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1.
J Agric Food Chem ; 68(1): 369-375, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31829586

RESUMO

A sensitive fluorescent DNA hydrogel aptasensor based on the self-assembly of rolling circle amplification (RCA) products was developed for ochratoxin A (OTA) detection in beer. A competitive binding mode of aptamer, complementary sequence, and target was integrated into the DNA hydrogel for OTA detection. The OTA aptamer first combined with the primer to form the hybridized product. Then, in the presence of OTA, the aptamer combined with OTA, which released the primer. The released primer hybridized with the padlock probe to form a circular template, and the RCA reaction was initiated by adding ligase, polymerase, and dNTPs. The fluorescent DNA hydrogel was obtained by adding Cy3-dUTP together with dNTPs, and the fluorescence (FL) intensity of the DNA hydrogel was positively correlated with OTA concentration. Under the optimal experimental conditions, the linear range of the relationship varied from 0.05 ng/mL to 100 ng/mL with a detection limit for OTA of 0.01 ng/mL. The fluorescent DNA hydrogel aptasensor showed good specificity and stability in beer samples. Therefore, the fabricated DNA hydrogel aptasensor shows considerable potential applications in detecting OTA for food safety.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , DNA/química , Hidrogéis/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Ocratoxinas/análise , Aptâmeros de Nucleotídeos/genética , Cerveja/análise , Técnicas Biossensoriais/instrumentação , DNA/genética , Fluorescência , Contaminação de Alimentos/análise , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/instrumentação
2.
J Sci Food Agric ; 100(1): 325-334, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31584699

RESUMO

BACKGROUND: Meat fraud and adulteration incidents occur frequently in almost all regions of the globe, especially with the increase in the world's population. To ensure the authenticity of meat products, we developed a 10-plex xMAP assay to simultaneously detect ten animal materials: bovine, caprine, poultry, swine, donkey, deer, horse, dog, fox and mink. RESULTS: This method was investigated by analyzing DNA extracts from raw muscle, muscle mixtures, meat products and animal feeds. Our results indicated that the species of interest can be identified, differentiated and detected down to 1 g kg-1 in binary mixtures or 0.01-0.001 ng of genomic DNA from specific species. Testing of 125 commercial samples showed a 97.4% coincidence rate with the method used in routine testing in our lab. CONCLUSION: These results indicated that the method established in this study could detect ten animal materials simultaneously within 3 h, which provides a new, useful tool for animal ingredient analysis in meat products and animal feeds. © 2019 Society of Chemical Industry.


Assuntos
DNA Mitocondrial/genética , Contaminação de Alimentos/análise , Produtos da Carne/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Ração Animal/análise , Animais , Bovinos , Cervos , Cães , Raposas , Cabras , Cavalos , Vison , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Aves Domésticas , Suínos
3.
Nature ; 574(7777): 228-232, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31597972

RESUMO

Microfluidic systems can deliver portable point-of-care diagnostics without the need for external equipment or specialist operators, by integrating all reagents and manipulations required for a particular assay in one device1. A key approach is to deposit picogram quantities of dried reagents in microchannels with micrometre precision using specialized inkjet plotters2-5. This means that reagents can be stored for long periods of time and reconstituted spontaneously when adding a liquid sample. But it is challenging to carry out complex operations using multiple reagents, because shear flow enhances their dispersion and they tend to accumulate at moving liquid fronts, resulting in poor spatiotemporal control over the concentration profile of the reconstituted reagents6. One solution is to limit the rate of release of reagents into the liquid7-10. However, this requires the fine-tuning of different reagents, conditions and targeted operations, and cannot readily produce the complex, time-dependent multireagent concentration pulses required for sophisticated on-chip assays. Here we report and characterize a capillary flow phenomenon that we term self-coalescence, which is seen when a confined liquid with a stretched air-liquid interface is forced to 'zip' back onto itself in a microfluidic channel, thereby allowing reagent reconstitution with minimal dispersion. We provide a comprehensive framework that captures the physical underpinning of this effect. We also fabricate scalable, compact and passive microfluidic structures-'self-coalescence modules', or SCMs-that exploit and control this phenomenon in order to dissolve dried reagent deposits in aqueous solutions with precise spatiotemporal control. We show that SCMs can reconstitute multiple reagents so that they either undergo local reactions or are sequentially delivered in a flow of liquid. SCMs are easily fabricated in different materials, readily configured to enable different reagent manipulations, and readily combined with other microfluidic technologies, so should prove useful for assays, diagnostics, high-throughput screening and other technologies requiring efficient preparation and manipulation of small volumes of complex solutions.


Assuntos
Indicadores e Reagentes/análise , Microfluídica/métodos , Técnicas de Química Analítica/instrumentação , Técnicas de Química Analítica/métodos , Testes Diagnósticos de Rotina , Ensaios Enzimáticos/instrumentação , Ensaios Enzimáticos/métodos , Fluorometria , Glucosefosfato Desidrogenase/análise , Glucosefosfato Desidrogenase/metabolismo , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/isolamento & purificação , Humanos , Microfluídica/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos
4.
Biosens Bioelectron ; 142: 111485, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31301578

RESUMO

Rolling circle amplification (RCA) combined with padlock probe recognition of a DNA target is attractive for on-chip nucleic acid testing due to its high specificity and isothermal reaction conditions. However, the integration of RCA on an automated chip platform is challenging due to the different reagents needed for the reaction steps and the temperature sensitivity of the phi29 polymerase. Here, we describe the integration of an RCA assay on a single-use polymer chip platform where magnetic microbeads are used as solid support to transport the DNA target between three connected reaction chambers for (i) padlock probe annealing and ligation, (ii) RCA, and (iii) optomagnetic detection of RCA products. The three chambers were loaded with reagents by sequential filling combined with passive microfluidic structures. After loading, the on-chip assay steps were automated. For an assay in which all steps but the padlock probe annealing on the target were performed on-chip, we found a limit of detection (LOD) for a synthetic influenza target of 2 pM after 45 min of RCA, which is comparable to the corresponding laboratory assay. The entire assay, including padlock probe annealing, could be performed on-chip with an LOD of 20 pM after 45 min of RCA. This LOD can likely be reduced by further optimizing the microbead mixing. The results present important steps towards the integration and automation of RCA and potentially also other complex multi-step assays on a single-use polymer chip for molecular analysis.


Assuntos
Técnicas Biossensoriais/instrumentação , DNA/análise , Dispositivos Lab-On-A-Chip , Técnicas de Amplificação de Ácido Nucleico/instrumentação , DNA/genética , Desenho de Equipamento , Limite de Detecção , Magnetismo/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação
5.
Biosens Bioelectron ; 142: 111496, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31302395

RESUMO

Recent developments in microfluidics enable the lab-on-a-chip-based molecular diagnosis. Rapid and accurate diagnosis of infectious diseases is critical for preventing the transmission of the disease. Here, we characterize a Lamb wave-based device using various parameters including the contact angle and viscosity of the sample droplet, the applied voltage, and the temperature increase. Additionally, we demonstrate the functionality of the Lamb wave-based device in clinical application. Optimal temperature for rolling circle amplification (RCA) process is 30 °C, and it was achieved by Lamb wave generation at 17 V. Gene amplification due to RCA process could be detected by viscosity increase due to DNA hydrogel formation in a sample droplet, which induced the acoustic streaming velocity of suspended particles to be decreased. In our Lamb wave-based device, isothermal amplification of target nucleic acids could be successfully detected within 30 min using 10 µL of sessile droplet, and was validated by comparing that of commercial real-time fluorescence analysis. Our device enables simple and low-cost molecular diagnosis, which can be applied to resource-limited clinical settings.


Assuntos
DNA de Cadeia Simples/química , DNA Viral/análise , Vírus da Dengue/isolamento & purificação , Hidrogéis/química , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Replicação do DNA , DNA de Cadeia Simples/genética , DNA Viral/genética , Dengue/diagnóstico , Dengue/virologia , Vírus da Dengue/genética , Desenho de Equipamento , Fluorescência , Humanos , Dispositivos Lab-On-A-Chip , Temperatura Ambiente , Viscosidade
6.
Biosens Bioelectron ; 141: 111448, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31252258

RESUMO

Over the last decade, nucleic acid amplification tests (NAATs) including polymerase chain reaction (PCR) were an indispensable methodology for diagnosing cancers, viral and bacterial infections owing to their high sensitivity and specificity. Because the NAATs can recognize and discriminate even a few copies of nucleic acid (NA) and species-specific NA sequences, NAATs have become the gold standard in a wide range of applications. However, limitations of NAAT approaches have recently become more apparent by reason of their lengthy run time, large reaction volume, and complex protocol. To meet the current demands of clinicians and biomedical researchers, new NAATs have developed to achieve ultrafast sample-to-answer protocols for the point-of-care testing (POCT). In this review, ultrafast NA-POCT platforms are discussed, outlining their NA amplification principles as well as delineating recent advances in ultrafast NAAT applications. The main focus is to provide an overview of NA-POCT platforms in regard to sample preparation of NA, NA amplification, NA detection process, interpretation of the analysis, and evaluation of the platform design. Increasing importance will be given to innovative, ultrafast amplification methods and tools which incorporate artificial intelligence (AI)-associated data analysis processes and mobile-healthcare networks. The future prospects of NA POCT platforms are promising as they allow absolute quantitation of NA in individuals which is essential to precision medicine.


Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/análise , Animais , Inteligência Artificial/economia , Técnicas Biossensoriais/economia , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Desenho de Equipamento , Humanos , Técnicas de Amplificação de Ácido Nucleico/economia , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Ácidos Nucleicos/genética , Sistemas Automatizados de Assistência Junto ao Leito/economia , Reação em Cadeia da Polimerase/economia , Reação em Cadeia da Polimerase/instrumentação , Reação em Cadeia da Polimerase/métodos , Fatores de Tempo
7.
Biosens Bioelectron ; 135: 120-128, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-31004922

RESUMO

This study presents a slidable paper-embedded plastic microdevice fully integrated with DNA extraction, loop-mediated isothermal amplification (LAMP), and colorimetric detection functionalities. The developed microdevice consists of three layers that allow a sliding movement to mix the sample and reagents for DNA purification, amplification, and detection in a sequential manner. An FTA card was employed in the main chamber for DNA extraction and purification from intact bacterial cells. Subsequently, LAMP reagents and fuchsin-stored chambers were pulled toward the main chambers for DNA amplifications at 65 °C. After 30 min, the detection reagents-stored chambers were then moved to main chambers for result analysis. For the detection of LAMP amplicons, a novel colorimetric fuchsin-based method was employed. The wide applicability of the integrated microdevice was demonstrated by successfully screening three major foodborne pathogens, namely Salmonella spp., Staphylococcus aureus, and Escherichia coli O157:H7 in food, enabling highly sensitive detection of 3.0 × 101 CFU/sample of Gram-negative bacteria (Salmonella spp. and Escherichia coli O157:H7) and 3.0 × 102 CFU/sample of Gram-positive bacteria (Staphylococcus aureus) within 75 min. The portable and integrated microdevice presented in this study holds significant promise for point-of-care applications to accurately and rapidly diagnose and control diseases.


Assuntos
Escherichia coli O157/isolamento & purificação , Doenças Transmitidas por Alimentos/microbiologia , Testes Imediatos , Salmonella/isolamento & purificação , Staphylococcus aureus/isolamento & purificação , Técnicas Biossensoriais/instrumentação , DNA Bacteriano/análise , DNA Bacteriano/genética , Desenho de Equipamento , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/genética , Doenças Transmitidas por Alimentos/diagnóstico , Humanos , Dispositivos Lab-On-A-Chip , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Papel , Plásticos , Salmonella/genética , Infecções por Salmonella/diagnóstico , Infecções por Salmonella/microbiologia , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética
8.
Anal Chim Acta ; 1065: 71-78, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31005153

RESUMO

Whooping cough also called Pertussis is a highly contagious respiratory infection that affects all age populations. Given recent pertussis outbreaks, there is an urgent need for a point-of-care (POC) device for rapid diagnosis of pertussis. Herein, we report a low-cost microfluidic POC device integrated with loop-mediated isothermal amplification (LAMP) technique for the rapid and accurate diagnosis of pertussis. The 3D-printed bioanalyzer housed not only the biochip but also an in-house-developed portable and fully battery-powered heater for rapid POC detection of pertussis, without the need of external electricity. The fluorescence-based results could be rapidly visualized in about one hour by the naked eye without the need for any additional instrumentation. In addition, a simple centrifuge-free sample preparation process was optimized for the efficient lysis of pertussis samples and successfully used for direct detection of bacteria in nasopharyngeal samples. High sensitivity, with a limit of detection (LOD) of 5 DNA copies per LAMP zone, and high specificity were demonstrated. We envision that the microfluidic POC device can be used in various venues such as medical clinics, schools, and other low-resource settings for the fast detection of pertussis.


Assuntos
Bordetella pertussis/isolamento & purificação , Técnicas Analíticas Microfluídicas/economia , Técnicas de Amplificação de Ácido Nucleico/economia , Coqueluche/diagnóstico , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Sistemas Automatizados de Assistência Junto ao Leito , Coqueluche/microbiologia
9.
PLoS One ; 14(4): e0215756, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31009510

RESUMO

Nucleic acid amplification technologies (NAATs) are high-performance tools for rapidly and accurately detecting infectious agents. They are widely used in high-income countries to diagnose disease and improve patient care. The complexities associated with test methods, reagents, equipment, quality control and assurance require dedicated laboratories with trained staff, which can exclude their use in low-resource and decentralized healthcare settings. For certain diseases, fully integrated NAAT devices and assays are available for use in environmentally-controlled clinics or emergency rooms where relatively untrained staff can perform testing. However, decentralized settings in many low- and middle-income countries with large burdens of infectious disease are challenged by extreme environments, poor infrastructure, few trained staff and limited financial resources. Therefore, there is an urgent need for low-cost, integrated NAAT tools specifically designed for use in low-resource settings (LRS). Two essential components of integrated NAAT tools are: 1) efficient nucleic acid extraction technologies for diverse and complex sample types; and 2) robust and sensitive nucleic acid amplification and detection technologies. In prior work we reported the performance and workflow capacity for the nucleic acid extraction component. In the current study we evaluated performance of eight novel nucleic acid amplification and detection technologies from seven developers using blinded panels of RNA and/or DNA from three pathogens to assess both diagnostic accuracy and suitability as an essential component for low-cost NAAT in LRS. In this exercise, we noted significant differences in performance among these technologies and identified those most promising for potential further development.


Assuntos
Doenças Transmissíveis/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/genética , Sistemas Automatizados de Assistência Junto ao Leito/economia , Chlamydia/genética , Doenças Transmissíveis/microbiologia , Doenças Transmissíveis/virologia , Análise Custo-Benefício , HIV-1/genética , Recursos em Saúde/economia , Humanos , Neisseria gonorrhoeae/genética , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Reprodutibilidade dos Testes , Zika virus/genética
10.
Biosens Bioelectron ; 132: 271-278, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30878727

RESUMO

Vibrio parahaemolyticus is one of the most important foodborne pathogens that cause various life-threatening diseases in human and animals. Here, we present a rapid detection platform for V. parahaemolyticus by combining loop-mediated isothermal amplification (LAMP) and disposable electrochemical sensors based on screen-printed graphene electrodes (SPGEs). The LAMP reactions using primers targeting V. parahaemolyticus toxR gene were optimized at an isothermal temperature of 65 °C, providing specific detection of V. parahaemolyticus within 45 min at the detection limit of 0.3 CFU per 25 g of raw seafood. The LAMP amplicons can be effectively detected using unmodified SPGEs, redox active molecules namely Hoechst-33258 and a portable potentiostat. Therefore, the proposed system is particularly suitable as a point-of-care device for on-site detection of foodborne pathogens.


Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas Eletroquímicas/instrumentação , Análise de Alimentos/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Alimentos Marinhos/microbiologia , Vibrioses/microbiologia , Vibrio parahaemolyticus/isolamento & purificação , DNA Bacteriano/análise , DNA Bacteriano/genética , Eletrodos , Desenho de Equipamento , Grafite/química , Humanos , Limite de Detecção , Sistemas Automatizados de Assistência Junto ao Leito , Vibrio parahaemolyticus/genética
11.
Malar J ; 18(1): 98, 2019 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-30909912

RESUMO

BACKGROUND: Microscopic detection of malaria parasites is the standard method for clinical diagnosis of malaria in Brazil. However, malaria epidemiological surveillance studies specifically aimed at the detection of low-density infection and asymptomatic cases will require more sensitive and field-usable tools. The diagnostic accuracy of the colorimetric malachite green, loop-mediated, isothermal amplification (MG-LAMP) assay was evaluated in remote health posts in Roraima state, Brazil. METHODS: Study participants were prospectively enrolled from health posts (healthcare-seeking patients) and from nearby villages (healthy participants) in three different study sites. The MG-LAMP assay and microscopy were performed in the health posts. Two independent readers scored the MG-LAMP tests as positive (blue/green) or negative (clear). Sensitivity and specificity of local microscopy and MG-LAMP were calculated using results of PET-PCR as a reference. RESULTS: A total of 91 participants were enrolled. There was 100% agreement between the two MG-LAMP readers (Kappa = 1). The overall sensitivity and specificity of MG-LAMP were 90.0% (95% confidence interval (CI) 76.34-97.21%) and 94% (95% CI 83.76-98.77%), respectively. The sensitivity and specificity of local microscopy were 83% (95% CI 67.22-92.66%) and 100% (95% CI 93.02-100.00%), respectively. PET-PCR detected six mixed infections (infection with both Plasmodium falciparum and Plasmodium vivax); two of these were also detected by MG-LAMP and one by microscopy. Microscopy did not detect any Plasmodium infection in the 26 healthy participants; MG-LAMP detected Plasmodium in five of these and PET-PCR assay detected infection in three. Overall, performing the MG-LAMP in this setting did not present any particular challenges. CONCLUSION: MG-LAMP is a sensitive and specific assay that may be useful for the detection of malaria parasites in remote healthcare settings. These findings suggest that it is possible to implement simple molecular tests in facilities with limited resources.


Assuntos
Malária Falciparum/diagnóstico , Malária Vivax/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Plasmodium falciparum/isolamento & purificação , Plasmodium vivax/isolamento & purificação , Vigilância da População/métodos , Corantes de Rosanilina/química , Brasil , Humanos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Sensibilidade e Especificidade
12.
Lab Chip ; 19(8): 1397-1405, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30847458

RESUMO

In this study, we have developed a foldable microdevice fully integrating DNA purification, amplification, and detection processes for detecting multiple foodborne pathogens. Specifically, the loop-mediated isothermal amplification (LAMP) technique was combined with a fuchsin-based direct DNA colorimetric detection method. The microdevice was composed of three parts: a sample zone, reaction zone, and detection zone. A sealing film attached to the sample, reaction, and detection zones served as a bottom layer to make the microdevice foldable. The detection zone was made up of paper strips attached to the sticky side of the sealing film, and fuchsin-stained lines were drawn on the paper strips. The microdevice can be folded to directly transfer the DNA template solution from the sample chambers to the reaction chambers. In this manner, fluid manipulation was readily realized and the use of a bulky instrument such as a pump or rotator was completely dispensed with. After the LAMP reaction, the detection zone was folded so that the fuchsin-stained lines were completely soaked into the reaction chambers. Genomic DNAs of Salmonella spp. and Escherichia coli O157:H7 were first successfully purified from thermally-lysed milk using polydopamine-coated paper, amplified by LAMP, and directly identified by the naked eye using fuchsin within 65 min. Using this microdevice, approximately 102 CFU per mL of Salmonella spp. was detected. These results indicate the significant potential of this microdevice for the sample-in-answer-out genetic analysis of multiple foodborne pathogens in resource-limited environments.


Assuntos
Colorimetria/instrumentação , Escherichia coli O157/isolamento & purificação , Dispositivos Lab-On-A-Chip , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Corantes de Rosanilina/metabolismo , Salmonella/isolamento & purificação , Escherichia coli O157/genética , Escherichia coli O157/metabolismo , Salmonella/genética , Salmonella/metabolismo
14.
Anal Bioanal Chem ; 411(19): 4401-4414, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30707267

RESUMO

Rapid, low-cost, and sensitive nucleic acid detection and quantification assays enabled by microfluidic paper-based analytical devices (µPADs) hold great promise for point-of-care disease diagnostics and field-based molecular tests. Through the capillary action in µPAD, flexible manipulation of nucleic acid samples can be achieved without the need for external pumps or power supplies, making it possible to fabricate highly integrated sample-to-answer devices that streamline the nucleic acid extraction, separation, concentration, amplification, and detection. To detect minute amounts of genetic materials from clinical and biological samples, it is also critical to develop sensitive signal readouts that generate physically detectable signals for in-device nucleic acid detection and/or quantification. In this review, we will focus on µPAD approaches for the facile manipulation of nucleic acids and emerging signal transduction strategies allowing sensitive and specific nucleic acid detection in µPAD. Graphical abstract ᅟ.


Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Ácidos Nucleicos/análise , Papel , Corantes/química , Hibridização de Ácido Nucleico , Sistemas Automatizados de Assistência Junto ao Leito , Transdução de Sinais , Biologia Sintética
15.
Lab Chip ; 19(6): 1035-1040, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30734822

RESUMO

Digital nucleic acid amplification and detection methods provide excellent sensitivity and specificity and allow absolute quantification of target nucleic acids. Isothermal methods such as digital loop-mediated isothermal amplification (digital LAMP) have potential for use in rapid disease diagnosis in low-resource settings due to their speed and lack of thermal cycling. We previously developed a self-digitization (SD) chip, a simple microfluidics device that automatically digitizes a sample into an array of nanoliter wells, for use in digital LAMP. In this work, we improve the SD chip design to increase sample loading efficiency, speed, and completeness, and test a range of well volumes and numbers. We demonstrate the diagnostic capability of this platform by applying it to quantifying human papillomavirus 18 gene.


Assuntos
DNA Viral/análise , Papillomavirus Humano 18/genética , Dispositivos Lab-On-A-Chip , Técnicas de Amplificação de Ácido Nucleico/métodos , DNA Viral/metabolismo , Humanos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Reprodutibilidade dos Testes
16.
Anal Chim Acta ; 1055: 115-125, 2019 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-30782362

RESUMO

Owing to the pivotal function in post transcriptional gene modification, miRNA biomarkers are playing crucial role in tracking and diagnosing various forms of tumors in a short time period. Hence, the need to develop simple, sensitive and specific detection of miRNAs for precise diagnosis arises. This current study is aimed to develop a detecting platform by combining rolling circle amplification with AuNps-based lateral flow strip (LFS-RCA) for simultaneous detection of miRNA 21 and miRNA let-7a. The current methodology is simple, sensitive, specific and selective for miRNA let-7a and miRNA 21 with the limits of detection (LOD) as low as 20 pM and 40 pM, respectively. In this technique, rolling circle amplification is playing an essential role in increasing sensitivity and reducing experimental cost. Moreover, the padlock probe with high specificity can immediately identify the simultaneous amplification of multiple miRNAs targets, which may contribute in saving sample volume and detection time. Hopefully, in future with further development, this developed technique can be chosen as a potential tool for detection of miRNAs in clinical diagnosis.


Assuntos
Limite de Detecção , MicroRNAs/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Sequência de Bases , Sondas de DNA/genética , Sondas de DNA/metabolismo , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Fatores de Tempo
17.
Biomed Microdevices ; 21(1): 9, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30617586

RESUMO

A major goal in the development of point-of-care (POC) devices is to build them as portable to provide a rapid and effective determination for disease pathogens. In nucleic acid testing, an optical detection system used to monitor the product of nucleic acid amplification has always been a bulky accessory. In this work, we developed a handheld, automatic and detection system-free thermal digital microfluidic (DMF) device for DNA detection by loop-mediated isothermal amplification (LAMP). Droplet manipulation and real-time temperature control systems were integrated into a handheld device. The control software could be installed into any tablet and communicate with the device via Bluetooth. In the experimentation, we loaded 2-µl samples with an electrowetting force into sandwich-structured DMF chips, thereby considerably reducing reagent consumptions. After an on-chip LAMP reaction, we added a highly concentrated SYBR Green I droplet and mixed it with a reaction droplet to enable product detection with the naked eye. This step prevented aerosol contamination by avoiding the exposure of the reaction droplet to the air. Using a blood parasite Trypanosoma brucei as a model system, this system showed similar results as a commercial thermal cycler and could detect 40 copies per reaction of the DNA target. This low-cost, compact device removed the bulky optical system for DNA detection, thus enabling it to be well suited for POC testing.


Assuntos
DNA de Protozoário , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas , Técnicas de Amplificação de Ácido Nucleico , Trypanosoma brucei brucei/genética , Tripanossomíase Africana , Animais , DNA de Protozoário/sangue , DNA de Protozoário/genética , Humanos , Camundongos , Técnicas Analíticas Microfluídicas/métodos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Tripanossomíase Africana/sangue , Tripanossomíase Africana/genética
18.
Electrophoresis ; 40(6): 914-921, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30511768

RESUMO

We developed a smartphone-based on-site nucleic acid testing (NAT) platform that can image and analyze lateral flow nucleic acid assays at point-of-care settings. An inexpensive add-on was devised to run lateral flow assays while providing homogeneous ambient light for imaging. In addition, an Android app with a user-friendly interface was developed for the result analysis and management. Linear color calibration is implemented inside the app to minimize the colorimetric reaction difference between smartphones. A relationship function between nucleic acid concentration and colorimetric reaction was established and evaluated by leave-one-out cross validation. The predicted concentration and true concentration showed a good agreement with an R-squared value of 0.96. This smartphone-based NAT platform can be used to diagnose infectious diseases and monitor disease progression, and assess treatment efficacy, especially for resource-limited settings.


Assuntos
Aplicativos Móveis , Técnicas de Amplificação de Ácido Nucleico , Sistemas Automatizados de Assistência Junto ao Leito , Smartphone , Desenho de Equipamento , HIV/genética , HIV/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/análise
19.
Langmuir ; 35(1): 248-253, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30512960

RESUMO

We report a visualized quantitative detection method for nucleic acid amplification tests based on the coffee-ring effect on colloid-crystal substrates. The solution for loop-mediated isothermal amplification (LAMP) of DNA is drop cast on a colloid-crystal surface. After complete drying, a coffee ring containing the LAMP byproduct (i.e., magnesium pyrophosphate) is formed, and it is found that the width of the coffee ring is linearly correlated to the logarithm of the original DNA concentration before the isothermal amplification. Importantly, compared with other substrates, we found that the colloid-crystal substrate is an appropriate substrate for carrying out the assay of high sensitivity. On the basis of these findings, we develop a coffee-ring-based assay for quantitative readout of trace DNA in a sample. The assay requires 0.50 µL of the sample and is completed in 5 min in a homemade chamber with constant humidity. Semiquantitative detection of trace DNA is performed using naked eyes. With the use of a smartphone, the DNA in a sample can be quantitatively detected with a limit of detection of 20 copies.


Assuntos
DNA/análise , Difosfatos/química , Compostos de Magnésio/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Ação Capilar , Coloides/química , DNA/genética , Limite de Detecção , Nanopartículas/química , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Salmonella/genética , Dióxido de Silício/química , Smartphone
20.
Food Chem ; 274: 822-830, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30373016

RESUMO

With genetically modified (GM) food circulating on the market, a rapid transgenic food screening method is needed to protect consumer rights. The on-site screening efficiency of GM food testing is low. We report rapid sample-to-answer detection of GM papayas with loop-mediated isothermal amplification (LAMP) and a compact, portable, integrated microfluidic platform using microfluidic lab-on-a-disc (LOAD). GM samples were differentiated from non-GM papaya, based on the detection of a specific GM (P-35S (Cauliflower mosaic virus 35S promoter)) and non-GM DNA marker (papain) in 15 min. The detection limits for DNA and juice from papaya were 10 pg/µL and 0.02 µL, respectively. Our LOAD platform is a simple and robust solution for GM screening, which is anticipated to be a foundation for on-site testing of transgenic food.


Assuntos
Carica/genética , Análise de Alimentos/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Plantas Geneticamente Modificadas/genética , Análise de Alimentos/instrumentação , Sucos de Frutas e Vegetais , Marcadores Genéticos , Hong Kong , Dispositivos Lab-On-A-Chip , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Papaína/genética , Regiões Promotoras Genéticas , Smartphone
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