RESUMO
Adulteration of meat is a global issue, necessitating rapid, inexpensive, and simple on-site testing methods. Therefore, the present study aimed to develop a one-minute toothpick-based DNA extraction method, a handheld microfluidic chip, and a smartphone-controlled portable analyzer for detecting multiple meat adulterations. A toothpick was inserted into the meat to promote DNA release and adsorption. Furthermore, a handheld microfluidic chip was designed for DNA elution on toothpicks and fluid distribution. Finally, a smartphone-actuated portable analyzer was developed to function as a heater, signal detector, and result reader. The portable device comprises a microcontroller, a fluorescence detection module, a step scanning unit, and a heating module. The proposed device is portable, and the app is user-friendly. This simple design, easy operation, and fast-response system could rapidly detect as little as 1% of simulated adulterated samples (following UK standards) within 40 min at a cost of less than USD 1 per test.
Assuntos
DNA , Contaminação de Alimentos , Carne , Contaminação de Alimentos/análise , Carne/análise , DNA/isolamento & purificação , DNA/análise , Animais , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Smartphone , Dispositivos Lab-On-A-Chip , Técnicas de Diagnóstico MolecularRESUMO
Uracil-DNA glycosylase (UDG), an enzyme for repairing uracil-containing DNA damage, is crucial for maintaining genomic stability. Simple and fast quantification of UDG activity is essential for biological assay and clinical diagnosis, since its aberrant level is associated with DNA damage and various diseases. Herein, we developed a fully integrated "sample in-signal out" distance-based paper analytical device (dPAD) for visual quantification of UDG using a flow-controlled uracil-rich DNA hydrogel (URDH). The uracil base sites contained in the DNA hydrogel are mis-incorporated with dUTP by rolling circle amplification (RCA), which simplifies the preparation process of the functionalized hydrogel. In the presence of UDG, the uracil in URDH can be recognized and removed to induce the permeability change of URDH, resulting in the visible distance signal along the paper channel. Using dPAD, as low as 6.4 × 10-4 U/mL of UDG (within 80 min) is visually identified without any instruments and complicated operations. This integrated dPAD is advantageous for its simplicity, cost effectiveness, and ease of use. We envision that it has the great potential for point-of-care testing (POCT) in DNA damage testing, personalized healthcare assessment, and biomedical applications.
Assuntos
Técnicas Biossensoriais , DNA , Hidrogéis , Papel , Uracila-DNA Glicosidase , Uracila , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/instrumentação , Humanos , DNA/química , Uracila/química , Hidrogéis/química , Desenho de Equipamento , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Limite de Detecção , Dano ao DNARESUMO
Timely and accurate detection of viruses is crucial for infection diagnosis and treatment. However, it remains a challenge to develop a portable device that meets the requirement of being portable, powerless, user-friendly, reusable, and low-cost. This work reports a compact ∅30 × 48 mm portable powerless isothermal amplification detection device (material cost â¼$1 USD) relying on LAMP (Loop-Mediated Isothermal Amplification). We have proposed chromatographic-strip-based microporous permeation technology which can precisely control the water flow rate to regulate the exothermic reaction. This powerless heating combined with phase-change materials can maintain a constant temperature between 50 and 70 °C for a duration of up to 49.8 min. Compared with the conventional methods, it avoids the use of an additional insulation layer for heat preservation, greatly reducing the size and cost. We have also deployed a color card and a corresponding algorithm to facilitate color recognition, data analysis, and storage using a mobile phone. The experimental results demonstrate that our device exhibits the same limit of detection (LOD) as the ProFlex PCR for SARS-CoV-2 pseudovirus samples, with that for both being 103 copies/µL, verifying its effectiveness and reliability. This work offers a timely, low-cost, and easy way for respiratory infectious disease detection, which could provide support in curbing virus transmission and protecting the health of humans and animals, especially in remote mountainous areas without access to electricity or trained professionals.
Assuntos
COVID-19 , Técnicas de Amplificação de Ácido Nucleico , SARS-CoV-2 , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/genética , Humanos , COVID-19/diagnóstico , COVID-19/virologia , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/economia , Limite de DetecçãoRESUMO
A low-cost, handheld centrifugal microfluidic system for multiplexed visual detection based on recombinase polymerase amplification (RPA) was developed. A concise centrifugal microfluidic chip featuring four reaction units was developed to run multiplexed RPA amplification in parallel. Additionally, a significantly shrunk-size and cost-effective handheld companion device was developed, incorporating heating, optical, rotation, and sensing modules, to perform multiplexed amplification and visual detection. After one-time sample loading, the metered sample was equally distributed into four separate reactors with high-speed centrifugation. Non-contact heating was adopted for isothermal amplification. A tiny DC motor on top of the chip was used to drive steel beads inside reactors for active mixing. Another small DC motor, which was controlled by an elaborate locking strategy based on magnetic sensing, was adopted for centrifugation and positioning. Visual fluorescence detection was optimized from different sides, including material, surface properties, excitation light, and optical filters. With fluorescence intensity-based visual detection, the detection results could be directly observed through the eyes or with a smartphone. As a proof of concept, the handheld device could detect multiple targets, e.g., different genes of African swine fever virus (ASFV) with the comparable LOD (limit of detection) of 75 copies/test compared to the tube-based RPA.
Assuntos
Técnicas de Amplificação de Ácido Nucleico , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Vírus da Febre Suína Africana/isolamento & purificação , Vírus da Febre Suína Africana/genética , Dispositivos Lab-On-A-Chip , Limite de Detecção , Centrifugação/instrumentação , Animais , Smartphone , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Técnicas Analíticas Microfluídicas/economiaRESUMO
In the past, vast research has been conducted on biosensors and point-of-care (PoC) diagnostics. Despite rapid advances especially during the SARS-CoV-2 pandemic in this research field a low-cost molecular biosensor exhibiting the user-friendliness of a rapid antigen test, and also the sensitivity and specificity of a PCR test, has not been developed yet. To this end we developed a novel microfluidics based and handheld PoC device, that facilitates viral detection at PCR sensitivity and specificity in less than 40 min, including 15 min sample preparation. This was attained by incorporation of pulse controlled amplification (PCA), a method which uses short electrical pulses to rapidly increase the temperature of a small fraction of the sample volume. In this work, we present a low-cost PCA device with a microfluidic consumable intended for the use in a decentralized or home-setting. We used finite element analysis (FEA) simulations to display the fundamental principle and highlight the critical parameter dependency of PCA, such as pulse length and resistor shape. Furthermore, we integrated a simple and fast workflow for sample preparation and evaluated the limit of detection (LoD) for SARS-CoV-2 viral RNA, which is 0.88 copies/µL (=44 copies/reaction), and thus, comparable to conventional RT-qPCR. Additionally, target specificity of the device was validated. Our device and PCA approach enables cost-effective, rapid and mobile molecular diagnostics while remaining highly sensitive and specific.
Assuntos
Técnicas Biossensoriais , COVID-19 , SARS-CoV-2 , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/genética , Humanos , COVID-19/diagnóstico , COVID-19/virologia , Técnicas Biossensoriais/instrumentação , Desenho de Equipamento , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Teste de Ácido Nucleico para COVID-19/instrumentação , Teste de Ácido Nucleico para COVID-19/métodos , Dispositivos Lab-On-A-Chip , Sistemas Automatizados de Assistência Junto ao Leito , Limite de Detecção , Sensibilidade e Especificidade , RNA Viral/análise , RNA Viral/isolamento & purificaçãoRESUMO
With the rise in extreme weather due to global warming, coupled with globalization facilitating the spread of infectious diseases, there's a pressing need for portable testing platforms offering simplicity, low cost, and remote transmission, particularly beneficial in resource-limited and non-urban areas. We have developed a portable device using loop-mediated isothermal amplification (LAMP) with spectrometric detection to identify Salmonella Typhimurium DNA. The device utilizes the LinkIt 7697 microcontroller and a microspectrometer to capture and transmit spectral signals in real-time, allowing for improved monitoring and analysis of the reaction progress. We built a hand-held box containing a microspectrometer, thermoelectric cooler, ultraviolet LED, disposable reaction tube, and homemade thermal module, all powered by rechargeable batteries. Additionally, we conducted thorough experiments to ensure temperature accuracy within 1 °C under thermal control, developed a heating module with a LinkIt 7697 IoT development board to heat the DNA mixture to the reaction temperature within 3 min, and integrated foam insulation and a 3D-printed frame to enhance the device's thermal stability. We successfully demonstrated the amplification of Salmonella Typhimurium DNA with an impressive sensitivity of 2.83 × 10-4 ng/µL. A remote webpage interface allows for monitoring the temperature and fluorescence during the LAMP process, improving usability. This portable LAMP device with real-time detection offers a cost-effective solution for detecting Salmonella Typhimurium in food products. Its unique design and capabilities make it a promising tool for ensuring food safety.
Assuntos
DNA Bacteriano , Técnicas de Amplificação de Ácido Nucleico , Salmonella typhimurium , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Salmonella typhimurium/isolamento & purificação , Salmonella typhimurium/genética , DNA Bacteriano/análise , DNA Bacteriano/genética , Microbiologia de Alimentos , Técnicas de Diagnóstico MolecularRESUMO
Objective: To develop a highly sensitive and rapid nucleic acid detection method for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: We designed, developed, and manufactured an integrated disposable device for SARS-CoV-2 nucleic acid extraction and detection. The precision of the liquid transfer and temperature control was tested. A comparison between our device and a commercial kit for SARS-Cov-2 nucleic acid extraction was performed using real-time fluorescence reverse transcription polymerase chain reaction (RT-PCR). The entire process, from SARS-CoV-2 nucleic acid extraction to amplification, was evaluated. Results: The precision of the syringe transfer volume was 19.2 ± 1.9 µL (set value was 20), 32.2 ± 1.6 (set value was 30), and 57.2 ± 3.5 (set value was 60). Temperature control in the amplification tube was measured at 60.0 ± 0.0 °C (set value was 60) and 95.1 ± 0.2 °C (set value was 95) respectively. SARS-Cov-2 nucleic acid extraction yield through the device was 7.10 × 10 6 copies/mL, while a commercial kit yielded 2.98 × 10 6 copies/mL. The mean time to complete the entire assay, from SARS-CoV-2 nucleic acid extraction to amplification detection, was 36 min and 45 s. The detection limit for SARS-CoV-2 nucleic acid was 250 copies/mL. Conclusion: The integrated disposable devices may be used for SARS-CoV-2 Point-of-Care test (POCT).
Assuntos
COVID-19 , Equipamentos Descartáveis , RNA Viral , SARS-CoV-2 , SARS-CoV-2/isolamento & purificação , COVID-19/diagnóstico , COVID-19/virologia , Humanos , RNA Viral/isolamento & purificação , RNA Viral/análise , Teste de Ácido Nucleico para COVID-19/instrumentação , Teste de Ácido Nucleico para COVID-19/métodos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/instrumentaçãoRESUMO
Nucleic acid tests are key tools for the detection and diagnosis of many diseases. In many cases, the amplification of the nucleic acids is required to reach a detectable level. To make nucleic acid amplification tests more accessible to a point-of-care (POC) setting, isothermal amplification can be performed with a simple heating source. Although these tests are being performed in bulk reactions, the quantification is not as accurate as it would be with digital amplification. Here, we introduce the use of the vibrating sharp-tip capillary for a simple and portable system for tunable on-demand droplet generation. Because of the large range of droplet sizes possible and the tunability of the vibrating sharp-tip capillary, a high dynamic range (~2 to 6000 copies/µL) digital droplet loop-mediated isothermal amplification (ddLAMP) system has been developed. It was also noted that by changing the type of capillary on the vibrating sharp-tip capillary, the same mechanism can be used for simple and portable DNA fragmentation. With the incorporation of these elements, the present work paves the way for achieving digital nucleic acid tests in a POC setting with limited resources.
Assuntos
Técnicas de Amplificação de Ácido Nucleico , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/instrumentação , Vibração , Sistemas Automatizados de Assistência Junto ao Leito , Humanos , Ácidos Nucleicos/análise , DNA/análise , DNA/genética , DNA/químicaRESUMO
Rapid detection of plant diseases before they escalate can improve disease control. Our team has developed rapid nucleic acid extraction methods with microneedles and combined these with loop-mediated amplification (LAMP) assays for pathogen detection in the field. In this work, we developed LAMP assays for early blight (Alternaria linariae, A. alternata, and A. solani) and bacterial spot of tomato (Xanthomonas perforans) and validated these LAMP assays and two previously developed LAMP assays for tomato spotted wilt virus and late blight. Tomato plants were inoculated, and disease severity was measured. Extractions were performed using microneedles, and LAMP assays were run in tubes (with hydroxynaphthol blue) on a heat block or on a newly designed microfluidic slide chip on a heat block or a slide heater. Fluorescence on the microfluidic chip slides was visualized using EvaGreen and photographed on a smartphone. Plants inoculated with X. perforans or tomato spotted wilt virus tested positive prior to visible disease symptoms, whereas Phytophthora infestans and A. linariae were detected at the time of visual disease symptoms. LAMP assays were more sensitive than PCR, and the limit of detection was 1 pg of DNA for both A. linariae and X. perforans. The LAMP assay designed for early blight detected all three species of Alternaria that infect tomato and is thus an Alternaria spp. assay. This study demonstrates the utility of rapid microneedle extraction followed by LAMP on a microfluidic chip for rapid diagnosis of four important tomato pathogens.
Assuntos
Técnicas de Amplificação de Ácido Nucleico , Doenças das Plantas , Smartphone , Solanum lycopersicum , Solanum lycopersicum/virologia , Solanum lycopersicum/microbiologia , Doenças das Plantas/virologia , Doenças das Plantas/microbiologia , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Agulhas/virologia , Agulhas/microbiologia , Oomicetos/isolamento & purificação , Dispositivos Lab-On-A-Chip , Fungos/isolamento & purificação , Técnicas de Diagnóstico MolecularRESUMO
Here, a separation-free and label-free portable aptasensor is developed for rapid and sensitive analysis of tumor-derived exosomes (TEXs). It integrated a parallel rolling circle amplification (RCA) reaction, selective binding of metal ions or small molecules to nucleic acid-specific conformations, and a low-cost, highly sensitive handheld fluorometer. Lung cancer, for example, is targeted with two typical biomarkers (mucin 1 and programmed cell death ligand 1 (PD-L1)) on its exosomes. The affinity of aptamers to the targets modulated the amount of RCA products (T-Hg2+-T and cytosine (C)-rich single-stranded DNA), which in turn affected the fluorescence intensity of quantum dots (QDs) and methylene blue (MB). The results revealed that the limit of detection (LOD) of the handheld fluorometer for cell-derived exosomes can be as low as 30 particles mL-1. Moreover, its specificity, sensitivity, and area under the curve (AUC) are 93% (14/15), 92% (23/25), and 0.956, as determined by the analysis of 40 clinical samples. Retesting 16 of these samples with the handheld fluorometer yielded strong concordance between the fluorometer results and those acquired from clinical computed tomography (CT) and pathology.
Assuntos
Exossomos , Neoplasias Pulmonares , Técnicas de Amplificação de Ácido Nucleico , Humanos , Exossomos/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Biópsia Líquida/métodos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Aptâmeros de Nucleotídeos , Biomarcadores Tumorais/genética , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/instrumentaçãoRESUMO
Throughout the COVID-19 pandemic, individuals potentially infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were forcibly recalled to local or central hospitals, where the diagnostic results were obtained a couple of days after the liquid biopsies were subjected to conventional polymerase chain reaction (PCR). This slow output of such a complex and time-consuming laboratory procedure hindered its widespread application. To overcome the limitations associated with such a centralized diagnostic system, we developed a hand-held and all-in-one type test kit in which the analytical results can be obtained in only 30 min. The test kit consists of three major steps for on-site SARS-CoV-2 RNA detection: 1) virus lysis by heat, 2) RNA enrichment by membrane, and 3) real-time detection by colorimetric loop-mediated isothermal amplification (c-LAMP). The proposed device operates in a sample-to-answer format, is fully automated, and reduces dependence on traditional laboratory settings, facilitating large-scale population screening.
Assuntos
COVID-19 , Colorimetria , Técnicas de Amplificação de Ácido Nucleico , RNA Viral , SARS-CoV-2 , Humanos , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/genética , Colorimetria/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , COVID-19/diagnóstico , COVID-19/virologia , RNA Viral/análise , RNA Viral/genética , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/métodos , Teste de Ácido Nucleico para COVID-19/instrumentação , Teste de Ácido Nucleico para COVID-19/métodos , Kit de Reagentes para DiagnósticoRESUMO
Point-of-care testing of "sample in, answer out" is urgently needed for communicable diseases. Recently, rapid nucleic acid tests for infectious diseases have been developed for use in resource-limited areas, but they require types of equipment in central laboratories and are poorly integrated. In this work, a portable centrifugal microfluidic testing system is developed, integrated with magnetic bead-based nucleic acid extraction, recombinase-assisted amplification and CRISPR-Cas13a detection. The system, with the advantage of its power-supplied active rotating chip and highly programable flow control through integrated addressable active thermally-triggered wax valves, has a rapid turnaround time within 45 min, requiring only one user step. All reagents are preloaded into the chip and can be automatically released. By exploiting a multichannel chip, it is capable of simultaneously detecting 10 infectious viruses with limits of detection of 1 copy per reaction and 5 copies per reaction in plasmid samples and mock plasma samples, respectively. The system was used to analyse clinical plasma samples with good consistency compared to laboratory-based molecular testing. Moreover, the generalizability of our device is reported by successfully testing nasopharyngeal swabs and whole blood samples. The portable device does not require the operation of professional technicians, making it an excellent assay for on-site testing.
Assuntos
Sistemas CRISPR-Cas , Dispositivos Lab-On-A-Chip , Humanos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Desenho de Equipamento , Técnicas Analíticas Microfluídicas/instrumentação , Limite de DetecçãoRESUMO
Point-of-care (POC) diagnostics have emerged as a crucial technology for emerging pathogen detections to enable rapid and on-site detection of infectious diseases. However, current POC devices often suffer from limited sensitivity with poor reliability to provide quantitative readouts. In this paper, we present a self-powered digital loop-mediated isothermal amplification (dLAMP) microfluidic chip (SP-dChip) for the rapid and quantitative detection of nucleic acids. The SP-dChip utilizes a vacuum lung design to passively digitize samples into individual nanoliter wells for high-throughput analysis. The superior digitization scheme is further combined with reverse transcription loop-mediated isothermal amplification (RT-LAMP) to demonstrate dLAMP detection of Zika virus (ZIKV). Firstly, the LAMP assay is loaded into the chip and passively digitized into individual wells. Mineral oil is then pipetted through the chip to differentiate each well as an individual reactor. The chip did not require any external pumping or power input for rapid and reliable results to detect ZIKA RNA as low as 100 copies per µL within one hour. As such, this SP-dChip offers a new class of solutions for truly affordable, portable, and quantitative POC detections for emerging viruses.
Assuntos
Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas , Técnicas de Amplificação de Ácido Nucleico , Zika virus , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Zika virus/isolamento & purificação , Zika virus/genética , Técnicas Analíticas Microfluídicas/instrumentação , Desenho de Equipamento , Humanos , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/métodos , Infecção por Zika virus/diagnóstico , Infecção por Zika virus/virologia , Sistemas Automatizados de Assistência Junto ao Leito , RNA Viral/análise , RNA Viral/genéticaRESUMO
Automation of liquid handling is indispensable to improve throughput and reproducibility in biochemical assays. However, the incorporation of automated systems into laboratory workflows is often hindered by the high cost and complexity associated with building robotic liquid handlers. Here, we report a 3D-printed liquid handler based on a fluidic manifold, thereby obviating the need for complex robotic mechanisms. The fluidic manifold, termed a dispensing and aspirating (DA) device, comprises parallelized multi-pipette structures connected by distribution and aspiration channels, enabling the precise supply and removal of reagents, respectively. Leveraging the versatility of 3D printing, the DA device can be custom-designed and printed to fit specific applications. As a proof-of-principle, we engineered a 3D-printed liquid handler dedicated for 3D digital rolling circle amplification (4DRCA), an advanced biochemical assay involving multiple sample preparation steps such as antibody incubation, cell fixation, nucleic acid amplification, probe hybridization, and extensive washing. We demonstrate the efficacy of the 3D-printed liquid handler to automate the preparation of clinical samples for the simultaneous, in situ analysis of oncogenic protein and transcript markers in B-cell acute lymphoblastic leukemia cells using 4DRCA. This approach provides an effective and accessible solution for liquid handling automation, offering high throughput and reproducibility in biochemical assays.
Assuntos
Técnicas Biossensoriais , Técnicas de Amplificação de Ácido Nucleico , Impressão Tridimensional , Humanos , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Desenho de Equipamento , AutomaçãoRESUMO
Quantification of SARS-CoV-2 RNA copies in wastewater can be used to estimate COVID-19 prevalence in communities. While such results are important for mitigating disease spread, SARS-CoV-2 measurements require sophisticated equipment and trained personnel, for which a centralized laboratory is necessary. This significantly impacts the time to result, defeating its purpose as an early warning detection tool. The objective of this study was to evaluate a field portable device (called MINI) for detecting SARS-CoV-2 viral loads in wastewater using real-time reverse transcriptase loop-mediated isothermal amplification (real-time RT-LAMP). The device was tested using wastewater samples collected from buildings (with 430 to 1430 inhabitants) that had known COVID-19-positive cases. Results show comparable performance of RT-LAMP against reverse transcriptase polymerase chain reaction (RT-qPCR) when detecting SARS-CoV-2 copies in wastewater. Both RT-LAMP and RT-qPCR detected SARS-CoV-2 in wastewater from buildings with at least three positive individuals within a 6-day time frame prior to diagnosis. The large 96-well throughput provided by MINI provided scalability to multi-building detection. The portability of the MINI device enabled decentralized on-site detection, significantly reducing the time to result. The overall findings support the use of RT-LAMP within the MINI configuration as an early detection system for COVID-19 infection using wastewater collected at the building scale.
Assuntos
COVID-19 , Técnicas de Amplificação de Ácido Nucleico , SARS-CoV-2 , Águas Residuárias , Águas Residuárias/virologia , COVID-19/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Humanos , RNA Viral/análise , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/instrumentação , Carga ViralRESUMO
Microfluidic dispensing technologies often require additional equipment, posing challenges for their integration into point-of-care testing (POCT) applications. In response to this challenge, we have developed a pipette-operable microfluidic device fabricated using 3D printing technology for precise liquid dispensing. This device features three reaction chambers and three distinct hydrophobic valves to control the flow direction of liquids. Through these valves, the pipette-operable microfluidic device can sequentially dispense and isolate the liquid into the three reaction chambers, allowing for the individual conduction of three distinct reactions. These hydrophobic valves, with optimized flow resistance and burst pressure, can sustain a volumetric flow rate of up to 25 µL s-1, making them compatible with a standard pipette, a syringe, or a dropper operation. Furthermore, the device is successfully used to operate with various liquids, including BSA, DMEM, FBS, plasma, and blood, representing that the device has the potential to be used for various applications. Additionally, distinct RT-LAMP primer sets have been incorporated for diagnosing SARS-CoV-2, influenza A, and influenza B within each chamber through lyophilization. This pipette-operable microfluidic device serves as a versatile tool for diagnosing these three diseases using a single loading process, with results readable by the naked eye or image assay within 30 minutes of incubation. Finally, the design concepts are extended to engineer a microfluidic device with 20 reaction chambers, offering significant potential for multi-disease diagnostics.
Assuntos
Interações Hidrofóbicas e Hidrofílicas , Dispositivos Lab-On-A-Chip , SARS-CoV-2 , Humanos , SARS-CoV-2/isolamento & purificação , Desenho de Equipamento , COVID-19/diagnóstico , COVID-19/virologia , Testes Imediatos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Impressão TridimensionalRESUMO
Laboratory-based nucleic acid amplification tests (NAATs) are highly sensitive and specific, but they require the transportation of samples to centralized testing facilities and have long turnaround times. During the Coronavirus Disease 2019 (COVID-19) pandemic, substantial advancement has been achieved with the development of paper-based point-of-care (POC) NAATs, offering features such as low cost, being easy to use, and providing rapid sample-to-answer times. Although most of the POC NAATs innovations are towards clinical settings, we have developed a portable, paper-based loop-mediated isothermal amplification (LAMP) testing platform for on-farm applications, capable of detecting Bacteroidales as a fecal contamination biomarker. Our integrated platform includes a drop generator, a heating and imaging unit, and paper-based biosensors, providing sensitive results (limit of detection 3 copies of Bacteroidales per cm2) within an hour of sample collection. We evaluated this integrated platform on a commercial lettuce farm with a concordance of 100% when compared to lab-based tests. Our integrated paper-based LAMP testing platform holds great promise as a reliable and convenient tool for on-site NAATs. We expect that this innovation will encourage the fresh produce industry to adopt NAATs as a complementary tool for decision-making in growing and harvesting. We also hope that our work can stimulate further research in the development of on-farm diagnostic tools for other agricultural applications, leading to improved food safety and technology innovation.
Assuntos
Técnicas Biossensoriais , COVID-19 , Fezes , Técnicas de Amplificação de Ácido Nucleico , Papel , SARS-CoV-2 , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Fezes/microbiologia , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , COVID-19/diagnóstico , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/genética , Humanos , Lactuca/microbiologia , Fazendas , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/métodos , Desenho de EquipamentoRESUMO
Viral infections, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are some of the most dangerous threats to humans. SARS-CoV-2 has caused a global pandemic, highlighting the unprecedented demand for rapid and portable diagnostic methods. To meet these requirements, we designed a label-free colorimetric platform that combines the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated proteins (Cas) 12a system for naked-eye detection (named LFP). This method utilizes reverse transcription loop-mediated isothermal amplification (RT-LAMP) and the trans-cleavage activity of the CRISPR/Cas12a system to increase the sensitivity and specificity of the reaction. This platform can detect as few as 4 copies/µL of RNA and produces no false positive results when tested against the influenza virus. To better meet the requirements of point-of-care (POC) detection, we developed a portable device that can be applied in resource-poor and densely populated regions. The LFP assay holds great potential for application in resource-limited settings, and the label-free gold nanoparticle (AuNPs) probe can reduce costs, making it suitable for large-scale screening. We expect that the LFP assay will be promising for the POC screening of COVID-19.
Assuntos
Colorimetria , Ouro , Nanopartículas Metálicas , Técnicas de Amplificação de Ácido Nucleico , RNA Viral , SARS-CoV-2 , Ouro/química , Colorimetria/métodos , Colorimetria/instrumentação , Nanopartículas Metálicas/química , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , RNA Viral/análise , RNA Viral/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Humanos , COVID-19/diagnóstico , COVID-19/virologia , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Técnicas de Diagnóstico MolecularRESUMO
Clinical diagnostics of infectious diseases via nucleic acid amplification tests (NAATs) depend on a separate step of isolation of nucleic acids from cells/viruses embedded in complex biological matrices. The most recent example has been reverse transcription polymerase chain reaction (RT-PCR) for amplification and detection of SARS-CoV-2 RNA for COVID-19 diagnostics. Kits for RNA extraction and purification are commercially available; however, their integration with amplification systems is generally lacking, resulting in two separate steps, i.e., sample preparation and amplification. This makes NAATs more time-consuming, requiring skilled personnel, and can increase the likelihood of contamination. Here, we describe a setup and methodology to perform the quick extraction and detection of nucleic acids in an integrated manner. In particular, we focus on the use of an immiscible filtration device for capture, isolation, concentration, amplification, and colorimetric detection of SARS-CoV-2 RNA.
Assuntos
COVID-19 , Filtração , Técnicas de Amplificação de Ácido Nucleico , RNA Viral , SARS-CoV-2 , RNA Viral/isolamento & purificação , RNA Viral/análise , RNA Viral/genética , Humanos , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , COVID-19/diagnóstico , COVID-19/virologia , Filtração/instrumentação , Filtração/métodos , Teste de Ácido Nucleico para COVID-19/métodos , Teste de Ácido Nucleico para COVID-19/instrumentação , Colorimetria/métodos , Colorimetria/instrumentaçãoRESUMO
In this study, a microfluidic-based system utilizing colorimetric loop-mediated isothermal amplification (LAMP) is introduced for the quantitative analysis of nucleic acid targets. This system offers a user-friendly and cost-effective platform for the multiplexed genetic diagnosis of various infectious diseases across multiple samples. It includes time-lapse imaging equipment for capturing images of the microfluidic device during the LAMP assay and a hue-based quantitative analysis software to analyze the LAMP reaction, streamlining diagnostic procedures. An electric pipette was used to simplify the loading of samples and LAMP reagents into the device, allowing easy operation even by untrained individuals. The hue-based analysis software employs efficient image processing and post-processing techniques to calculate DNA amplification curves based on color changes in multiple reaction chambers. This software automates several tasks, such as identifying reaction chamber areas from time-lapse images, quantifying color information within each chamber, correcting baselines of DNA amplification curves, fitting experimental data to theoretical curves, and determining the threshold time for each curve. To validate the developed system, conventional off-chip LAMP assays were conducted with a 25 µL reaction mixture in 0.2 mL polymerase chain reaction (PCR) tubes using a real-time turbidimeter. The results indicated that the threshold time obtained using the colorimetric LAMP assay in the developed system is comparable to that obtained with real-time turbidity measurements in PCR tubes, demonstrating the system's capability for quantitative analysis of target nucleic acids, including those from human herpesviruses.