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In situ monitoring of the actions of correlated enzymes in living cells is crucial for expanding our understanding of disease progression and evaluating drug efficacy. However, due to the diverse functions of different enzymes, currently available methods for comprehensive analysis of these events are limited. Here, we present an in situ track-generated DNA walker for AND-gate logic imaging of telomerase (TE) and flap endonuclease 1 (FEN1) activities in live cells. TE is in charge of generating the tracks for the walking strands by extending the TE primer on a gold nanoparticle, while FEN1 is responsible for recognizing the overlapping structure formed by the walking strands and the tracks and then cleaving the fluorescent reporter to produce signals. By utilizing the DNA walker, we successfully determined the expression levels and activities of TE and FEN1 in various cancer cell lines, offering promising prospects for screening inhibitors and investigating the biomolecular mechanisms of diseases.
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Nanopartículas del Metal , Telomerasa , Endonucleasas de ADN Solapado/genética , Telomerasa/metabolismo , Oro/química , Nanopartículas del Metal/química , ADN/químicaRESUMEN
Liquid biopsy as well as genotyping plays important roles in guiding the use of tumor-targeted drugs and monitoring the generation of drug resistance. However, current methods, such as next-generation sequencing (NGS) and pyrosequencing, require long analysis time and complicated steps. To achieve ultrafast and highly specific detection of cell-free DNA (cfDNA) from blood, we improved our recently developed FEN1-aided RPA (FARPA), which combined flap endonuclease 1 (FEN1)-catalyzed invasive reactions with recombinase polymerase amplification (RPA) by inactivating the RPA enzymes before invasive reactions, designing short RPA primers, and changing invasive reaction conditions. Using the L858R and T790M mutations as examples, FARPA was sensitive to detect 5 copies of targeted mutants, specific to sense the mutants with an abundance as low as 0.01% from blood, and ultrafast to get results within 40 min. The method was readily expended to genotyping, and 15 min was enough to report the allele species directly from oral swab samples by coupling quick DNA extraction reagents. Validation was carried out by detecting clinical samples, including 20 cfDNA from patients with non-small cell lung cancer (NSCLC) for liquid biopsy and 43 human genomic DNA (gDNA) purified from blood (33) or lysed from oral swabs (10) for genotyping, giving 100% agreement with NGS and pyrosequencing, respectively. Furthermore, a portable battery-driven device with dual-channel fluorescence detection was successfully constructed to facilitate point-of-care testing (POCT) of liquid biopsy and genotyping, providing doctors with a potential tool to achieve genotyping- or mutant-guided personalized medicine at emergency or source-limited regions.
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Carcinoma de Pulmón de Células no Pequeñas , Ácidos Nucleicos Libres de Células , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Neoplasias Pulmonares/diagnóstico , Receptores ErbB/genética , Mutación , Inhibidores de Proteínas Quinasas , ADN/genéticaRESUMEN
Rapid and accurate detection of pathogens and antimicrobial-resistant (AMR) genes of the pathogens are crucial for the clinical diagnosis and effective treatment of infectious diseases. However, the time-consuming steps of conventional culture-based methods inhibit the precise and early application of anti-infection therapy. For the prompt treatment of pathogen-infected patients, we have proposed a novel tube array strategy based on our previously reported FARPA (FEN1-aided recombinase polymerase amplification) principle for the ultra-fast detection of antibiotic-resistant pathogens on site. The entire process from "sample to result" can be completed in 25 min by combining quick DNA extraction from a urine sample with FARPA to avoid the usually complicated DNA extraction step. Furthermore, a 36-tube array made from commercial 384-well titre plates was efficiently introduced to perform FARPA in a portable analyser, achieving an increase in the loading sample throughput (from several to several tens), which is quite suitable for the point-of-care testing (POCT) of multiple pathogens and multiple samples. Finally, we tested 92 urine samples to verify the performance of our proposed method. The sensitivities for the detection of E. coli, K. pneumoniae, E. faecium, and E. faecalis were 92.7%, 93.8%, 100% and 88.9%, respectively. The specificities for the detection of the four pathogens were 100%. Consequently, our rapid, low-cost and user-friendly POCT method holds great potential for guiding the rational use of antibiotics and reducing bacterial resistance.
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ADN Bacteriano , Humanos , ADN Bacteriano/orina , ADN Bacteriano/genética , ADN Bacteriano/análisis , ADN Bacteriano/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico/métodos , Farmacorresistencia Bacteriana , Antibacterianos/farmacología , Pruebas en el Punto de Atención , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Escherichia coli/efectos de los fármacos , Recombinasas/metabolismoRESUMEN
BACKGROUND: Ambient air pollution might serve as a prognostic factor for ovarian cancer (OC) survival, yet the relationships between plant-based diet indices (PDIs) and OC survival remain unclear. We aimed to investigate the associations of comprehensive air pollution and PDIs with OC survival and explored the effects of air pollution-diet interactions. METHODS: The present study encompassed 658 patients diagnosed with OC. The overall plant-based diet index (PDI), the healthful PDI (hPDI), and the unhealthful PDI (uPDI) were evaluated by a self-reported validated food frequency questionnaire. In addition, an air pollution score (APS) was formulated by summing the concentrations of particulate matter with a diameter of 2.5 microns or less, ozone, and nitrogen dioxide. Cox proportional hazard models were applied to calculate hazard ratios (HRs) and 95â¯% confidence intervals (CIs). The potential interactions of APS with PDIs in relation to overall survival (OS) were assessed on both multiplicative and additive scales. RESULTS: Throughout a median follow-up of 37.60 (interquartile: 24.77-50.70) months, 123 deaths were confirmed. Comparing to the lowest tertiles, highest uPDI was associated with lower OS of OC (HR = 2.06, 95â¯% CI = 1.30, 3.28; P-trend < 0.01), whereas no significant associations were found between either overall PDI or hPDI and OC survival. Higher APS (HR for per interquartile range = 1.27, 95â¯% CI = 1.01, 1.60) was significantly associated with worse OC survival, and the association was exacerbated by adherence to uPDI. Notably, an additive interaction was identified between combined air pollution and uPDI (P < 0.005 for high APS and high uPDI). We also found that adherence to overall PDI aggravated associations of air pollution with OC survival (P-interaction = 0.006). CONCLUSIONS: Joint exposure to various ambient air pollutants was significantly associated with lower survival among patients with OC, particularly for those who predominantly consumed unhealthy plant-based foods.
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The potentially different genetics and epigenetics in the primary tumors and metastases affect the efficacy of treatment in breast cancer patients. Nevertheless, the cellular and molecular mechanisms of breast cancer lymph node metastasis still remain elusive. Here, we employed single-cell RNA sequencing to acquire the transcriptomic profiles of individual cells from primary tumors, negative lymph nodes (NLs) and positive lymph nodes (PLs). We also performed a single-cell assay for transposase-accessible chromatin (ATAC) sequencing (scATAC-seq) of the positive and NL samples to get the chromatin accessibility profile. We identified a novel cell subpopulation with an abnormally high expression level of CXCL14 in the PL of breast cancer patients. Cell trajectory analysis also revealed that CXCL14 was increased expressed in the late pseudo-time. Moreover, based on a tissue microarray of 55 patients and the Oncomine database, we validated that CXCL14 expression was significantly higher in breast cancer patients with lymph node metastasis. Furthermore, scATAC-seq identified several transcription factors that may be potential regulation factors for the lymph node metastasis of breast cancer. Thus, our findings will improve our current understanding of the mechanism for lymph node metastasis, and they are potentially valuable in providing novel prognosis markers for the lymphatic metastasis of breast cancer.
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Neoplasias de la Mama/genética , Quimiocinas CXC/genética , Cromatina/genética , Regulación Neoplásica de la Expresión Génica , Metástasis Linfática/genética , Biomarcadores de Tumor , Neoplasias de la Mama/metabolismo , Quimiocinas CXC/metabolismo , Secuenciación de Inmunoprecipitación de Cromatina/métodos , Femenino , Humanos , Análisis por Micromatrices , Persona de Mediana Edad , Pronóstico , ARN Citoplasmático Pequeño , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual , Factores de Transcripción/genética , TranscriptomaRESUMEN
In situ observation of changes in the activity of marker proteins in living cells is crucial for both biomarker-based disease diagnosis and drug screening. Flap endonuclease 1 (FEN1) has been recognized as a broad-spectrum cancer biomarker and therapeutic target. However, simple and reliable methods for in situ studying the FEN1 activity changes in living cells are limited. Here, we introduce a nano firework as a fluorescent sensor to sense and report FEN1 activity changes in living cells through FEN1 recognizing the substrates on the surface of the nano firework to release and restore the fluorescence of the prequenched fluorophores. We verified the high selectivity, anti-interference ability, stability, and quantitative performance of the nano firework in tubes and living cells, respectively. A series of controlled experiments have demonstrated that the nano firework could accurately report changes in FEN1 activity in different cells, enabling "sensors in, results out" in the manner of simple addition to the cell culture medium. Using an in silico molecular docking study and experiments, we also explored the ability of the nano firework for rapid screening of FEN1 inhibitors and found two new candidate compounds myricetrin and neoisoliquritin, which could be used as FEN1 inhibitors for further research. These performances of the nano firework suggest that it can be used in high-throughput screening applications, providing a promising tool for biomarker-based new drug discovery.
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Endonucleasas de ADN Solapado , Ensayos Analíticos de Alto Rendimiento , Endonucleasas de ADN Solapado/genética , Simulación del Acoplamiento Molecular , Biomarcadores de Tumor , ADN/químicaRESUMEN
A simple, cost-effective and reliable diagnosis of pathogen nucleic acids assay is much required for controlling a pandemic of a virus disease, such as COVID-19. Our previously developed visualized detection of pathogen DNA in a single closed tube is very suitable for POCT. However, virus RNA could not be detected directly and should be reverse-transcribed into cDNA in advance. To enable this visualized assay to detect virus RNA directly, various types of reverse transcriptase were investigated, and finally we found that HiScript II reverse transcriptase could keep active and be well adapted to the one-pot visualized assay in optimized conditions. Reverse transcription, template amplification and amplicon identification by PCR coupled with invasive reaction, as well as visualization by self-assembling of AuNP probes could be automatically and sequentially performed in a closed tube under different temperature conditions, achieving "sample (RNA)-in-result (red color)-out" only by a simple PCR engine plus the naked eye. The visualized RT-PCR is sensitive to unambiguous detection of 5 copies of the N and ORFlab genes of SARS-CoV-2 RNA comparing favourably with qPCR methods (commercialized kit), is specific to genotype 3 variants (Alpha, Beta and Omicron) of SARS-CoV-2, and is very accurate for picking up 0.01% Omicron variant from a large amount of sequence-similar backgrounds. The method is employed in detecting 50 clinical samples, and 10 of them were detected as SARS-CoV-2 positive samples, identical to those by conventional RT-PCR, indicating that the method is cost-effective and labor-saving for pathogen RNA screening in resource-limited regions.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ARN Viral/genética , ARN Viral/análisis , Técnicas de Amplificación de Ácido Nucleico/métodos , ADN Polimerasa Dirigida por ARN/genética , Sensibilidad y Especificidad , Prueba de COVID-19RESUMEN
Recent evidence advises particles with a diameter of 2.5 µm or less (PM2.5) might be a prognostic factor for ovarian cancer (OC) survival. The oxidative balance score (OBS) incorporates diet-lifestyle factors to estimate individuals' anti-oxidant exposure status which may be relevant to cancer prognosis. We aimed to investigate the roles of PM2.5, and OBS and their interaction in OC prognosis. 663 patients with OC were enrolled in the current study. Satellite-derived annual average exposures to PM2.5 based on patients' residential locations. The OBS was calculated based on 16 different diet-lifestyle components derived using an acknowledged self-reported questionnaire. The Cox regression model was performed to estimate the hazard ratios (HRs) and 95% confidence intervals (CIs) for overall survival (OS). We also assessed the effect of modification between PM2.5 and OS by OBS via interaction terms. During a median follow-up of 37.57 (interquartile:35.27-40.17) months, 123 patients died. Compared to low-concentration PM2.5 exposure, high PM2.5 during 1 year before diagnosis was associated with worse OC survival (HR= 1.19, 95% CI = 1.01-1.42). We observed an improved OS with the highest compared with the lowest OBS (HR = 0.46, 95% CI = 0.27-0.79, P for trend < 0.05). Notably, we also found an additive interaction between low OBS and high exposure to PM2.5, with the corresponding associations of PM2.5 being more pronounced among participants with lower OBS (HR = 1.42, 95% CI = 1.09-1.86). PM2.5 may blunt OC survival, but high OBS represented an antioxidative performance that could alleviate the adverse association of PM2.5 and OS.
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Contaminantes Atmosféricos , Contaminación del Aire , Neoplasias Ováricas , Humanos , Femenino , Material Particulado , Estudios Prospectivos , Antioxidantes , Estrés Oxidativo , Exposición a Riesgos AmbientalesRESUMEN
Digital nucleic acid analysis technology has shown great application potential due to its excellent performance. However, most current digital nucleic acid detection methods are based on PCR or other template amplification strategies. Here, we present an alternative analysis platform based on digital nucleic acid signal amplification in droplets termed dNASA. Using a bead-based controllable extension bridged cascade signal amplification reaction, we achieved an ultralow background, high efficiency, and highly specific nucleic acid signal amplification analysis. As a "proof of concept", we demonstrated the feasibility of the proposed dNASA platform in single-base DNA mutation analysis using artificially synthesized samples. This platform provides innovative ideas for the field of digital nucleic acid analysis.
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Técnicas de Amplificación de Ácido Nucleico , Ácidos Nucleicos , ADN/genética , Análisis Mutacional de ADN , Mutación , Técnicas de Amplificación de Ácido Nucleico/métodosRESUMEN
A lateral flow strip (LFS) is an ideal tool for point-of-care testing (POCT), but traditional LFSs cannot be used for multiplex detection. Herein, a multiplex and versatile LFS based on flap endonuclease 1 (FEN1)-induced steric hindrance change (FISH-LFS) is proposed. In this method, multiplex PCR coupled with cascade invasive reactions was employed to yield single-stranded flaps, which were target-specific but independent of target sequences. Then, the amplicons were applied for FISH-LFS, and the single-stranded flaps would be efficiently captured by the complementary LFS-probes at different test lines. As flaps were cleaved from the specially designed hairpin probes, competition among flaps and hairpin probes would occur in capturing the probes at test lines. We enabled the hairpin probes to flow through the test lines while the flaps to stay at the test lines by making use of the difference in steric hindrance between hairpin probes and flaps. The assay is able to detect as low as two copies of blood pathogens (HBV, HCV, and HIV), to pick up as low as 0.1% mutants from wild-type gDNA, and to genotype 200 copies of SARS-CoV-2 variants α and ß within 75 min at a conventional PCR engine. As the method is free of dye, a portable PCR engine could be used for a cost-effective multiplex detection on site. Results using an ultrafast mobile PCR system for FISH-LFS showed that as fast as 30 min was achieved for detecting three pathogens (HBV, HCV, and HIV) in blood, very suitable for POCT of pathogen screening. The method is convenient in operation, simple in instrumentation, specific in genotyping, and very easy in setting up multiplex POCT assays.
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COVID-19 , Infecciones por VIH , Hepatitis C , Humanos , SARS-CoV-2 , Endonucleasas de ADN Solapado , ADN , Sensibilidad y EspecificidadRESUMEN
Antimicrobial susceptibility testing (AST) is an effective way to guide antibiotic selection. However, conventional culture-based phenotypic AST is time-consuming. The key point to shorten the test is to quantify the small change in the bacterial number after the antibiotic exposure. To achieve rapid AST, we proposed a combination of multiplexed PCR with barcoded pyrosequencing to significantly shorten the time for antibiotic exposure. First, bacteria exposed to each antibiotic were labeled with a unique barcode. Then, the pool of the barcoded products was amplified by PCR with a universal primer pair. Finally, barcodes in the amplicons were individually and quantitatively decoded by pyrosequencing. As pyrosequencing is able to discriminate as low as 5% variation in target concentrations, as short as 7.5 min was enough for cultivation to detect the susceptibility of Escherichia coli to an antibiotic. The barcodes enable more than six kinds of drugs or six kinds of concentrations of a drug to be tested at a time. The susceptibility of 6 antibiotics to 43 E. coli-positive samples from 482 clinical urine samples showed a consistency of 99.3% for drug-resistant samples and of 95.7% for drug-sensitive samples in comparison with the conventional method. In addition, the minimum inhibitory concentration (MIC) of 29 E. coli samples was successfully measured. The proposed AST is dye free (pyrosequencing), multiplexed (six antibiotics), fast (a half-working day for reporting the results), and able to detect the MIC, thus having a great potential for clinical use in quick antibiotic selection.
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Antibacterianos , Infecciones por Escherichia coli , Antibacterianos/farmacología , Bacterias , Escherichia coli/genética , Infecciones por Escherichia coli/tratamiento farmacológico , Infecciones por Escherichia coli/microbiología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Pruebas de Sensibilidad MicrobianaRESUMEN
Cancer-derived exosomes carry a variety of important biomarkers specific to the formation, invasion and metastasis of tumor tissue. Dynamic monitoring of exosomes originated from cancer cells has clinical significance. Here we proposed a novel method to employ zirconium-metal-organic frameworks (Zr-MOFs) for extracting and identifying exosomes from blood. At first UiO-66 was magnetically modified as the adsorbent to anchor exosomes by forming Zr-O-P bonds. Then UiO-66-NH2 modified with anti-EpCAM was used to construct the fluorescent probe to recognize the extracted EpCAM-positive exosomes by forming a "MOF-exosome-MOF" structure. The proposed fluorescence detection method was evaluated by quantifying MCF-7 cell-derived exosomes at the concentration as low as 16.72 particles/µl. This method was successfully applied to analyze exosomes in the plasma samples from healthy donors and breast cancer patients, demonstrating that our method might have a great potential in assisting the early diagnosis and in dynamically monitoring the efficacy of cancer treatment. We believe that the method could be extended to the detection of other biomarkers in exosomes derived from cancer cell.
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Exosomas , Estructuras Metalorgánicas , Neoplasias , Fluorescencia , Humanos , Lípidos , Estructuras Metalorgánicas/química , Ácidos Ftálicos , Circonio/químicaRESUMEN
Here, we characterized a flap endonuclease 1 (FEN1) plus hairpin DNA probe (hpDNA) system, designated the HpSGN system, for both DNA and RNA editing without sequence limitation. The compact size of the HpSGN system make it an ideal candidate for in vivo delivery applications. In vitro biochemical studies showed that the HpSGN system required less nuclease to cleave ssDNA substrates than the SGN system we reported previously by a factor of â¼40. Also, we proved that the HpSGN system can efficiently cleave different RNA targets in vitro. The HpSGN system cleaved genomic DNA at an efficiency of â¼40% and â¼20% in bacterial and human cells, respectively, and knocked down specific mRNAs in human cells at a level of â¼25%. Furthermore, the HpSGN system was sensitive to the single base mismatch at the position next to the hairpin both in vitro and in vivo. Collectively, this study demonstrated the potential of developing the HpSGN system as a small, effective, and specific editing tool for manipulating both DNA and RNA without sequence limitation.
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Archaeoglobus fulgidus/enzimología , Endonucleasas de ADN Solapado/metabolismo , Edición Génica/métodos , Secuencias Invertidas Repetidas , Edición de ARN , Archaeoglobus fulgidus/genética , Disparidad de Par Base , ADN/química , Sondas de ADN/química , Sondas de ADN/genética , ADN de Cadena Simple , Escherichia coli/genética , Endonucleasas de ADN Solapado/química , Endonucleasas de ADN Solapado/genética , Endonucleasas de ADN Solapado/aislamiento & purificación , Células HEK293 , Humanos , Técnicas In Vitro , Conformación de Ácido Nucleico , ARN/química , Especificidad por SustratoRESUMEN
Cervical cancer is the fourth leading cause of death in women, especially in developing countries. Specific and economic methodologies for HPV typing are crucial in cancer diagnosis and further disease control. However, routine assays based on real-time polymerase chain reaction (qPCR) or DNA-chip hybridization are either incapable of offering detailed subtype information or involve tedious open-tube operations with the risk of cross-contamination from PCR amplicons. Herein, we proposed a multiplex visualized closed-tube PCR (Multi-Vision) for HPV typing. Using gold nanoparticle probes (AuNPs) as a color change indicator combined with a Hamming distance 2 coding scheme, 13 high-risk HPVs and two subtypes associated with high-incidence benign lesions were successfully typed by performing six closed-tube PCRs. The assay demonstrates high specificity with no cross-reaction among different subtypes under several artificial sample concentrations (from 100 to 103 copies per reaction) and enables highly sensitive detection of as low as 0.5 copies/µL. Further, 105 clinical samples were successfully analyzed using our method with a high concordance rate of 99.05% (104/105) compared to a HPV typing kit. The inconsistent sample was confirmed by sequencing to be consistent with the typing results determined by our method, indicating that Multi-Vision could be a useful tool for HPV detection, especially in resource-limited regions.
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Nanopartículas del Metal , Infecciones por Papillomavirus , ADN Viral/genética , Femenino , Oro , Humanos , Papillomaviridae/genética , Infecciones por Papillomavirus/diagnóstico , Sensibilidad y EspecificidadRESUMEN
DNA walkers have shown superior performance in biosensing due to their programmability to design molecular walking behaviors with specific responses to different biological targets. However, it is still challenging to make DNA walkers capable of distinguishing DNA targets with single-base differences, so that DNA walkers that can be used for circulating tumor DNA sensing are rarely reported. Herein, a flap endonuclease 1 (FEN 1)-assisted DNA walker has been proposed to achieve mutant biosensing. The target DNA is captured on a gold nanoparticle (AuNP) as a walking strand to walk by hybridizing to the track strands on the surface of the AuNP. FEN 1 is employed to report the walking events by cleaving the track strands that must form a three-base overlapping structure recognized by FEN 1 after hybridizing with the captured target DNA. Owing to the high specificity of FEN 1 for structure recognition, the one-base mutant DNA target can be discriminated from wild-type DNA. By constructing a sensitivity-enhanced DNA walker system, as low as 1 fM DNA targets and 0.1% mutation abundance can be sensed, and the theoretical detection limits for detecting the DNA target and mutation abundance achieve 0.22 fM and 0.01%, respectively. The results of epidermal growth factor receptor (EGFR) L858R mutation detection on cell-free DNA samples from 15 patients with nonsmall cell lung cancer were completely consistent with that of next-generation sequencing, indicating that our DNA walker has potential for liquid biopsy.
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Técnicas Biosensibles , Carcinoma de Pulmón de Células no Pequeñas , ADN Tumoral Circulante/análisis , Neoplasias Pulmonares , Nanopartículas del Metal , Endonucleasas de ADN Solapado , Oro , HumanosRESUMEN
Detection of blood-borne pathogens such as hepatitis C virus (HCV), hepatitis B virus (HBV) and human immunodeficiency virus (HIV) is essential to ensure the safety of blood transfusion. However, traditional PCR-based pathogen nucleic acid detection methods require relatively high experimental facilities and are difficult to apply in areas with limited resources. In this study, a self-driven microfluidic chip was designed to carry out multiplex detection of HBV, HCV and HIV by using loop-mediated isothermal amplification (LAMP). Benefitting from the air permeability of the polydimethylsiloxane material, the chip could accomplish sample loading within 12 min driven by the pressure difference between the reaction chambers and vacuum chambers in the chip without using pumps or any injection devices. Multiplex detection is achieved by presetting LAMP primers specific to different targets in different reaction chambers. Calcein was used as an indicator to indicate the positive amplification reaction, and the result can be recorded by a smartphone camera. After 50 min of isothermal amplification at 63 °C, 2 copies/µL of HBV, HCV and HIV target nucleic acids could be detected. The results of HBV detection of 20 clinical plasma samples by using the chip are consistent with that of the qPCR-based kit, indicating that the LAMP-based self-driven chip has the clinical application potential for blood-borne pathogen detection, especially in resource-limited areas.
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Patógenos Transmitidos por la Sangre/aislamiento & purificación , Dispositivos Laboratorio en un Chip , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , VIH/aislamiento & purificación , Hepacivirus/aislamiento & purificación , Virus de la Hepatitis B/aislamiento & purificación , Límite de Detección , Técnicas Analíticas Microfluídicas/instrumentaciónRESUMEN
Nucleic acid detection is important for clinical diagnostics; however, it is challenging to perform genetic testing at the point-of-care due to the tedious steps involved in DNA extraction and the risk of cross-contamination from amplicons. To achieve a fully-automated and contamination-free nucleic acid detection, we propose a closed-type cassette system which enables the following steps to be operated automatically and sequentially: sample preparation based on magnetic beads, target amplification using multiplex polymerase chain reaction, and colorimetric detection of amplicons using a serial invasive reaction coupled with the aggregation of gold nanoparticle probes. The cassette was designed to be round and closed, and 10 targets in a sample could be simultaneously detected by the naked eye or using a spectrophotometer in the system. In addition, a cassette-driven device was fabricated to transfer reagents between wells, to control the temperature of each reaction, and to sense the colour in the detection wells. The cassette system was sensitive enough to detect 10 genotypes at 5 single nucleotide polymorphism sites related to the anticoagulant's usage, by using a 0.5⯵L blood sample. The accuracy of the system was evaluated by detecting 12 whole blood samples, and the results obtained were consistent with those obtained using pyrosequencing. The cassette is airtight and the whole system is fully automatic; the only manual operation is the addition of the sample to the cassette, performing point-of-care genetic testing in a sample-in/answer-out way.
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Individual variation in pharmacokinetics of faropenem is significant. We attempted to predict drug response of faropenem using a pharmacometabonomic approach. Metabolic profiling was performed on predose plasma samples from 36 healthy volunteers by gas chromatography-mass spectrometry (GC/MS), with 204 endogenous metabolites detected. Plasma concentration was measured after drug administration, using high pressure liquid chromatography-tandem mass spectroscopy (LC/MS/MS), and the pharmacokinetic parameters were calculated. Then a two-stage partial least squares strategy was employed to screen potential biomarkers and predict the pharmacokinetic parameters of faropenem. The results showed a good prediction of AUC and Cmax with the screened biomarkers, and metabolites such as valine, proline, aspartic acid, gluconic acid, glucuronic acid, and 2-ketoisocaproic acid were indicated as candidate biomarkers. Finally, we explored the mechanism of individual variation by pathway enrichment analysis, and it suggested that organic anion transporter 1 (OAT1) and 3 (OAT3) might be responsible for individual variation of faropenem, and this hypothesis was verified by an experiment in rats.
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Metabolómica , Espectrometría de Masas en Tándem , Animales , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Ratas , beta-LactamasRESUMEN
INTRODUCTION: Pharmacogenetics and pharmacometabolomics are the common methods for personalized medicine, either genetic or metabolic biomarkers have limited predictive power for drug response. OBJECTIVES: In order to better predict drug response, the study attempted to integrate genetic and metabolic biomarkers for drug pharmacokinetics prediction. METHODS: The study chose celecoxib as study object, the pharmacokinetic behavior of celecoxib was assessed in 48 healthy volunteers based on UPLC-MS/MS platform, and celecoxib related single nucleotide polymorphisms (SNPs) were also detected. Three mathematic models were constructed for celecoxib pharmacokinetics prediction, the first one was mainly based on celecoxib-related SNPs; the second was based on the metabolites selected from a pharmacometabolomic analysis by using GC-MS/MS method, the last model was based on the combination of the celecoxib-related SNPs and metabolites above. RESULTS: The result proved that the last model showed an improved prediction power, the integration model could explain 71.0% AUC variation and predict 62.3% AUC variation. To facilitate clinical application, ten potential celecoxib-related biomarkers were further screened, which could explain 68.3% and predict 54.6% AUC variation, the predicted AUC was well correlated with the measured values (r = 0.838). CONCLUSION: This study provides a new route for personalized medicine, the integration of genetic and metabolic biomarkers can predict drug response with a higher accuracy.
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Antiinflamatorios no Esteroideos/metabolismo , Celecoxib/metabolismo , Polimorfismo de Nucleótido Simple/genética , Adulto , Antiinflamatorios no Esteroideos/farmacocinética , Biomarcadores/análisis , Celecoxib/farmacocinética , Cromatografía Líquida de Alta Presión , Voluntarios Sanos , Humanos , Masculino , Metabolómica , Medicina de Precisión , Espectrometría de Masas en Tándem , Adulto JovenRESUMEN
Accurately quantifying hepatitis B virus DNA (HBV-DNA) in serum is important in dynamic monitoring and prognosis evaluation for patients with hepatitis B. Routine assays based on real-time polymerase chain reaction (qPCR) for HBV-DNA quantification usually require laborious calibration curves and may bring bias from the biological samples. To enable absolute quantification of HBV-DNA in a single tube, we described a modification of the conventional Q-Invader assay by separately encoding targeted DNA and artificially designed internal quantitative-standard DNA (QS-DNA) at the flaps of the corresponding downstream probes. Quantification of targeted HBV-DNA was readily achieved by the difference in the quantification cycle value (Ct) between itself and QS-DNA. Furthermore, spiked-in QS-DNA before DNA extraction allowed errors caused by DNA extraction to be corrected. Two different gene regions covering eight genotypes were encoded with the same flap to avoid false-negative results. The method demonstrates a high sensitivity, which enables accurate detection of as low as 2 copies of the HBV-DNA plasmid or 20 IU mL-1 HBV-DNA in serum in a single tube. Successful quantification of 50 clinical samples indicates that our method is cost-effective, labor-saving and reproducible, and promising for the ultra-sensitive quantification analysis of many types of pathogens other than HBV.