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1.
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
2.
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
3.
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
4.
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
7.
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
8.
Parasit Vectors ; 12(1): 360, 2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31340841

RESUMO

BACKGROUND: Perkinsosis, a disease caused by the protist Perkinsus, is responsible for mass mortalities of many molluscan species worldwide. The rapid, early and accurate detection of Perkinsus infection is necessary to react to outbreaks, and manage disease transmission. Current methods for diagnosis of Perkinsus spp. are time-consuming or require professional equipment and experienced personnel, rendering them unsuitable for field application. Recombinase polymerase amplification (RPA) assay is a highly sensitive and selective isothermal amplification technique that operates at temperatures of 37-42 °C, requires minimal sample preparation, and is capable of amplifying as low as 1-10 target DNA copies in less than 20 minutes. METHODS: We report a novel RPA assay that amplifies the internal transcriber spacer (ITS) region of P. beihaiensis, which, followed by rapid detection of amplicons using a lateral flow (LF) strip, enables easy visualization of results by the naked eye. RESULTS: The LF-RPA assay successfully amplified P. beihaiensis DNA using a set of primers of 20-25 bp in length. After incubation at 37 °C for 25 min, results were read within 5 min by the naked eye on a lateral flow strip. Our LF-RPA assay was comparably sensitive to qPCR assay, and capable of detecting as few as 26 copies of P. beihaiensis DNA. Cross-amplification occurred with other two Perkinsus species, P. olseni and P. chesapeaki, but not with other potential pathogen taxa in culture environments. We compared the performance of LF-RPA, conventional PCR and qPCR assays on 60 oyster samples. While LF-RPA assay results were 86.2% as sensitive, 77.4% as specific, and generally in agreement with those of conventional PCR results, they were more (93.3%) sensitive, (86.7%) specific, and agreed better with qPCR assay results. Future research should focus on developing simple DNA extraction methods that do not require professional laboratories and complicated extraction procedures, to facilitate application of this LF-RPA assay in the field. CONCLUSIONS: Our LF-RPA assay provides a rapid and efficient method for detecting species of Perkinsus. This novel assay has potential to be used in field applications.


Assuntos
Alveolados/isolamento & purificação , Crassostrea/parasitologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Infecções Protozoárias em Animais/diagnóstico , Alveolados/genética , Animais , Primers do DNA/genética , DNA Intergênico/genética , Visualização de Dados , Eletroforese em Gel de Ágar , Reação em Cadeia da Polimerase/métodos , Infecções por Protozoários , Recombinases/genética , Sensibilidade e Especificidade
9.
Adv Clin Chem ; 91: 31-98, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31331490

RESUMO

Specific nucleic acid detection in vitro or in vivo has become increasingly important in the discovery of genetic diseases, diagnosing pathogen infection and monitoring disease treatment. One challenge, however, is that the amount of target nucleic acid in specimens is limited. Furthermore, direct sensing methods are also unable to provide sufficient sensitivity and specificity. Fortunately, due to advances in nanotechnology and nanomaterials, nanotechnology-based bioassays have emerged as powerful and promising approaches providing ultra-high sensitivity and specificity in nucleic acid detection. This chapter presents an overview of strategies used in the development and integration of nanotechnology for nucleic acid detection, including optical and electrical detection methods, and nucleic acid assistant recycling amplification strategies. Recent 5 years representative examples are reviewed to demonstrate the proof-of-concept with promising applications for DNA/RNA detection and the underlying mechanism for detection of DNA/RNA with the higher sensitivity and selectivity. Furthermore, a brief discussion of common unresolved issues and future trends in this field is provided both from fundamental and practical point of view.


Assuntos
DNA/química , RNA/química , Técnicas Biossensoriais/métodos , Humanos , Nanotecnologia , Técnicas de Amplificação de Ácido Nucleico/métodos
10.
Virol J ; 16(1): 86, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31262315

RESUMO

BACKGROUND: Human adenoviruses are a common group of viruses that cause acute infectious diseases. Human adenovirus (HAdV) 3 and HAdV 7 cause major outbreaks of severe pneumonia. A reliable and practical method for HAdV typing in clinical laboratories is lacking. A simple, rapid and accurate molecular typing method for HAdV may facilitate clinical diagnosis and epidemiological control. METHODS: We developed and evaluated duplex real-time recombinase-aided amplification (RAA) assays incorporating competitive internal controls for detection of HAdV 3 and HAdV 7, respectively. The assays were performed in a one-step in a single tube reaction at 39° for 20 min. RESULTS: The analytical sensitivities of the duplex RAA assays for HAdV 3 and HAdV 7 were 5.0 and 14.8 copies per reaction, respectively (at 95% probability by probit regression analysis). No cross-reaction was observed with other types of HAdV or other common respiratory viruses. The duplex RAA assays were used to detect 152 previously-defined HAdV-positive samples. These results agreed with those obtained using a published triplex quantitative real-time PCR protocol. CONCLUSIONS: We provide the first report of internally-controlled duplex RAA assays for the detection of HAdV 3 and HAdV 7. These assays effectively reduce the rate of false negative results and may be valuable for detection of HAdV 3 and HAdV 7 in clinical laboratories, especially in resource-poor settings.


Assuntos
Adenovírus Humanos/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/normas , Recombinases/genética , Adenovírus Humanos/genética , Humanos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Infecções Respiratórias/epidemiologia , Sensibilidade e Especificidade , Sorogrupo , Temperatura Ambiente
11.
Arch Virol ; 164(10): 2581-2584, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31359148

RESUMO

Maize chlorotic mottle virus (MCMV), an important quarantine virus, causes lethal necrosis in maize when coinfected with a potyvirid, which is seriously threatening the production of maize worldwide. In this study, recombinase polymerase amplification (RPA), a novel isothermal DNA amplification and detection technique, was developed to detect MCMV in maize crops. A pair of specific primers was designed based on the conserved sequences of the MCMV coat protein region. The RT-RPA assay was carried out as an isothermal reaction at 38 °C that was complete within 30 min, and no cross-reactivity was detected with other viruses infecting maize in China. The limit of detection of the RT-RPA assay was tenfold lower than that of ordinary RT-PCR. Moreover, this method was successfully applied to test field-collected samples. The newly developed RT-RPA assay offers a reliable, sensitive and efficient method for rapid detection of MCMV in maize in equipment-limited diagnostic laboratories and on-site facilities.


Assuntos
Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Doenças das Plantas/virologia , Tombusviridae/isolamento & purificação , Proteínas do Capsídeo/genética , China , Primers do DNA/genética , Sensibilidade e Especificidade , Temperatura Ambiente , Fatores de Tempo , Tombusviridae/classificação , Tombusviridae/genética
12.
Food Chem ; 298: 125031, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31260975

RESUMO

Hyperbranched rolling circle amplification (HRCA) with a padlock probe (PLP) targeting the α-amanitin (α-AMA) gene, as a screening tool for the universal identification of lethal amanitas, was established in this study. With the isothermal HRCA assay, all of the lethal Amanita species tested from Phalloideae (10) were positive, while the non-Phalloideae Amanita species (15) and three amanitin-containing Lepiota and Galerina species were negative. Furthermore, the PLP based on α-AMA sequences from lethal Amanita species was effective for Amanita α-AMA, but not Amanita ß-AMA or non-Amanita α-AMA. HRCA sensitivity was 100-fold higher than conventional PCR with a detection limit of 100 copies (recombinant plasmid containing α-AMA), and 0.2% lethal amanitas could be detected in dry mushroom blends. The HRCA method presented provided a rapid, specific, sensitive and low-cost identification tool for lethal amanitas.


Assuntos
Amanitinas/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Agaricales/genética , Alfa-Amanitina/genética , Amanita/genética , Limite de Detecção , Intoxicação Alimentar por Cogumelos/genética , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade
13.
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
14.
BMC Infect Dis ; 19(1): 542, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221109

RESUMO

BACKGROUND: Tuberculosis rapid molecular assays, including GeneXpert MTB/RIF® and Loopamp MTBC Detection Kit®, are highly sensitive and specific. Such performance does not automatically translate in improved disease control and highly depends on their use, local epidemiology and the diagnostic algorithms they're implemented within. We evaluate the performance of both assays and assess their impact on additional cases notification when implemented within WHO recommended tuberculosis diagnostic algorithms in Madagascar. METHODS: Five hundred forty eight presumptive pulmonary tuberculosis patients were prospectively recruited between November 2013 and December 2014 in Antananarivo, Madagascar, a high TB incidence sub-Saharan African urban setting. Both molecular assays were evaluated as first line or add-on testing following negative smear microscopy. Based on locally defined assay performance characteristics we measure the impact of both assays and WHO-recommended diagnostic algorithms on additional tuberculosis case notifications. RESULTS: High sensitivity and specificity was confirmed for both GeneXpert MTB/RIF® (86.6% (95% CI 81.1-90.7%) and 97.4% (95% CI 94.9-98.8%)) and Loopamp MTBC Detection Kit® (84.6% (95% CI 78.9-89.0%) and 98.4% (95% CI 96.2-99.4%)). Implementation of GeneXpert MTB/RIF® and Loopamp MTBC Detection Kit® increased tuberculosis diagnostic algorithms sensitivity from 73.6% (95% CI 67.1-79.3%) up to 88.1% (95% CI 82.8-91.9%). This increase was highest when molecular assays were used as add-on testing following negative smear microscopy. As add-on testing, GeneXpert MTB/RIF® and Loopamp MTBC Detection Kit® respectively improved case detection by 23.8 and 21.2% (p < 0.05). CONCLUSION: Including GeneXpert MTB/RIF® or Loopamp MTBC Detection Kit® molecular assays for TB detection on sputum samples from presumptive TB cases can significantly increase case notification in TB diagnostic centers. The TB case detection rate is further increased when those tests are use as second-line follow-on testing following negative smear microscopy results. A country wide scale-up and digital integration of molecular-based TB diagnosis assays shows promises for TB control in Madagascar.


Assuntos
Mycobacterium tuberculosis/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Tuberculose Pulmonar/diagnóstico , Adulto , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Feminino , Humanos , Madagáscar , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/isolamento & purificação , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade , Escarro/microbiologia , Tuberculose Pulmonar/microbiologia
15.
Biochimie ; 163: 137-141, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31181235

RESUMO

RNA amplification has extensive applications on biochemistry and its related fields. Here, we present an isothermal strategy named rolling circle reverse transcription-mediated RNA amplification (RCRT-MRA) to amplify small RNAs with accurate length and sequence. The target RNA and complementary DNA were circularized to serve as amplicons replicated via the rolling circle mechanism. The transcription product consisting of tandemly repeated RNA units, was monomerized by site-specific cleavage to generate amplified RNA with authentic length and sequence. T4 DNA ligase was chosen to circularize RNA template for its high efficiency and low cost. SuperScript IV reverse transcriptase was found to be able to catalyze the RCRT reaction on the circular RNA template, and the reaction efficiency was enhanced by adding the nicking enzyme, Nb.BbvCI to the RCRT system. E. Coli RNA polymerase, instead of the commonly used T7 RNA polymerase, was applied to synthesize long-strand RNA product for its high universality and processivity. Under the optimized conditions, small RNAs can be precisely amplified by 105∼6 folds. The fidelity of the established method was demonstrated by the accordance of the sequencing result and the initial RNA sequences. Free from expensive thermal cycler (necessary for RT-PCR-based amplification), precise replication of the initial RNA and high fidelity will enable the established strategy to be applied in RNA-seq, mRNA profiling, microarray analysis and RNA-based SELEX as well.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA/metabolismo , Transcrição Reversa , DNA Circular/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo
16.
BMC Infect Dis ; 19(1): 553, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31234780

RESUMO

BACKGROUND: Kazakhstan remains a high-burden TB prevalence country with a concomitent high-burden of multi-drug resistant tuberculosis. For this reason, we performed an in depth genetic diversity and population structure characterization of Mycobacterium tuberculosis complex (MTC) genetic diversity in Kazakhstan with both patient and community benefit. METHODS: A convenience sample of 700 MTC DNA cultures extracts from 630 tuberculosis patients recruited from 12 out of 14 regions in Kazakhstan, between 2010 and 2015, was independently studied by high-throughput hybridization-based methods, TB-SPRINT (59-Plex, n = 700), TB-SNPID (50-Plex, n = 543). DNA from 391 clinical isolates was successfully typed by two methods. To resolve the population structure of drug-resistant clades in more detail two complementary assays were run on the L2 isolates: an IS6110-NTF insertion site typing assay and a SigE SNP polymorphism assay. RESULTS: Strains belonged to L2/Beijing and L4/Euro-American sublineages; L2/Beijing prevalence totaled almost 80%. 50% of all samples were resistant to RIF and to INH., Subtyping showed that: (1) all L2/Beijing were "modern" Beijing and (2) most of these belonged to the previously described 94-32 sublineage (Central Asian/Russian), (3) at least two populations of the Central Asian/Russian sublineages are circulating in Kazakhstan, with different evolutionary dynamics. CONCLUSIONS: For the first time, the global genetic diversity and population structure of M. tuberculosis genotypes circulating in Kazakhstan was obtained and compared to previous local studies. Results suggest a region-specific spread of a very limited number of L2/Beijing clonal complexes in Kazakhstan many strongly associated with an MDR phenotype.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Adulto , Idoso , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Evolução Molecular , Variação Genética , Genótipo , Humanos , Cazaquistão/epidemiologia , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico/métodos , Fenótipo , Prevalência , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Adulto Jovem
17.
Chem Commun (Camb) ; 55(58): 8386-8389, 2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-31231732

RESUMO

A highly sensitive triple-amplification assay for the detection of microRNA (miRNA) let-7a is reported in this work. The assay relies on the formation of magnetic two-dimensional DNA/Fe3O4 nanosheet networks initiated by the target miRNA-associated hybridization chain reaction. The Fe3O4 nanosheets in the DNA/Fe3O4 networks display peroxidase-like catalytic activity towards a colorimetric reaction, thereby producing a highly sensitive signal for the quantification of let-7a. Under optimal conditions, the assay achieved a detection limit of 13 aM at a signal-to-noise ratio of 3 and a linear calibration plot between 0.05 fM and 12 nM. Successful attempts were made in the quantification of let-7a in serum samples.


Assuntos
Materiais Biomiméticos/química , DNA/química , Óxido Ferroso-Férrico/química , MicroRNAs/sangue , Humanos , Limite de Detecção , Fenômenos Magnéticos , Nanoestruturas/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Peroxidase/química
18.
World J Microbiol Biotechnol ; 35(6): 95, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31187258

RESUMO

Recombinase polymerase amplification (RPA) is an isothermal amplification technique. Because of its short detection cycle and high specificity, it has been applied in various fields. However, the design of probe on the efficiency of RPA is not well understood and the effect of sequence mismatches of oligonucleotides on the performance of RPA is rarely discussed. In this study, we found that different primers with the same probe have a slight effect on the efficiency of fluorescent RPA, and different probes with the same amplified region have a great influence on the efficiency of fluorescent RPA. We summarized the design rules of probes suitable for fluorescent RPA by analyzing the experimental data. The rule is that the best distance between fluorescent groups in the probe is 1-2 bases, and the G content should be reduced as far as possible. In addition, we verified this rule by designing a series of probes. Furthermore, we found the base mismatches of the probe had a significant effect on RPA, which can lead to false positives and can change the amplification efficiency. However, 1-3 mismatches covering the center of the primer sequence only affect the amplification efficiency of RPA, not its specificity. And with an increase in the number of primer mismatches, the efficiency of RPA will decrease accordingly. This study suggests that the efficiency of fluorescent RPA is closely related to the probe. We recommend that when designing a fluorescent probe, one must consider the presence of closely related non-targets and specific bases.


Assuntos
Pareamento Incorreto de Bases , Técnicas de Amplificação de Ácido Nucleico/métodos , Recombinases , Bactérias , Primers do DNA/genética , Sensibilidade e Especificidade
19.
Anal Bioanal Chem ; 411(17): 3789-3800, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31161320

RESUMO

MicroRNAs (miRNAs) in a blood sample are usually measured by quantitative reverse transcription PCR (qRT-PCR), microarray, and next-generation sequencing (NGS) which requires time-consuming pre-treatment, manual operation, and a stand-alone instrument. To overcome these disadvantages, miRNA testing has been developed using the automated analyzers routinely used in clinical laboratories. An isothermal DNA amplification reaction was adapted to a fully automated immunoassay analyzer that conducts extraction, amplification, and detection processes at 37 °C in 44 min. In a reaction vessel, a pre-designed single-stranded signal DNA was amplified in the presence of miRNA, using DNA templates, DNA polymerase, and nicking endonuclease. Then, the amplified signal DNA was hybridized by one DNA probe attached to a magnetic particle and another DNA probe labeled with acridinium ester. After the chemiluminescence reaction, luminescence intensity was automatically measured. The automated assays of cancer-related miRNAs were implemented on the analyzer with throughput of 66 tests per hour. In the assays with one-step amplification, three miRNAs (miR-21-5p, miR-18a-5p, and miR-500a-3p) at concentrations lower than 100 fM were automatically detected and the cross reactivity for miR-21-5p with fifteen similar miRNAs was not higher than 0.02%. In the assay with two-step amplification, detection sensitivity and amplification rate for miR-21-5p were 3 fM and 103-fold, respectively. The coefficient of variations (CVs) in the measurement at the target concentrations from 5 fM to 1000 pM were less than 8%. Furthermore, we also achieved automated nucleic acid detection in human serum. The proposed fully automated miRNA assays showed high sensitivity, low cross reactivity, and reproducibility suitable for clinical use. Graphical abstract.


Assuntos
Imunoensaio/métodos , MicroRNAs/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Automação , Humanos , Luminescência
20.
Anal Chim Acta ; 1076: 138-143, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31203958

RESUMO

The detection and quantification of microRNA (miRNA) plays essential roles in clinical and biomedical research. Yet, it is of major challenge to sense miRNA with high degree of selectivity and sensitivity due to its unique characteristics of short length, similarity of sequence among family members and low abundance. Here, with the design of a new hairpin/DNA ring ternary probe, we describe the development of a rolling circle amplification (RCA) method for sensitively and selectively sensing miRNA from cancer cells. The target miRNA binds the hairpin/DNA ring probes through toehold-mediated strand displacement (TSD) to form the ternary structures, in which the bound miRNA and DNA ring are respectively used as the primer and template to realize RCA, leading to the generation of many repeated metal ion-dependent DNAzyme sequences. The fluorescently quenched hairpin signal probes can be cyclically cleaved by these DNAzyme sequences with co-existence of the corresponding metal ions in buffer to show drastically enhanced fluorescence recovery for highly sensitive sensing of miRNA in the range between 10 fM and 10 nM with a detection limit of 1.51 fM. Besides, owing to the high base variation discrimination ability of TSD, selective detection of the target miRNA among the corresponding family members can be achieved by this method. Moreover, such a method can also be employed to differentiate miRNA expression variations in cancer cells for screening potential therapeutic drugs.


Assuntos
Sondas de DNA/química , DNA/química , MicroRNAs/análise , Sequência de Bases , Linhagem Celular Tumoral , DNA/genética , Sondas de DNA/genética , DNA Catalítico/química , Fluorescência , Corantes Fluorescentes/química , Humanos , Sequências Repetidas Invertidas , Limite de Detecção , MicroRNAs/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Espectrometria de Fluorescência/métodos
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