Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 9.687
Filtrar
2.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 31(4): 388-392, 2019 Sep 19.
Artigo em Chinês | MEDLINE | ID: mdl-31612673

RESUMO

OBJECTIVE: To establish a recombinase-aided isothermal amplification (RAA) assay for detection of Cryptosporidium. METHODS: Based on Cryptosporidium-specific 18S rRNA selected as the target gene to be detected, and the primer sequences and fluorescent probes designed using the software Amplfix, and a fluorescent RAA assay was established and optimized. The fluorescent RAA assay was performed to detect 18S RNA target sequence-contained recombinant plasmids at various copies, genomic DNA of Cryptosporidium oocysts at various concentrations, and genomic DNA extracted from various numbers of Cryptosporidium oocysts to assess the sensitivity of the assay, and to detect genomic DNA extracted from Cryptosporidium oocysts, Giardia lamblia cysts, Schistosoma japonicum eggs, Ascaris lumbricoides eggs, Clonorchis sinensis eggs, Salmonella and Shigella to determine the specificity of the assay. RESULTS: A fluorescent RAA assay was successfully established, which was effective to amplify the specific 18S RNA gene fragments of Cryptosporidium within 20 min at 39 ℃. The lowest limits of the fluorescent RAA assay were 102 copies/µL for detection of 18S RNA target sequence-contained recombinant plasmids at various copies, 1 pg/µL for detection of genomic DNA of Cryptosporidium oocysts at various concentrations, and one Cryptosporidium oocyst/µL for detection of genomic DNA extracted from various numbers of Cryptosporidium oocysts, and the fluorescent RAA assay was all negative for detection of genomic DNA from G. lamblia cysts, S. japonicum eggs, A. lumbricoides eggs, C. sinensis eggs, Salmonella and Shigella. CONCLUSIONS: A novel fluorescent RAA assay is successfully established, which is simple, rapid, sensitive and specific to detect genomic DNA of Cryptosporidium oocysts.


Assuntos
Cryptosporidium , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/normas , Cryptosporidium/genética , DNA de Protozoário/genética , Limite de Detecção , Oocistos , RNA Ribossômico 18S/genética , Recombinases/metabolismo , Sensibilidade e Especificidade
3.
N Engl J Med ; 381(14): 1347-1357, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31577876

RESUMO

BACKGROUND: The World Health Organization has set ambitious targets for the global elimination of tuberculosis. However, these targets will not be achieved at the current rate of progress. METHODS: We performed a cluster-randomized, controlled trial in Ca Mau Province, Vietnam, to evaluate the effectiveness of active community-wide screening, as compared with standard passive case detection alone, for reducing the prevalence of tuberculosis. Persons 15 years of age or older who resided in 60 intervention clusters (subcommunes) were screened for pulmonary tuberculosis, regardless of symptoms, annually for 3 years, beginning in 2014, by means of rapid nucleic acid amplification testing of spontaneously expectorated sputum samples. Active screening was not performed in the 60 control clusters in the first 3 years. The primary outcome, measured in the fourth year, was the prevalence of microbiologically confirmed pulmonary tuberculosis among persons 15 years of age or older. The secondary outcome was the prevalence of tuberculosis infection, as assessed by an interferon gamma release assay in the fourth year, among children born in 2012. RESULTS: In the fourth-year prevalence survey, we tested 42,150 participants in the intervention group and 41,680 participants in the control group. A total of 53 participants in the intervention group (126 per 100,000 population) and 94 participants in the control group (226 per 100,000) had pulmonary tuberculosis, as confirmed by a positive nucleic acid amplification test for Mycobacterium tuberculosis (prevalence ratio, 0.56; 95% confidence interval [CI], 0.40 to 0.78; P<0.001). The prevalence of tuberculosis infection in children born in 2012 was 3.3% in the intervention group and 2.6% in the control group (prevalence ratio, 1.29; 95% CI, 0.70 to 2.36; P = 0.42). CONCLUSIONS: Three years of community-wide screening in persons 15 years of age or older who resided in Ca Mau Province, Vietnam, resulted in a lower prevalence of pulmonary tuberculosis in the fourth year than standard passive case detection alone. (Funded by the Australian National Health and Medical Research Council; ACT3 Australian New Zealand Clinical Trials Registry number, ACTRN12614000372684.).


Assuntos
Doenças Endêmicas/prevenção & controle , Programas de Rastreamento/métodos , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Pulmonar/diagnóstico , Adolescente , Adulto , Criança , Serviços de Saúde Comunitária , Feminino , Humanos , Análise de Intenção de Tratamento , Masculino , Mycobacterium tuberculosis/genética , Técnicas de Amplificação de Ácido Nucleico , Prevalência , Escarro/microbiologia , Tuberculose Pulmonar/epidemiologia , Tuberculose Pulmonar/prevenção & controle , Vietnã/epidemiologia , Adulto Jovem
4.
Chem Commun (Camb) ; 55(83): 12463-12466, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31576854

RESUMO

Herein, an ultrasensitive electrochemical biosensor is proposed for the quantification of the Flu A virus biomarker DNA (fDNA), and is based on loop-mediated isothermal amplification-generated hydrogen ions (LAMP-H+) which induce the formation of the dimer i-motif structure (DiMS) for signal transduction, coupled with exonuclease III (ExoIII)-assisted DNA walking for signal dual-amplification.


Assuntos
Técnicas Biossensoriais , DNA Viral/análise , Técnicas Eletroquímicas , Vírus da Influenza A/química , Técnicas de Amplificação de Ácido Nucleico , Prótons , Biomarcadores/análise , Dimerização , Íons/química
5.
Chem Commun (Camb) ; 55(86): 12980-12983, 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31603440

RESUMO

Nuclear factor kappa B p50 (NF-κB p50) induces various biological processes. In this study, a highly selective and sensitive electrochemiluminescence (ECL) biosensor for the detection of NF-κB p50 has been developed, which combines the high selectivity of the proximity hybridization assay (PHA) with the high efficiency of the hybridization chain reaction (HCR).


Assuntos
Técnicas Biossensoriais/métodos , Subunidade p50 de NF-kappa B/análise , DNA/química , Técnicas Eletroquímicas , Eletrodos , Humanos , Medições Luminescentes , Técnicas de Amplificação de Ácido Nucleico , Hibridização de Ácido Nucleico
6.
Klin Lab Diagn ; 64(9): 571-577, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31610111

RESUMO

This study presents the results of laboratory trials of the reagent kit for the rapid detection of RNA of the Crimean-Congo hemorrhagic fever virus (CCHFV) using loop-mediated isothermal amplification with reverse transcription (RT-LAMP). The developed RT-LAMP reagent kit was used to detect the CCHFV and showed a sensitivity of 103 GE/ml of viral RNA, which is sufficient for detection of the CCHFV in the early stage of human infections. The kit showed high specificity and no cross-reactivity with viral panel from the State collection of viruses of the FBRI SRC VB «Vector¼ (arboviruses and hemorrhagic fever viruses). Laboratory trials of the RT-LAMP kit are showed a high analytical and diagnostic sensitivity and specificity for RNA detection of the CCHFV and high speed of the analysis (60-70 min with sample preparation) compared to real-time PCR. Approbation of the kit field version has showed the possibility of setting the RT-LAMP reaction and viral RNA detection without the using of analytical equipments.


Assuntos
Vírus da Febre Hemorrágica da Crimeia-Congo/isolamento & purificação , Febre Hemorrágica da Crimeia/diagnóstico , Técnicas de Amplificação de Ácido Nucleico , Kit de Reagentes para Diagnóstico , Humanos , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Reversa , Sensibilidade e Especificidade
7.
Chem Commun (Camb) ; 55(79): 11932-11935, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31531427

RESUMO

An enzyme-free DNA circuit was designed for the amplified detection of Cd2+ based on hairpin probe-mediated toehold binding and branch migration. A Cd2+-specific aptamer was used to recognize Cd2+ and a G-quadruplex was used to report the detection signal. The assay is sensitive, with a detection limit of 5 pM.


Assuntos
Aptâmeros de Nucleotídeos/química , Cádmio/análise , DNA/química , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Quadruplex G , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico , Sensibilidade e Especificidade , Espectrometria de Fluorescência
9.
Zhonghua Liu Xing Bing Xue Za Zhi ; 40(8): 1018-1022, 2019 Aug 10.
Artigo em Chinês | MEDLINE | ID: mdl-31484272

RESUMO

Nucleic acid sequence-based amplification and recombinase polymerase amplification are the recently developed thermostatic amplification techniques based on PCR. This paper briefly summarizes the principle of reaction, design principle of primer and probe, advantage of these two techniques (simple, accurate, highly sensitive and rapid) and introduces the application of the techniques in the detection of pathogenic bacteria.


Assuntos
Bactérias/genética , DNA Bacteriano/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Replicação de Sequência Autossustentável/métodos , Primers do DNA , Humanos , Sensibilidade e Especificidade
10.
Chem Commun (Camb) ; 55(77): 11551-11554, 2019 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-31490470

RESUMO

A novel ratiometric electrochemiluminescence (ECL)-electrochemical (EC) hybrid biosensor with a high accuracy and reproducibility was fabricated for the ultrasensitive detection of miRNA-133a. With the help of a DNA tetrahedron nanostructure and hybridization chain reaction dual amplification strategy, the detection limit of 12.17 aM for miRNA-133a was obtained.


Assuntos
Técnicas Biossensoriais/métodos , DNA/química , MicroRNAs/análise , Nanoestruturas/química , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/química , Técnicas Biossensoriais/instrumentação , Técnicas Eletroquímicas , Corantes Fluorescentes/química , Humanos , Limite de Detecção , Medições Luminescentes , Azul de Metileno/química , Técnicas de Amplificação de Ácido Nucleico , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Compostos Organometálicos/química
11.
J Med Microbiol ; 68(10): 1431-1437, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31385779

RESUMO

Purpose. Rapid and accurate detection of carbapenem resistance is a critical requirement for the selection of appropriate therapy and initiation of infection control measures. Although several tests are available, their use is limited by one or more factors. Phenotypic tests are lengthy, have variable sensitivity and specificity and do not generally identify the carbapenemase. Molecular assays overcome many of these issues but cost can be a barrier to adoption, particularly in low-resource settings. To address the need for affordable, molecular tools, we have assessed the performance characteristics of loop-mediated isothermal amplification (LAMP)-based assays for the major carbapenemase genes, blaNDM, blaKPC, blaOXA-48, blaOXA-23 blaVIM and blaIMP.Methodology. The assays were validated using 1849 Gram-negative Indian clinical isolates obtained from seven hospitals and diagnostic centres.Results. The assays had diagnostic sensitivities of 98.14 %, 98.92 %, 100 %, 98.48 %, and diagnostic specificities of 98.94 %, 99.61 %, 97.42 %, 99.38 % for blaNDM, blaOXA-48, blaOXA-23 and blaVIM, respectively. Due to a low number of isolates positive for blaKPC and blaIMP, the performance characteristics of assays for these two genes could not be suitably evaluated.Conclusion. The performance characteristics suggest suitability for diagnostic and surveillance purposes.


Assuntos
Proteínas de Bactérias/genética , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/isolamento & purificação , Infecções por Bactérias Gram-Negativas/microbiologia , Técnicas de Amplificação de Ácido Nucleico/métodos , beta-Lactamases/genética , Proteínas de Bactérias/metabolismo , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/diagnóstico , Humanos , beta-Lactamases/metabolismo
12.
BMC Infect Dis ; 19(1): 676, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370782

RESUMO

BACKGROUND: In addition to seasonal influenza viruses recently circulating in humans, avian influenza viruses (AIVs) of H5N1, H5N6 and H7N9 subtypes have also emerged and demonstrated human infection abilities with high mortality rates. Although influenza viral infections are usually diagnosed using viral isolation and serological/molecular analyses, the cost, accessibility, and availability of these methods may limit their utility in various settings. The objective of this study was to develop and optimized a multiplex detection system for most influenza viruses currently infecting humans. METHODS: We developed and optimized a multiplex detection system for most influenza viruses currently infecting humans including two type B (both Victoria lineages and Yamagata lineages), H1N1, H3N2, H5N1, H5N6, and H7N9 using Reverse Transcriptional Loop-mediated Isothermal Amplification (RT-LAMP) technology coupled with a one-pot colorimetric visualization system to facilitate direct determination of results without additional steps. We also evaluated this multiplex RT-LAMP for clinical use using a total of 135 clinical and spiked samples (91 influenza viruses and 44 other human infectious viruses). RESULTS: We achieved rapid detection of seasonal influenza viruses (H1N1, H3N2, and Type B) and avian influenza viruses (H5N1, H5N6, H5N8 and H7N9) within an hour. The assay could detect influenza viruses with high sensitivity (i.e., from 100 to 0.1 viral genome copies), comparable to conventional RT-PCR-based approaches which would typically take several hours and require expensive equipment. This assay was capable of specifically detecting each influenza virus (Type B, H1N1, H3N2, H5N1, H5N6, H5N8 and H7N9) without cross-reactivity with other subtypes of AIVs or other human infectious viruses. Furthermore, 91 clinical and spiked samples confirmed by qRT-PCR were also detected by this multiplex RT-LAMP with 98.9% agreement. It was more sensitive than one-step RT-PCR approach (92.3%). CONCLUSIONS: Results of this study suggest that our multiplex RT-LAMP assay may provide a rapid, sensitive, cost-effective, and reliable diagnostic method for identifying recent influenza viruses infecting humans, especially in locations without access to large platforms or sophisticated equipment.


Assuntos
Colorimetria/métodos , Vírus da Influenza A/genética , Influenza Humana/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Reações Cruzadas , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Virus da Influenza A Subtipo H5N1/genética , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/isolamento & purificação , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza A/patogenicidade , Transcrição Reversa
13.
BMC Infect Dis ; 19(1): 680, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370795

RESUMO

BACKGROUND: A major challenge in dengue management in resource limited settings is the confirmation of diagnosis. Clinical features of dengue often overlap with other infections and molecular diagnostic tools are not readily accessible to clinicians at hospitals. In addition, the prediction of plasma leakage in dengue is also difficult. Hematocrit level and ultrasound scans (combined with clinical parameters) are helpful to detect plasma leakage once it has happened, not before. METHODS: Colombo Dengue Study (CDS) is a prospective cohort study of clinically suspected adult dengue patients recruited from the National hospital of Sri Lanka (within the first 3 days of fever) that aimed to a) identify clinical and basic laboratory test parameters to differentiate dengue from non-dengue fever, b) evaluate the comparative efficacy of loop-mediated isothermal amplification (LAMP) for dengue diagnosis (vs. NS1 antigen test and RT-qPCR) and c) identify early associations that are predictive of plasma leakage or severe dengue. The basic laboratory tests considered here included hematological parameters, serum biochemistry and inflammatory markers. RESULTS: Only 70% of clinically suspected patients were confirmed as having dengue by either the NS1 antigen test or RT-qPCR. On a Bayesian latent class model which assumes no "gold standard", LAMP performed equally or better than RT-qPCR and NS1 antigen test respectively. When confirmed dengue patients were compared with others, the earlier group had significantly lower lymphocyte counts and higher aspartate aminotransferase levels (AST) within the first 3 days of fever. Confirmed dengue patients with plasma leakage had a lower mean age and a higher median baseline AST level compared to those without plasma leakage (p < 0.05). CONCLUSION: Clinical suspicion overestimates the true number of dengue patients. RT-LAMP is a potentially useful low-cost diagnostic tool for dengue diagnosis. Confirmed dengue patients had significantly higher AST levels and lower lymphocyte counts in early disease compared to others. In confirmed dengue patients, younger age and a higher AST level in early infection were associated with subsequent plasma leakage.


Assuntos
Aspartato Aminotransferases/sangue , Técnicas de Amplificação de Ácido Nucleico/métodos , Dengue Grave/diagnóstico , Dengue Grave/etiologia , Adulto , Teorema de Bayes , Biomarcadores/sangue , Estudos de Coortes , Dengue/diagnóstico , Vírus da Dengue/genética , Feminino , Febre/virologia , Humanos , Testes Imunológicos , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Medição de Risco , Sensibilidade e Especificidade , Dengue Grave/sangue , Sri Lanka
15.
Chem Commun (Camb) ; 55(72): 10677-10680, 2019 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-31424057

RESUMO

Beacon-mediated Exponential Amplification Reaction (BEAR) enables isothermal, exponential signal amplification. BEAR uses only a single enzyme and a single primer. Detection of 0.2 amol of a mitochondrial DNA with a point mutation in less than an hour demonstrates an application of the BEAR technique for nucleic acid research.


Assuntos
Técnicas Biossensoriais , DNA Mitocondrial/análise , DNA Polimerase Dirigida por DNA/química , Técnicas de Amplificação de Ácido Nucleico , DNA Mitocondrial/genética , DNA Polimerase Dirigida por DNA/metabolismo , Mutação Puntual
16.
Arch Virol ; 164(11): 2683-2690, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31428915

RESUMO

Current antiviral therapies against hepatitis B virus (HBV) infections, such as treatment with nucleos(t)ide analogs (NAs) and interferon alpha, can significantly lower HBV DNA titers, eventually to undetectable levels. However, it is still difficult to completely eliminate the stable template of HBV, the covalently closed circular DNA (cccDNA), and this contributes to viral rebound when treatment is discontinued. HBV pregenomic RNA (pgRNA), which was recently found to be present in the enveloped mature HBV viral particle in blood, is tentatively regarded, with still accumulating clinical evidence, as a novel bona fide virological marker reflecting the amount and status of cccDNA when serum HBV DNA becomes undetectable. HBV pgRNA and DNA share almost identical sequences, and it is therefore difficult to differentiate pgRNA from viral DNA using normal PCR methods. To exclude interference from viral DNA, methods for measuring pgRNA usually require a selective DNA degradation step, which is complicated and time-consuming and also compromises the accuracy of detection. In this study, we developed a simplified quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay with improved accuracy achieved by probing the polyA tail of pgRNA. Using clinical serum samples, we observed that not all patients share the same 3' sequence, suggesting slight differences between HBV strains in the way they end transcription. We then designed and evaluated a universal primer and probe set for distinguishing HBV pgRNA from HBV DNA. Our results demonstrated that a one-step qRT-PCR assay could selectively amplify HBV pgRNA from a mixture of HBV RNA and DNA, which is valuable for clinical applications.


Assuntos
Vírus da Hepatite B/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , DNA Viral/análise , DNA Viral/genética , Hepatite B/virologia , Humanos , RNA Viral/análise , RNA Viral/genética
17.
Vet Parasitol ; 273: 17-23, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31442888

RESUMO

The protozoan parasite Tritrichomonas foetus may cause severe diarrhea in cats all over the world. In order to evaluate the methodology in coprological molecular diagnosis of feline tritrichomonosis, we compared previously published ("old") and newly developed ("novel") loop-mediated isothermal amplification (LAMP) (targeted to the T. foetus ß-tubulin and the elf1α 1 gene, respectively) as well as an old conventional and an old and novel real-time PCR (all targeted to overlapping regions of T. foetus rDNA) assays regarding their diagnostic sensitivities and specificities. Here, the novel real-time PCR yielded the best methodical performance in that a sensitivity with a detection limit of <0.1 trophozoites (corresponding to ca.<0.13 trophozoites per mg feces) and a maximal specificity for diagnosis of Tritrichomonas spp. was achieved. The other test systems exhibited either an approximately 10-times lower sensitivity (<1 trophozoite corresponding to ca.<1.3 trophozoites per mg feces) (conventional PCR and both LAMP assays) or a lower specificity (old real-time PCR). Conversely, the diagnostic performance assessed with clinical fecal samples from cats demonstrated identical sensitivities (8 of 20 samples tested were positive) for the novel PCR and both LAMP assays. Diagnostic sensitivities were significantly higher than those found for the old real-time (5 positive samples) and conventional PCR (6 positive samples), respectively. Accordingly, our data suggested the novel PCR and both LAMP assays to be well suited molecular tools for direct (i.e. without including an in vitro cultivation step) coprological diagnosis of tritrichomonosis in cats. Interestingly, relative high (novel LAMP, 7 positive samples) to at least moderate (old LAMP, 6 positive samples and 1 sample with equivocal score) diagnostic sensitivities were also achieved by testing clinical samples upon simple visual inspection of colorimetric changes during the LAMP amplification reactions. Accordingly, both LAMP assays may serve as practical molecular tools to perform epidemiological studies on feline (and bovine as well as porcine) tritrichomonosis under simple laboratory conditions.


Assuntos
Doenças do Gato/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/veterinária , Reação em Cadeia da Polimerase/veterinária , Infecções Protozoárias em Animais/diagnóstico , Tritrichomonas foetus , Animais , Doenças do Gato/parasitologia , Gatos , Fezes/parasitologia , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/normas , Reação em Cadeia da Polimerase/normas , Sensibilidade e Especificidade
18.
Anal Bioanal Chem ; 411(23): 6039-6047, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31304564

RESUMO

Circulating tumor DNA (ctDNA) is a tumor-derived fragmented DNA in the bloodstream that is not associated with cells. It has been greatly focused in the recent decade because of its potential clinical utility for liquid biopsies. Development of ctDNA analytical techniques with high sensitivity and cost-efficiency will undoubtedly promote the clinical spread of ctDNA testing. In this paper, we propose a novel flow cytometry-based ctDNA sensing strategy which combines enzyme-free amplification and magnetic separation. The target DNA is capable of triggering a hybridization chain reaction, producing a fluorescent long linear assembly of DNA, which can be further captured by magnetic beads to present fluorescent signals using flow cytometry. In comparison with some conventional methods, our strategy has the advantages of easy operation and cost-efficiency, and thereby shows a promising application in clinical diagnosis. Graphical abstract.


Assuntos
DNA Tumoral Circulante/sangue , Citometria de Fluxo/métodos , Imãs/química , Técnicas de Amplificação de Ácido Nucleico/métodos , DNA Tumoral Circulante/análise , Humanos , Limite de Detecção , Biópsia Líquida/métodos , Hibridização de Ácido Nucleico/métodos
19.
Analyst ; 144(16): 4917-4924, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31313769

RESUMO

MicroRNAs (miRNAs) are attractive candidates for biomarkers for early cancer diagnosis, and play vital roles in physiological and pathological processes. In this work, we developed a colorimetric and fluorescent dual-mode sensor for miRNA detection based on the optical properties of gold nanoparticles (AuNPs) and the duplex-specific nuclease (DSN)-assisted signal amplification technique. In brief, FAM labelled hairpin probes (HPs) were immobilized on AuNPs, and fluorescence was efficiently quenched by the vicinity of the fluorophores to the AuNPs surface. In the presence of target miRNAs, the HPs could specifically hybridize with miRNAs and the DNA strand in the DNA/RNA heteroduplexes could be subsequently hydrolyzed by DSN. As a result, numbers of fluorophores were released into the solution, resulting in obvious fluorescence signal recovery. Meanwhile, the target miRNAs were able to participate in other hybridization reactions. With the DSN-assisted signal amplification technique, lots of gold nanoparticles were produced with short-chain DNA on their surface, which could aggregate in salt solution and result in a colorimetric detection. The proposed dual-mode strategy offers a sensitive, accurate and selective detection method for miRNAs. One reason is that the stem of the HPs was elaborately designed to avoid hydrolyzation by DSN under optimal conditions, which ensures a relatively low background and high sensitivity. The other is that the dual-mode strategy is more beneficial for enhancing the accuracy and reproducibility of the measurements. Moreover, the unique selective-cutting ability and single-base mismatch differentiation capability of the DSN also give rise to a satisfactory selectivity. This demonstrated that the developed method could quantitatively detect miR-21 down to 50 pM with a linear calibration range from 50 pM to 1 nM, and the analytical assay of target miRNAs in cell lysate samples revealed its great potential for application in biomedical research and clinical diagnostics.


Assuntos
Corantes/química , Endonucleases/química , Ouro/química , Nanopartículas Metálicas/química , MicroRNAs/análise , Técnicas Biossensoriais/métodos , Linhagem Celular , Colorimetria , DNA/química , Humanos , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/métodos , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/química , Hibridização de Ácido Nucleico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Fluorescência
20.
Anal Chim Acta ; 1078: 24-31, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31358225

RESUMO

A novel electrochemical DNA biosensor was developed and MON89788 of soybean transgenic gene sequence was detected based on a strategy of rolling circle amplification (RCA) and gold nanoparticle cube (AuNPC)-labeled multiple probes. First, the mercapto-modified capture DNA was immobilized on the surface of the Fe3O4@Au magnetic nanoparticles via an Au-S bond, and the capture DNA was opened and complementarily hybridized with the target DNA to form a double-stranded DNA. In the 10 × reaction buffer, Exonuclease III (ExoIII) specifically recognized and sheared the double-stranded DNA to release the target DNA, which led to the next round of reaction. Afterward, AuNP cube-loaded ssDNA (AuNPC/DNA) was added with the rolling circle reaction with the help of Phi29 DNA polymerase and T4 ligase. Finally, [Ru(NH3)6]3+ was attracted directly by the anionic phosphate of ssDNA via electrostatic interaction. The determination was carried out by using chronocoulometry (CC), and the CC signal was recorded. The mass amount of DNA strands extended infinitely on the AuNPs cube and numerous [Ru(NH3)6]3+ were absorbed, thus the detected signal was highly amplified. The corresponding CC signal showed a good linear relationship with the logarithm of the target DNA concentration in the range of 1 × 10-16 to 1 × 10-7 mol L-1, with a detection limit of 4.5 × 10-17 mol L-1. Specific gene sequence of MON89788 in soybean samples was determined, and the recoveries ranged from 97.3% to 102.0%. This sensor is one of the most sensitive sensors for genetic sequence assessment at present. Moreover, it demonstrates good selectivity, stability, and reproducibility.


Assuntos
Técnicas Biossensoriais/métodos , DNA de Plantas/análise , Técnicas Eletroquímicas/métodos , Plantas Geneticamente Modificadas/genética , Soja/genética , Sequência de Bases , Calibragem , Sondas de DNA/química , Sondas de DNA/genética , DNA de Plantas/química , DNA de Plantas/genética , Exodesoxirribonucleases/química , Ouro/química , Limite de Detecção , Nanopartículas de Magnetita/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Reprodutibilidade dos Testes , Compostos de Rutênio/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA