RESUMEN
The lack of hepatitis C virus (HCV) diagnostic tests designed for use in decentralized settings is a major obstacle for providing access to treatment and prevention services particularly in low and middle income countries. Here we describe the development and validation of two building blocks of the HCV Quant Assay, a test in development for point-of-care use: 1) an RT-qPCR assay with noncompetitive internal control that equivalently detects the 6 major HCV genotypes and 2) an automated sample prep method using immiscible phase filter technology. This novel assay has wide dynamic range of HCV quantification and a limit of detection of 30IU/ml with 200µl specimen volume. In a preliminary study of 61 clinical specimens, the HCV Quant Assay demonstrated 100% sensitivity and specificity and gave comparable viral load results across 4 logs of IU/ml when compared to the Abbott RealTime HCV Assay.
Asunto(s)
Hepacivirus/genética , Hepatitis C/diagnóstico , Sistemas de Atención de Punto , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Genotipo , Hepacivirus/aislamiento & purificación , Hepatitis C/prevención & control , Hepatitis C/virología , Humanos , ARN Viral/aislamiento & purificación , Juego de Reactivos para Diagnóstico/estadística & datos numéricos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Pruebas Serológicas , Carga Viral/métodosRESUMEN
Nucleic acid amplification tests for Mycobacterium tuberculosis (MTB) detection from sputum are highly sensitive and specific with smear microscopy positive specimens, but their sensitivity with smear-negative/culture-positive specimens is much lower; therefore, these tests cannot rule out a tuberculosis diagnosis. Co-extraction of PCR inhibitors may be a cause of decreased test sensitivity. Here the design and early validation of a MTB screening assay with sample preparation and qPCR methods designed to specifically address this diagnostic gap is reported. First, human genomic DNA is identified as a significant qPCR inhibitor. To circumvent this problem, a novel, streamlined sample preparation method utilizing detergent and proteolysis to thin the sputum and DNA sequence specific MTB DNA isolation was developed. Additionally, a multiplexed qPCR assay targeting two MTB complex-specific loci: the potentially multi-copy IS6110 and the single-copy senX3-regX3, combined with the cotJC gene from Bacillus atrophaeus spores amplified as a process control was developed. The limit of detection of the test was estimated to be 20 cfu/ml which is significantly lower than the Xpert® MTB/RIF assay. In a preliminary field study of 60 de-identified blinded sputa, a test sensitivity of 96% and specificity of 100% was observed when compared to the Xpert® MTB/RIF assay.
Asunto(s)
Mycobacterium tuberculosis/aislamiento & purificación , Esputo/microbiología , Tuberculosis Pulmonar/diagnóstico , ADN Bacteriano/análisis , Humanos , Tamizaje Masivo/métodos , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Mycobacterium tuberculosis/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sensibilidad y Especificidad , Manejo de Especímenes/métodosRESUMEN
Early diagnosis and access to treatment for infants with human immunodeficiency virus-1 (HIV-1) is critical to reduce infant mortality. The lack of simple point-of-care tests impedes the timely initiation of antiretroviral therapy. The development of FINA, filtration isolation of nucleic acids, a novel DNA extraction method that can be performed by clinic personnel in less than 2 min has been reported previously. In this report, significant improvements in the DNA extraction and amplification methods are detailed that allow sensitive quantitation of as little as 10 copies of HIV-1 proviral DNA and detection of 3 copies extracted from 100 µl of whole blood. An internal control to detect PCR inhibition was also incorporated. In a preliminary field evaluation of 61 South African infants, the FINA test demonstrated 100% sensitivity and specificity. The proviral copy number of the infant specimens was quantified, and it was established that 100 microliters of whole blood is required for sensitive diagnosis of infants.
Asunto(s)
Sangre/virología , ADN Viral/aislamiento & purificación , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , Provirus/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Manejo de Especímenes/métodos , ADN Viral/genética , VIH-1/genética , Humanos , Lactante , Recién Nacido , Provirus/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Estándares de Referencia , Sensibilidad y Especificidad , Manejo de Especímenes/normas , Factores de Tiempo , Carga Viral/métodosRESUMEN
A low-cost, fully integrated sample-to-answer, quantitative PCR (qPCR) system that can be used for detection of HIV-1 proviral DNA in infants at the point-of-care in resource-limited settings has been developed and tested. The system is based on a novel DNA extraction method, which uses a glass fiber membrane, a disposable assay card that includes on-board reagent storage, provisions for thermal cycling and fluorescence detection, and a battery-operated portable analyzer. The system is capable of automated PCR mix assembly using a novel reagent delivery system and performing qPCR. HIV-1 and internal control targets are detected using two spectrally separated fluorophores, FAM and Quasar 670. In this report, a proof-of-concept of the platform is demonstrated. Initial results with whole blood demonstrate that the test is capable of detecting HIV-1 in blood samples containing greater than 5000 copies of HIV-1. In resource-limited settings, a point-of-care HIV-1 qPCR test would greatly increase the number of test results that reach the infants caregivers, allowing them to pursue anti-retroviral therapy.
Asunto(s)
Técnicas Biosensibles , ADN Viral/aislamiento & purificación , VIH-1/aislamiento & purificación , ADN Viral/química , Infecciones por VIH/diagnóstico , Infecciones por VIH/virología , VIH-1/química , Humanos , Recién Nacido , Reacción en Cadena de la PolimerasaRESUMEN
A major requirement for the development of point-of-care tests for the detection of disease analytes is the need to separate plasma from whole blood in an efficient and rapid manner. Furthermore, the separated plasma must be able to elute efficiently the analyte of interest and serve effectively as a physical matrix to deliver the equivalent of neat plasma for downstream diagnostic analysis. Additionally, many applications require the use of heat shock to liberate immunocomplexed antigen found in the collected plasma. A membrane-based filter method is reported for rapid and efficient collection of plasma from a whole blood sample that is compatible with heat shock. Using pediatric human immunodeficiency virus as an example, this device elutes 100% of the input p24 core antigen post-collection and enables heat shock of plasma samples identical to neat plasma treatment.
Asunto(s)
Filtración/métodos , Infecciones por VIH/diagnóstico , Plasma/virología , Sistemas de Atención de Punto , Manejo de Especímenes/métodos , Virología/métodos , HumanosRESUMEN
In this report, we demonstrate the purification of DNA and RNA from a 10% serum sample using an oligonucleotide capture matrix. This approach provides a one-stage, completely aqueous system capable of purifying both RNA and DNA for downstream PCR amplification. The advantages of utilizing the polymer capture matrix method in place of the solid-phase extraction method is that the capture matrix eliminates both guanidine and the 2-propanol wash that can inhibit downstream PCR and competition with proteins for the binding sites that can limit the capacity of the device. This method electrophoreses a biological sample (e.g., serum) containing the nucleic acid target through a polymer matrix with covalently bound oligonucleotides. These capture oligonucleotides selectively hybridize and retain the target nucleic acid, while the other biomolecules and reagents (e.g., SDS) pass through the matrix to waste. Following this purification step, the solution can be heated above the melting temperature of the capture sequence to release the target molecule, which is then electrophoresed to a recovery chamber for subsequent PCR amplification. We demonstrate that the device can be applied to purify both DNA and RNA from serum. The gag region of HIV at a starting concentration of 37.5 copies per microliter was successfully purified from a 10% serum sample demonstrating the applicability of this method to detect viruses present in low copy numbers.
Asunto(s)
Resinas Acrílicas/química , VIH/química , Técnicas Analíticas Microfluídicas/métodos , ARN Viral/aislamiento & purificación , Técnicas Analíticas Microfluídicas/instrumentación , Reacción en Cadena de la Polimerasa/métodos , ARN Viral/sangreRESUMEN
Currently, the majority of HIV-infected infants are found within limited-resource settings, where inadequate screening for HIV due to the lack of access to simple and affordable point-of-care tests impedes implementation of antiretroviral therapy. Here we report development of a low-cost dipstick p24 antigen assay using a visual readout format that can facilitate the diagnosis of HIV for infants in resource-poor conditions. A heat shock methodology was developed to optimize disruption of immune complexes present in the plasma of infected infants. The analytical sensitivity of the assay using recombinant p24 antigen is 50 pg/mL (2 pM) with whole virus detection as low as 42.5k RNA copies per milliliter plasma. In a blinded study comprising 51 archived infant samples from the Women and Infants Transmission Study, our assay demonstrated an overall sensitivity and specificity of 90% and 100%, respectively. In field evaluations of 389 fresh samples from South African infants, a sensitivity of 95% and specificity of 99% was achieved. The assay is simple to perform, requires minimal plasma volume (25 µL), and yields a result in less than 40 minutes making it ideal for implementation in resource-limited settings.
Asunto(s)
Proteína p24 del Núcleo del VIH/sangre , Infecciones por VIH/diagnóstico , VIH/aislamiento & purificación , África del Sur del Sahara , Anticuerpos Monoclonales/inmunología , Femenino , Anticuerpos Anti-VIH/inmunología , Proteína p24 del Núcleo del VIH/inmunología , Humanos , Inmunoensayo , Lactante , Recién Nacido , Tiras Reactivas , Proteínas Recombinantes/sangre , Proteínas Recombinantes/inmunología , Sensibilidad y Especificidad , Estados UnidosRESUMEN
Extraction and purification of nucleic acids from complex biological samples for PCR are critical steps because inhibitors must be removed that can affect reaction efficiency and the accuracy of results. This preanalytical processing generally involves capturing nucleic acids on microparticles that are then washed with a series of buffers to desorb and dilute out interfering substances. We have developed a novel purification method that replaces multiple wash steps with a single pass of paramagnetic particles (PMPs) though an immiscible hydrophobic liquid. Only two aqueous solutions are required: a lysis buffer, in which nucleic acids are captured on PMPs, and an elution buffer, in which they are released for amplification. The PMPs containing the nucleic acids are magnetically transported through a channel containing liquid wax that connects the lysis chamber to the elution chamber in a specially designed cartridge. Transporting PMPs through the immiscible phase yielded DNA and RNA as pure as that obtained after extensive wash steps required by comparable purification methods. Our immiscible-phase process has been applied to targets in whole blood, plasma, and urine and will enable the development of faster and simpler purification systems.
Asunto(s)
Ácidos Nucleicos/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Infecciones por Chlamydia/diagnóstico , Infecciones por Chlamydia/microbiología , Chlamydia trachomatis/genética , Chlamydia trachomatis/aislamiento & purificación , Gonorrea/diagnóstico , Infecciones por VIH/diagnóstico , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/aislamiento & purificaciónRESUMEN
RNA remains the most informative and accurate biomarker for human immunodeficiency virus type 1 load diagnostics and for surveillance of drug resistance markers. Viral load testing by nucleic acid amplification currently is a complex and expensive test that is restricted to centralized laboratory testing. Successful extension of centralized viral load testing to rural or remote settings is a major challenge. Emerging nucleic acid-based technologies are progressing rapidly toward platforms appropriate for field use in low-resource settings, leaving a growing gap for sample processing technologies that complement them. One area in which new technologies could be applied to improve access is clinical specimen preservation and processing. Novel technologies that extract nucleic acid from clinical specimens and stabilize it at the point of specimen collection could fill this gap. In addition, these technologies may provide alternative viral load detection and surveillance solutions to the current centralized laboratory testing paradigm.
Asunto(s)
Infecciones por VIH/virología , VIH/aislamiento & purificación , Manejo de Especímenes/métodos , Carga Viral/métodos , Humanos , ARN Viral/sangreRESUMEN
Ekins' ambient analyte theory predicts, counterintuitively, that an immunoassay's limit of detection can be improved by reducing the amount of capture antibody. In addition, it also anticipates that results should be insensitive to the volume of sample as well as the amount of capture antibody added. The objective of this study was to empirically validate all of the performance characteristics predicted by Ekins' theory. Flow cytometric analysis was used to detect binding between a fluorescent ligand and capture microparticles because it can directly measure fractional occupancy, the primary response variable in ambient analyte theory. After experimentally determining ambient analyte conditions, comparisons were carried out between ambient and nonambient assays in terms of their signal strengths, limits of detection, and sensitivity to variations in reaction volume and number of particles. The critical number of binding sites required for an assay to be in the ambient analyte region was estimated to be 0.1 VK(d). As predicted, such assays exhibited superior signal/noise levels and limits of detection and were not affected by variations in sample volume and number of binding sites. When the signal detected measures fractional occupancy, ambient analyte theory is an excellent guide to developing assays with superior performance characteristics.
Asunto(s)
Inmunoensayo/métodos , Algoritmos , Anticuerpos Inmovilizados/inmunología , Anticuerpos Inmovilizados/metabolismo , Sitios de Unión , Citometría de Flujo , Colorantes Fluorescentes , Cinética , Límite de DetecciónRESUMEN
To push the 100-plex envelope of suspension array technology, we have developed fully automated methods to acquire multispectral images of multiplexed quantum-dot (QD) encoded microspheres, to segment them in the images, to classify them based on their color code, and to quantify the multiplexed assays. Instead of coding microspheres with two colors and n levels, microspheres were coded with n colors and two levels (present or absent), thus transforming the classification problem from analog to digital. Images of multiplexed microspheres, sedimented at the bottom of microwells, were acquired through a tunable filter at the peak luminescence wavelength of each QD coding species in the system and the assay label wavelength. Another image of the light scattered from microspheres was captured in the excitation bandwidth that was utilized to localize microspheres in multispectral luminescence images. Objects in the acquired images are segmented and luminescence from each identified microsphere in each channel is recorded, based on which the "color code" of each microsphere is determined by applying a mathematical model and a classification algorithm. Our image analysis procedures could identify and classify microspheres with more than 97% accuracy, and the assay CVs were under 20%. These proof-of-principle results demonstrate that highly multiplexed quantification of specific proteins is possible with this rapid, small-sample volume format.
Asunto(s)
Bioensayo/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Microesferas , Calibración , Puntos Cuánticos , Estándares de Referencia , Programas Informáticos , SuspensionesRESUMEN
PCR detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA is the method recommended for use for the diagnosis of HIV-1 infection in infants in limited-resource settings. Currently, testing must be performed in central laboratories, which are usually located some distance from health care facilities. While the collection and transportation of samples, such as dried blood spots, has improved test accessibility, the results are often not returned for several weeks. To enable PCR to be performed at the point of care while the mothers wait, we have developed a vertical filtration method that uses a separation membrane and an absorbent pad to extract cellular DNA from whole blood in less than 2 min. Cells are trapped in the separation membrane as the specimen is collected, and then a lysis buffer is added. The membrane retains the DNA, while the buffer washes away PCR inhibitors, which get wicked into the absorbent blotter pad. The membrane containing the entrapped DNA is then added to the PCR mixture without further purification. The method demonstrates a high degree of reproducibility and analytical sensitivity and allows the quantification of as few as 20 copies of HIV-1 proviral DNA from 100 microl of blood. In a blinded study with 182 longitudinal samples from infants (ages, 0 to 72 weeks) obtained from the Women and Infants Transmission Study, our assay demonstrated a sensitivity of 99% and a specificity of 100%.
Asunto(s)
Sangre/virología , ADN Viral/aislamiento & purificación , Infecciones por VIH/diagnóstico , VIH-1/genética , Sistemas de Atención de Punto , Reacción en Cadena de la Polimerasa/métodos , Provirus/genética , Femenino , Filtración/métodos , Infecciones por VIH/virología , Humanos , Lactante , Recién Nacido , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Here we report proof-of-principle for a microsphere-based genotyping assay that detects single nucleotide polymorphisms (SNPs) directly from human genomic DNA samples. This assay is based on a structure-specific cleavage reaction that achieves single base discrimination with a 5'-nuclease which recognizes a tripartite substrate formed upon hybridization of target DNA with probe and upstream oligonucleotides. The assay is simple with two easy steps: a cleavage reaction, which generates fluorescent signal on microsphere surfaces, followed by flow cytometry analysis of the microspheres. Genomic DNA samples were genotyped for the SNP in the Apolipoprotein E gene at amino acid position 158. The assay successfully scored wild type, heterozygous and homozygous mutants. To our knowledge, this is the first report of a solid-support assay for detection of SNPs directly from genomic DNA without PCR amplification of the target.
Asunto(s)
Citometría de Flujo , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN/métodos , Apolipoproteínas E/genética , Colorantes Fluorescentes/química , Genoma Humano , Genotipo , Humanos , Microesferas , Oligonucleótidos/químicaRESUMEN
Combining positive attributes of planar arrays and suspension arrays, immobilized particle arrays offer a new format in which immobilized submicrometer particles are arrayed on hydrogel-coated slides, providing 100+ assay replicates within each spot. This research describes how to prepare immobilized protein arrays and how to assay the binding of labeled target molecules to the arrayed capture probes. The assay system exhibits an intrinsic dynamic range of two to three decades, with coefficients of variation from 5 to 10%. For antibody-antigen binding, target capture appears to be reaction rate limited. For labeled antibody binding to antigen on the immobilized particles, the detection limit is approximately 0.5 ng/mL. When antibodies on the immobilized particles exhibit multivalent binding of target molecules, the detection limit is approximately 0.01 ng/mL. For protein arrays, potential advantages of this format are improved coating of the capture reagent, an increased number of options for protein presentation, reduced mass transport effects, and higher density multiplexing.
RESUMEN
An automated imaging system was developed to quantify fluorescence signals from particles immobilized on hydrogel-coated slides. Arrays of submicrometer-diameter particles were printed with up to 600 particles/spot. The slides were read under 20x magnification without cover slips. Software was written to image individual spots and measure the median particle fluorescence in each spot. To locate array spots, an alignment program made use of two fiducial grids of fluorescent reference particles at either end of the slide. Focusing was adjusted locally using spots of reference particles located at the centers of focusing neighborhoods. The response was linear across a two-decade range, and the precision of readings was better than 5% down to approximately 1000 fluors/particle. Exposure times varied with signal intensity, reaching 1 s at the lowest levels of fluorescence. Data demonstrate feasibility for measuring fluorescence from immobilized particle arrays on an automated microscope with accuracy and precision similar to fluorescence measurements of microparticles with a flow cytometer. This work provides automation of imaging and analysis procedures necessary for development of immobilized particle arrays as an analytical platform that combines advantageous features of both planar and suspension arrays.
RESUMEN
The structure-specific invasive cleavage reaction is a useful means for sensitive and specific detection of single nucleotide polymorphisms, or SNPs, directly from genomic DNA without a need for prior target amplification. A new approach integrating this invasive cleavage assay and surface DNA array technology has been developed for potentially large-scale SNP scoring in a parallel format. Two surface invasive cleavage reaction strategies were designed and implemented for a model SNP system in codon 158 of the human ApoE gene. The upstream oligonucleotide, which is required for the invasive cleavage reaction, is either co-immobilized on the surface along with the probe oligonucleotide or alternatively added in solution. The ability of this approach to unambiguously discriminate a single base difference was demonstrated using PCR-amplified human genomic DNA. A theoretical model relating the surface fluorescence intensity to the progress of the invasive cleavage reaction was developed and agreed well with experimental results.
Asunto(s)
ADN/química , ADN/genética , ADN/metabolismo , Genoma Humano , Humanos , Modelos Genéticos , Polimorfismo de Nucleótido Simple , Especificidad por Sustrato , Propiedades de Superficie , TemperaturaAsunto(s)
Ingeniería Biomédica/educación , Evaluación Educacional , Aprendizaje Basado en Problemas/métodos , Enseñanza , Acreditación/organización & administración , Acreditación/normas , Humanos , Evaluación de Necesidades , Aprendizaje Basado en Problemas/normas , Aprendizaje Basado en Problemas/tendencias , Sociedades Científicas , Enseñanza/normas , Enseñanza/tendencias , Estados Unidos , Universidades , Recursos HumanosRESUMEN
The structure-specific invasive cleavage of single-stranded DNA by 5' nucleases is a useful means for sensitive detection of single-nucleotide polymorphisms or SNPs. The solution-phase invasive cleavage reaction has sufficient sensitivity for direct detection of as few as 600 target molecules with no prior target amplification. One approach to the parallelization of SNP analysis is to adapt the invasive cleavage reaction to an addressed array format. Two surface invasive cleavage reaction strategies were designed and tested using the polymorphic site in codon 158 of the human ApoE gene as a model system, with a synthetic oligonucleotide as target. The upstream oligonucleotide, which is required for the invasive cleavage reaction, was either added in solution (strategy 1), or co-immobilized on the surface along with the probe oligonucleotide (strategy 2). Both strategies showed target-concentration and time-dependent amplification of signal. Parameters that govern the rate of the surface-invasive cleavage reactions are discussed.