021 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-34608447

RESUMO

The global COVID-19 pandemic has created an urgent demand for large numbers of inexpensive, accurate, rapid, point-of-care diagnostic tests. Analyte-based assays are suitably rapid and inexpensive and can be rapidly mass-produced, but for sufficiently accurate performance, they require highly optimized antibodies and assay conditions. We used an automated liquid handling system, customized to handle arrays of lateral flow (immuno)assays (LFAs) in a high-throughput screen, to identify anti-nucleocapsid antibodies that will perform optimally in an LFA. We tested 1021 anti-nucleocapsid antibody pairs as LFA capture and detection reagents with the goal of highlighting pairs that have the greatest affinity for the nucleocapsid protein of SARS-CoV-2 within the LFA format. In contrast to traditional antibody screening methods (e.g., ELISA, bio-layer interferometry), the method described here integrates real-time reaction kinetics with transport in, and immobilization directly onto, nitrocellulose. We have identified several candidate antibody pairs that are suitable for further development of an LFA for SARS-CoV-2.

7.
Expert Rev Mol Diagn ; 18(1): 19-26, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29200322

RESUMO

INTRODUCTION: Biomarkers are objective indications of a medical state that can be measured accurately and reproducibly. Traditional biomarkers enable diagnosis of disease through detection of disease-specific molecules, disease-mediated molecular changes, or distinct physiological or anatomical signatures. Areas covered: This work provides a framework for selecting biomarkers that are most likely to provide useful information about a patient's disease state. Though the authors emphasize markers related to disease, this work is also applicable to biomarkers for monitoring physiological changes such as ovulation or pregnancy. Additionally, the scope was restricted to biomarkers that are amenable to analytical detection across a range of health care levels, including low resource settings. The authors describe trade-offs between biomarkers' sensitivity/specificity for a disease-causing agent, the complexity of detection, and how this knowledge can be applied to the development of diagnostic tests. This report also details additional assessment criteria for successful tests. Expert commentary: Biomarker selection should primarily be driven by an attempt to answer an explicit clinical question (preferably causative relationship of the biomarker to disease-state), and only then by test development expediency (ease of detection). This framework is useful for stakeholders from test developers to clinicians to identify the trade-offs for diagnostic biomarkers for any use case.


Assuntos
Biomarcadores , Técnicas de Diagnóstico Molecular , Humanos , Técnicas de Diagnóstico Molecular/economia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
8.
Lab Chip ; 16(19): 3777-87, 2016 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-27549897

RESUMO

The prototype demonstrated here is the first fully integrated sample-to-result diagnostic platform for performing nucleic acid amplification tests that requires no permanent instrument or manual sample processing. The multiplexable autonomous disposable nucleic acid amplification test (MAD NAAT) is based on two-dimensional paper networks, which enable sensitive chemical detection normally reserved for laboratories to be carried out anywhere by untrained users. All reagents are stored dry in the disposable test device and are rehydrated by stored buffer. The paper network is physically multiplexed to allow independent isothermal amplification of multiple targets; each amplification reaction is also chemically multiplexed with an internal amplification control. The total test time is less than one hour. The MAD NAAT prototype was used to characterize a set of human nasal swab specimens pre-screened for methicillin-resistant Staphylococcus aureus (MRSA) bacteria. With qPCR as the quantitative reference method, the lowest input copy number in the range where the MAD NAAT prototype consistently detected MRSA in these specimens was ∼5 × 10(3) genomic copies (∼600 genomic copies per biplexed amplification reaction).


Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Desenho de Equipamento , Humanos , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Nariz/microbiologia , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Papel , Fatores de Tempo
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