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1.
Sci Rep ; 9(1): 16374, 2019 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-31705044

RESUMO

Here, we describe a simple, universal protocol for use in nucleic acid testing-based pathogen diagnostics, which requires only hand-powered sample preparation, including the processes of pathogen enrichment and nucleic acid isolation. The protocol uses low-cost amine-functionalized diatomaceous earth with a 1-µm Teflon filter as a reaction matrix in both stages of the process, using homobifunctional imidoesters. Using a simple syringe as a pump, the capture efficiency for a large sample volume (<50 mL) was enhanced by up to 98.3%, and the detection limit was 1 CFU/mL, 100-fold better than that of common commercial nucleic acid isolation kit. This protocol can also be combined with commercialized 96-well filter plates for robust sample preparation. Our proposed system is robust, simple, low-cost, universal, and rapid (taking <20 min), and it works regardless of the ambient environment and sample pretreatment, requiring no electricity or instruments. Its benefits include the simplicity of producing its components and its ease of operation, and it can be readily integrated with other assays for point-of-care diagnostics.

2.
Biosens Bioelectron ; 111: 66-73, 2018 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-29653418

RESUMO

Diseases caused by pathogenic microorganisms including bacteria and viruses can cause serious medical issues including death and result in huge economic losses. Despite the myriad of recent advances in the rapid and accurate detection of pathogens, large volume clinical samples with a low concentration of pathogens continue to present challenges for diagnosis and surveillance. We here report a simple and label-free approach via homobifunctional imidoesters (HIs) with a microfluidic platform (SLIM) to efficiently enrich and extract pathogens at low concentrations from clinical samples. The SLIM system consists of an assembled double microfluidic chip for streamlining large volume processing and HIs for capturing pathogens and isolating nucleic acids by both electrostatic and covalent interaction without a chaotropic detergent or bulky instruments. The SLIM system significantly increases the enrichment and extraction rate of pathogens (up to 80% at 10 CFU (colony forming unit) in a 1 mL volume within 50 min). We demonstrated its clinical utility in large sample volumes from 46 clinical specimens including environmental swabs, saliva, and blood plasma. The SLIM system showed higher sensitivity with these samples and could detect pathogens that were below the threshold of detection with other methods. Finally, by combining our SLIM approach with an isothermal optical sensor, pathogens could be detected at a very high sensitivity in blood plasma samples within 80 min via enrichment, extraction and detection steps. Our SLIM system thus provides a simple, reliable, cost-effective and ultrasensitive pathogen diagnosis platform for use with large volume clinical samples and would thus have significant utility for various infectious diseases.


Assuntos
Técnicas Biossensoriais/instrumentação , Doenças Transmissíveis/microbiologia , Doenças Transmissíveis/virologia , Imidoésteres/química , Dispositivos Lab-On-A-Chip , Doenças Transmissíveis/sangue , Doenças Transmissíveis/diagnóstico , Desenho de Equipamento , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/sangue , Infecções por Escherichia coli/diagnóstico , Herpes Zoster/sangue , Herpes Zoster/diagnóstico , Herpesvirus Humano 3/isolamento & purificação , Humanos , Sistemas Automatizados de Assistência Junto ao Leito , Saliva/microbiologia , Saliva/virologia
3.
Anal Chem ; 90(8): 5108-5115, 2018 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-29561136

RESUMO

Nucleic acid-based diagnostics are widely used for clinical applications due to their powerful recognition of biomolecule properties. Isolation and purification of nucleic acids such as DNA and RNA in the diagnostic system have been severely hampered in point-of-care testing because of low recovery yields, degradation of nucleic acids due to the use of chaotropic detergent and high temperature, and the requirement of large instruments such as centrifuges and thermal controllers. Here, we report a novel large instrument- and detergent-free assay via binary nanomaterial for ultrasensitive nucleic acid isolation and detection from cells (eukaryotic and prokaryotic). This binary nanomaterial couples a zinc oxide nanomultigonal shuttle (ZnO NMS) for cell membrane rupture without detergent and temperature control and diatomaceous earth with dimethyl suberimidate complex (DDS) for the capture and isolation of nucleic acids (NA) from cells. The ZnO NMS was synthesized to a size of 500 nm to permit efficient cell lysis at room temperature within 2 min using the biological, chemical, and physical properties of the nanomaterial. By combining the ZnO NMS with the DDS and proteinase K, the nucleic acid extraction could be completed in 15 min with high quantity and quality. For bacterial cells, DNA isolation with the binary nanomaterial yielded 100 times more DNA, than a commercial spin column based reference kit, as determined by the NanoDrop spectrophotometer. We believe that this binary nanomaterial will be a useful tool for rapid and sensitive nucleic acid isolation and detection without large instruments and detergent in the field of molecular diagnostics.


Assuntos
Nanoestruturas/química , Ácidos Nucleicos/análise , Reação em Cadeia da Polimerase/métodos , Bactérias/genética , DNA Bacteriano/análise , DNA Bacteriano/isolamento & purificação , Detergentes/química , Dimetil Suberimidato/química , Endopeptidase K/metabolismo , Células HCT116 , Humanos , Ácidos Nucleicos/isolamento & purificação , Tamanho da Partícula , Testes Imediatos , Temperatura Ambiente , Óxido de Zinco/química
4.
Anal Biochem ; 544: 87-92, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29289485

RESUMO

Rapid and sensitive detection of low amounts of pathogen in large samples is needed for early diagnosis and treatment of patients and surveillance of pathogen. In this study, we report a microfluidic platform for detection of low pathogen levels in a large sample volume that couples an Magainin 1 based microfluidic platform for pathogen enrichment and a recombinase polymerase amplification (RPA) sensor for simultaneous pathogenic DNA amplification and detection in a label-free and real-time manner. Magainin 1 is used as a pathogen enrichment agent with a herringbone microfluidic chip. Using this enrichment platform, the detection limit was found to be 20 times more sensitive in 10 ml urine with Salmonella and 10 times more sensitive in 10 ml urine with Brucella than that of real-time PCR without the enrichment process. Furthermore, the combination system of the enrichment platform and an RPA sensor that based on an isothermal DNA amplification method with rapidity and sensitivity for detection can detect a pathogen at down to 50 CFU in 10 ml urine for Salmonella and 102 CFU in 10 ml urine for Brucella within 60 min. This system will be useful as it has the potential for better diagnosis of pathogens by increasing the capture efficiency of the pathogen in large samples, subsequently enhancing the detection limit of pathogenic DNA.


Assuntos
Doenças Transmissíveis/diagnóstico , DNA Bacteriano/genética , Técnicas Analíticas Microfluídicas , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase em Tempo Real , Doenças Transmissíveis/genética
5.
Anal Chem ; 90(3): 1725-1733, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29313337

RESUMO

In this study, we developed an amine-functionalized, diatomaceous earth-based, dimethyl suberimidate assisted (ADD) system as a novel binding strategy to improve the solid-phase extraction method for rapid and simple purification of RNA from biological samples including human cells and pathogenic bacteria. This ADD system is based on reversible cross-linking reactions between RNA and the silica matrix. The formation of robust covalent bonds protects RNA from both the sufferance of washing steps and isolation with ribonuclease (RNase)-rich samples, leading to the extraction of higher quality RNA. This improved RNA extraction system integrated with quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is evaluated for pathogen diagnostics. Compared to standard solid-phase extraction based commercial kits, this improved method shows highly enhanced sensitivity with 1000-fold higher sensitivity for human cells and 100-fold higher sensitivity for Brucella bacteria, according to the cycle threshold value of RT-qPCR. We envision that the ADD system can be tailored for commercial applications for RNA expression analysis in forensics studies, as well as for disease diagnostics in clinical applications.


Assuntos
Reagentes para Ligações Cruzadas/química , Terra de Diatomáceas/química , Dimetil Suberimidato/química , RNA Bacteriano/isolamento & purificação , Extração em Fase Sólida/métodos , Animais , Brucella/genética , Brucelose/diagnóstico , Células HCT116 , Humanos , RNA Bacteriano/química , RNA Bacteriano/urina , Reação em Cadeia da Polimerase em Tempo Real , Ovinos
6.
Clin Ther ; 35(4): 431-9, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23474153

RESUMO

BACKGROUND: Diacerein is a drug used in osteoarthritis (OA) that elicits an inhibitory effect on interleukin-1 and metalloproteases. Although diacerein has shown modest efficacy and safety in the treatment of knee and hip OA, there have been no placebo-controlled clinical trials for hand OA. OBJECTIVE: The aim of the current study was to investigate the efficacy and tolerability of diacerein in patients with hand OA. METHODS: Patients fulfilling the American College of Rheumatology criteria for hand OA participated in this randomized, double-blind, placebo-controlled study. Eligible patients were >40 years of age, had at least 1 tender joint, and had a joint pain visual analog scale of >30 mm. Patients received diacerein (50 mg) or placebo BID for 12 weeks. The primary end point was the Australian/Canadian Osteoarthritis Hand Index (AUSCAN) pain score at 4 weeks. Secondary end points were AUSCAN pain score at 12 weeks and AUSCAN physical function and stiffness score, patient and physician global assessment, functional index of hand OA scores, and multidimensional health assessment questionnaire results at 4 weeks and 12 weeks. RESULTS: Eighty-six Korean patients were enrolled (42 diacerein, 44 placebo). The intention-to-treat and per-protocol analyses revealed no significant differences between the 2 groups in terms of change in AUSCAN pain score at 4 weeks, except for improvement in physician global assessment at 4 weeks (per-protocol analysis, P = 0.004). The safety profile of diacerein was comparable to placebo, except for frequent discoloration of the urine (88% vs 20%). CONCLUSION: These results suggest that diacerein 50 mg BID may be ineffective in controlling the symptoms of hand OA. ClinicalTrials.gov identifier: NCT00685542.


Assuntos
Antraquinonas/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Mãos/patologia , Osteoartrite/tratamento farmacológico , Antraquinonas/efeitos adversos , Anti-Inflamatórios/efeitos adversos , Método Duplo-Cego , Humanos , Placebos
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