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Sleep deprivation (SD) is prevalent throughout the world, which has negative effects on cognitive abilities, and causing mood alterations. 8-O-acetyl shanzhiside methylester (8-OaS), a chief component in Lamiophlomis rotata (L. rotata) Kudo, possesses potent neuroprotective properties and analgesic effects. Here, we evaluated the alleviative effects of 8-OaS on memory impairment and anxiety in mice subjected to SD (for 72-h). Our results demonstrated that 8-OaS (0.2, 2, 20 mg/kg) administration dose-dependently ameliorated behavioral abnormalities in SD mice, accompanied with restored synaptic plasticity and reduced shrinkage and loss of hippocampal neurons. 8-OaS reduced the inflammatory response and oxidative stress injury in hippocampus caused by SD, which may be related to inhibition of NLRP3 inflammasome-mediated inflammatory process and activation of the Nrf2/HO-1 pathway. SD also led to increases in the expressions of TLR-4/MyD88, active NF-κB, pro-IL-1ß, TNFα and MDA, as well as a decrease in the level of SOD in mice hippocampus, which were reversed by 8-OaS administration. Moreover, our molecular docking analyses showed that 8-OaS also has good affinity for NLRP3 and Nrf2 signaling pathways. These results suggested that 8-OaS could be used as a novel herbal medicine for the treatment of sleep loss and for use as a structural base for developing new drugs.
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Proteína 3 que Contém Domínio de Pirina da Família NLR , Privação do Sono , Animais , Camundongos , Ansiedade/tratamento farmacológico , Ansiedade/etiologia , Cognição , Simulação de Acoplamento Molecular , Fator 2 Relacionado a NF-E2/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Privação do Sono/complicações , Privação do Sono/tratamento farmacológicoRESUMO
Nucleic acid detection is widely used in pathogen screening and detection due to its high sensitivity and specificity. With the increase of detection requirements and the development of amplification technology, nucleic acid detection methods are gradually developing towards simple, fast and low-cost. Quantitative polymerase chain reaction (qPCR), as the "gold standard" for nucleic acid detection, relies on expensive equipment and professional operators, which is not suitable for rapid on-site detection of pathogens. The visual detection method without relying on excitation light source or complex equipment can present the detection results in a more intuitive and portable way after combining with rapid and efficient amplification technology, which has the potential of point-of-care testing (POCT). This paper focuses on the reported application of amplification technology and CRISPR/Cas technology in visual detection and compares their advantages and disadvantages, so as to provide reference for POCT strategy based on pathogen nucleic acid.
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Técnicas de Amplificação de Ácido Nucleico , Ácidos Nucleicos , Técnicas de Amplificação de Ácido Nucleico/métodos , Tecnologia , Sistemas CRISPR-CasRESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused an ongoing pandemic of new coronavirus pneumonia (corona virus disease 2019, COVID-19). The virus has a long incubation period and strong infectivity, which poses a major threat to global health and safety. Detection of SARS-CoV-2 nucleic acid lies at the center of rapid detection of COVID-19, which is instrumental for mitigation of the ongoing pandemic. As of August 17, 2020, The National Medical Products Administration in China has approved 15 new coronavirus nucleic acid detection kits, 10 kits of which are based on reverse transcription-real-time quantitative PCR (RT-qPCR) technology. The remaining kits use five molecular diagnostic technologies different from RT-qPCR. This article reviews the principles, reaction time, advantages and disadvantages of above 15 detection kits, in order to provide references for rapid screening, diagnosis, prevention and control of COVID-19 and similar infectious diseases.
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Betacoronavirus , Infecções por Coronavirus , Pandemias , Pneumonia Viral , COVID-19 , Teste para COVID-19 , China , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Humanos , Patologia Molecular , Pneumonia Viral/diagnóstico , SARS-CoV-2RESUMO
Rapid detection of pathogenic microorganisms is key to the epidemiologic identification, prevention and control of disease in the field of public health. PCR-based pathogen detection methods have been widely used because they overcome the time-consuming issues that traditional culture-based methods required including the limited window required by immunological detection. However, the requirement on precision temperature-controlled thermal cyclers severely limits their use in resource-limited areas. The detection methods of pathogenic microorganisms based on isothermal amplification of nucleic acids are free of dependence on high-precision temperature control equipment, but requirements for nucleic acids extraction, amplification and detection must be defined. In recent years, a number of alternative methods for pathogenic microorganism detection have been developed by combining microfluidic technology with nucleic acid isothermal amplification technology. By designing the chip structures, optimizing the injection modes, and utilizing multiple detection and quantitative methods, the integration of pathogen nucleic acid extraction, amplification and detection is achieved. The method provides advantages of less instrument dependence, decreased operator requirements, smaller sample size, and higher automation which are suitable for the rapid detection of pathogenic microorganisms in various environments. In this review, we summarize several microfluidic detection methods based on nucleic acid isothermal amplification for pathogens including amplification principles, injection methods and detection methods. These methods provide more capability for the rapid screening of pathogenic microorganisms which enhances the management of infectious diseases in the field of public health.
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Bactérias/isolamento & purificação , Dispositivos Lab-On-A-Chip , Técnicas de Amplificação de Ácido Nucleico , Ácidos Nucleicos/análise , Vírus/isolamento & purificação , Bactérias/patogenicidade , Reação em Cadeia da Polimerase , Vírus/patogenicidadeRESUMO
Loop-mediated isothermal amplification (LAMP) has been widely applied in nucleic acid diagnostics due to its high sensitivity and specificity, high speed and low requirement of equipment. In order to fully leverage these merits, achieve high efficiency and reliability in diagnostics, and expand the applicable fields while keeping low reagent cost, multiplex LAMP technology has been extensively explored in recent years. Common methods for LAMP products detection are mostly based on the double-stranded DNA amplicons or byproducts from the polymerization reaction, so they can only identify the occurrence of amplification reaction but not the origins or specificity of the products. To achieve specific LAMP products detection, researchers developed various multiplex methods by improving the conventional LAMP technology or coupling LAMP with other assays. However, the interference and/or the different amplification efficiencies among different primer sets often lead to biased amplification and thus limited multiplexing level. We here defined these methods as narrow-sensed multiplex LAMP. The research on miniaturized amplification technology which is booming in recent years has given rise to the novel general-sensed multiplex LAMP technology that breaks this limitation by its capability to perform highly parallel and miniaturized simplex reactions in independent compartments. Methods of this type have additional benefits such as lower reagent cost, higher level of automation, lower risk of cross-contamination and better suitability for on-site detection of multiple targets. In this review, we summarize the recent research progress in multiplex LAMP technology from the following aspects: the principle and design of narrow-sensed LAMP and its amplification optimization, the general-sensed LAMP, and the various applications of all multiplex LAMP technologies in diagnostics.
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Técnicas de Amplificação de Ácido Nucleico/métodos , HumanosRESUMO
Pharmacometabonomics, as an emerging branch of system biology, has been increasingly used in personalized medicine and showed broad prospects. By means of metabonomics, the complicated and detailed metabolic profile of the patient is described, thus providing more detailed description of the disease phenotype. With this understanding, response of different individuals to the drugs are predicted or evaluated through inherent genetic information of the individual combined with the environmental factors. As a result, appropriate drugs and dosage are chosen, which greatly promotes the realization of the individualized therapy goals. This article describes the emerging field of pharmacometabonomics, and the research results of personalized medicine based on the pharmacometabonomics in recent years are reviewed in detail.
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Metabolômica , Farmacogenética , Medicina de Precisão/métodos , Humanos , MetabolomaRESUMO
The quaternion cubature Kalman filter (QCKF) algorithm has emerged as a prominent nonlinear filter algorithm and has found extensive applications in the field of GNSS/SINS integrated attitude determination and positioning system (GNSS/SINS-IADPS) data processing for unmanned aerial vehicles (UAV). However, on one hand, the QCKF algorithm is predicated on the assumption that the random model of filter algorithm, which follows a white Gaussian noise distribution. The noise in actual GNSS/SINS-IADPS is not the white Gaussian noise but rather a ubiquitous non-Gaussian noise. On the other hand, the use of quaternions as state variables is bound by normalization constraints. When applied directly in nonlinear non-Gaussian system without considering normalization constraints, the QCKF algorithm may result in a mismatch phenomenon in the filtering random model, potentially resulting in a decline in estimation accuracy. To address this issue, we propose a novel Gaussian sum quaternion constrained cubature Kalman filter (GSQCCKF) algorithm. This algorithm refines the random model of the QCKF by approximating non-Gaussian noise with a Gaussian mixture model. Meanwhile, to account for quaternion normalization in attitude determination, a two-step projection method is employed to constrain the quaternion, which consequently enhances the filtering estimation accuracy. Simulation and experimental analyses demonstrate that the proposed GSQCCKF algorithm significantly improves accuracy and adaptability in GNSS/SINS-IADPS data processing under non-Gaussian noise conditions for Unmanned Aerial Vehicles (UAVs).
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We have developed a dual color fluorescence quantitative detection method for specific single-stranded DNA with molecular beacons (MBs) and nucleic acid dye SYBR Green I by synchronous scanning fluorescence spectrometry. It is demonstrated by a reverse-transcription oligonucleotide sequence (target DNA, 33 bases) of RNA fragment of human immunodeficiency virus (HIV) as a model system. In the absence of target DNA, the MBs are in the stem-closed state, the fluorescence of 5-carboxy-X-rhodamine (ROX) is quenched by black hole quencher-2 (BHQ-2), and the interaction between SYBR Green I and the MBs is very weak. At this time the fluorescence signals of ROX and SYBR Green I are all very weak. In the presence of target DNA, MBs hybridize with target DNA and form a double-strand structure, the fluorophore ROX is separated from the quencher BHQ-2, and the fluorescence of ROX recovers. At the same time, SYBR Green I binds to hybridized dsDNA, whose fluorescence intensity is significantly enhanced. Thus, dual color fluorescence quantitative detection for the target DNA can be realized by synchronous scanning fluorescence spectrometry. In this strategy, the fluorescence signal of SYBR Green I is far larger than that of ROX, so the quantitative analysis of target DNA with the fluorescence intensity of SYBR Green I can significantly improve the detection sensitivity. In addition, the false-positive signals of MBs do not affect the fluorescence signals of nucleic acid dye SYBR Green I. Thereby, in the analysis of complex samples, quantitative analysis of target DNA with SYBR Green I can avoid the false-positive signals of MBs and improve the detection accuracy.
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DNA de Cadeia Simples/análise , DNA de Cadeia Simples/química , Corantes Fluorescentes/química , Sondas de Oligonucleotídeos/química , Compostos Orgânicos/química , Espectrometria de Fluorescência/métodos , Sequência de Bases , Benzotiazóis , Soluções Tampão , Cor , DNA de Cadeia Simples/genética , Diaminas , Estudos de Viabilidade , Concentração de Íons de Hidrogênio , Limite de Detecção , Modelos Moleculares , Conformação de Ácido Nucleico , Sondas de Oligonucleotídeos/genética , Concentração Osmolar , Polimorfismo de Nucleotídeo Único , Quinolinas , Temperatura , Fatores de TempoRESUMO
OBJECTIVE: To establish a high-sensitivity, high-specificity and low-cost hydrogel chip platform for the clinical screening of Y chromosome microdeletions. METHODS: Site-specific extended primers with a common sequence at the 5' end were used for hybridizing with the target. The Cy5-dUTP was incorporated into the products by primer extension, and the products were labeled with fluorescence. Then the extended products were added to the chip for hybridizing with acrylamide-modified common probes immobilized on the chip. After removal of the free Cy5-dUTP by electrophoresis, the signals were obtained by fluorescence scanning. And the detecting conditions of this method were optimized. RESULTS: SY254 of 9 samples was successfully detected with the hydrogel chip. The results showed that 3 were normal and the other 6 with microdeletions (1 female sample as a negative control), which coincided with the results of conventional multiplex PCR-electrophoresis. CONCLUSION: The hydrogel chip platform we established has provided a new technique for the detection of Y chromosome microdeletions, and is beneficial to the diagnosis and treatment of male infertility.
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Análise de Sequência com Séries de Oligonucleotídeos/métodos , Transtornos do Cromossomo Sexual no Desenvolvimento Sexual/genética , Carbocianinas , Deleção Cromossômica , Cromossomos Humanos Y/genética , Nucleotídeos de Desoxiuracil , Humanos , Hidrogéis , Infertilidade Masculina , Masculino , Aberrações dos Cromossomos Sexuais , Transtornos do Cromossomo Sexual no Desenvolvimento Sexual/diagnósticoRESUMO
Paris spp. are important medicinal plant and main raw material for many Chinese patent medicines, but viral diseases have became serious problems in cultivation of this group of important medicinal plants in China. In this study, eight viruses were identified in the diseased plants of Paris yunnanensis by high-throughput sequencing (HTS) and RT-PCR. These viruses include three novel viruses (two potyviruses and one nepovirus), Hippeastrum chlorotic ringspot virus (HCRV), Lychnis mottle virus (LycMoV), Paris mosaic necrosis virus (PMNV), Paris virus 1 and pepper mild mottle virus. The three new viruses were tentatively named Paris potyvirus 3 (ParPV-3), Paris potyvirus 4 (ParPV-4), Paris nepovirus 1 (ParNV-1) and their complete genome sequences were determined. Sequence analyses showed ParPV-3 and ParPV-4 shared the highest amino acid (aa) sequence identities of 54.3% to each other and 53.0-57.8% to other known potyviruses. ParNV-1 had aa sequence identities of 28.8-63.7% at protease-polymerase (Pro-Pol) with other nepoviruses. Phylogenetic analyses further support that the three viruses are new members of their corresponding genera. Analyses of the partial sequences of HCRV and LycMoV infecting P. yunnanensis revealed they diverged from existing isolates by aa sequence identities of 97.1% at glycoprotein precursor of HCRV and 93.3% at polyprotein of LycMoV. These two viruses are reported for the first time in Paris spp. A total of 123 field samples collected from P. yunnanensis in four counties of Yunnan, Southwest China were tested by RT-PCR for detecting each of the eight viruses. Results showed that nearly half of the samples were positive for at least one of the eight viruses. Two potyviruses, ParPV-3 (26.8%) and PMNV (24.4%), were predominant and widely distributed in the fields, while other viruses occurred in low rates and/or had limited distribution. This study insights into the virome infecting P. yunnanensis and provides valuable information for diagnosis and control of viral diseases in P. yunnanensis.
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BACKGROUND: Breast neuroendocrine carcinoma (NEC) is a rare malignancy with unclear treatment options and prognoses. This study aimed to construct a high-quality model to predict overall survival (OS) and breast cancer-specific survival (BCSS) and help clinicians choose appropriate breast NEC treatments. PATIENTS AND METHODS: A total of 378 patients with breast NEC and 349,736 patients with breast invasive ductal carcinoma (IDC) were enrolled in the Surveillance, Epidemiology, and End Results (SEER) database between 2010 and 2018. Propensity score matching (PSM) was performed to balance the clinical baseline. Prognostic factors determined by multivariate Cox analysis were included in the nomogram. C-index and calibration curves were used to verify the performance of the nomogram. RESULTS: Nomograms were constructed for the breast NEC and breast IDC groups after PSM. The C-index of the nomograms ranged from 0.834 to 0.880 in the internal validation and 0.818-0.876 in the external validation, indicating that the nomogram had good discrimination. The risk stratification system showed that patients with breast NEC had worse prognoses than those with breast IDC in the low-risk and intermediate-risk groups but had a similar prognosis that those in the high-risk group. Moreover, patients with breast NEC may have a better prognosis when undergoing surgery plus chemotherapy than when undergoing surgery alone or chemotherapy alone. CONCLUSIONS: We established nomograms with a risk stratification system to predict OS and BCSS in patients with breast NEC. This model could help clinicians evaluate prognosis and provide individualized treatment recommendations for patients with breast NEC.
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Intractable functional constipation (IFC) is the most severe form of constipation, but its etiology has long been unknown. We hypothesized that IFC is caused by refractory infection by a pathogenic bacterium. Here, we isolated from patients with IFC a Shigella species - peristaltic contraction-inhibiting bacterium (PIB) - that significantly inhibited peristaltic contraction of the colon by production of docosapentenoic acid (DPA). PIB colonized mice for at least 6 months. Oral administration of PIB was sufficient to induce constipation, which was reversed by PIB-specific phages. A mutated PIB with reduced DPA was incapable of inhibiting colonic function and inducing constipation, suggesting that DPA produced by PIB was the key mediator of the genesis of constipation. PIBs were detected in stools of 56% (38 of 68) of the IFC patients, but not in those of non-IFC or healthy individuals (0 of 180). DPA levels in stools were elevated in 44.12% (30 of 68) of the IFC patients but none of the healthy volunteers (0 of 97). Our results suggest that Shigella sp. PIB may be the critical causative pathogen for IFC, and detection of fecal PIB plus DPA may be a reliable method for IFC diagnosis and classification.
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Motilidade Gastrointestinal , Shigella , Animais , Colo , Constipação Intestinal/diagnóstico , Constipação Intestinal/genética , Fezes , Humanos , Camundongos , Shigella/genéticaRESUMO
Our previous study revealed that spaceflight induced biological changes in human cervical carcinoma Caski cells. Here, we report that 48A9 cells, which were subcloned from Caski cells, experienced significant growth suppression and exhibited low tumorigenic ability after spaceflight. To further understand the potential mechanism at the transcriptional level, we compared gene expression between 48A9 cells and ground control Caski cells with suppression subtractive hybridization (SSH) and reverse Northern blotting methods, and analyzed the relative gene network and molecular functions with the Ingenuity Pathways Analysis (IPA) program. We found 5 genes, SUB1, SGEF, MALAT-1, MYL6, and MT-CO2, to be up-regulated and identified 3 new cDNAs, termed B4, B5, and C4, in 48A9 cells. In addition, we also identified the two most significant gene networks to indicate the function of these genes using the IPA program. To our knowledge, our results show for the first time that spaceflight can reduce the growth of tumor cells, and we also provide a new model for oncogenesis study.
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Perfilação da Expressão Gênica , Voo Espacial , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Northern Blotting/métodos , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Biblioteca Gênica , Redes Reguladoras de Genes , Humanos , Hibridização de Ácido Nucleico/métodos , Regulação para CimaRESUMO
PURPOSE: The aim of this study was to establish individualized nomograms to predict survival outcomes in older female patients with stage IV breast cancer who did or did not undergo local surgery, and to determine which patients could benefit from surgery. METHODS: A total of 3,129 female patients with stage IV breast cancer aged ≥70 years between 2010 and 2015 were included in the Surveillance, Epidemiology, and End Results program. Multivariate Cox regression analysis was used to identify risk factors for overall survival (OS) and breast cancer-specific survival (BCSS). Survival analysis was performed using the Kaplan-Meier plot and log-rank test. Nomograms and risk stratification models were constructed. RESULTS: Patients who underwent surgery had better OS (HR = 0.751, 95% CI [0.668-0.843], P < 0.001) and BCSS (HR = 0.713, 95% CI [0.627-0.810], P < 0.001) than patients who did not undergo surgery. Patients with human epidermal growth factor receptor 2-positive, lung or liver metastases may not benefit from surgery. In the stratification model, low-risk patients benefited from surgery (OS, HR = 0.688, 95% CI [0.568-0.833], P < 0.001; BCSS, HR = 0.632, 95% CI [0.509-0.784], P < 0.001), while patients in the high-risk group had similar outcomes (OS, HR = 0.920, 95% CI [0.709-1.193], P = 0.509; BCSS, HR = 0.953, 95% CI [0.713-1.275], P = 0.737). CONCLUSION: Older female patients with stage IV breast cancer who underwent surgery had better OS and BCSS than those who did not in each specific subgroup. Patients in low- or intermediate-risk group benefit from surgery while those in the high-risk group do not.
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Neoplasias da Mama , Idoso , Mama/patologia , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Feminino , Humanos , Estimativa de Kaplan-Meier , Estadiamento de Neoplasias , Nomogramas , Prognóstico , Programa de SEERRESUMO
MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs that play an important role in the control of developmental process of different cells by negative regulation of protein-coding gene expression. Analyzing miRNA expression in tissues or cells can supply valuable information for investigating the biological function of these molecules. Recently, researchers had proposed a number of approaches for analyzing the differences of miRNA expression among different physiological or pathological conditions, and found that aberrant expression of miRNA was related to cancers, neurological disorders and heart diseases, etc. This review focuses on newly developed strategies for miRNA quantification, and elucidates in detail the probe-hybridization based methods including Northern blotting, microarray, gold nanoparticle labelling, and splinted ligation with radioactive labels. The amplification-based methods including quantitative PCR, rolling cycle amplification, invader assay, and the next generation sequencing methods were also discussed. The advantages and disadvantages of these methods were compared.
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Técnicas Genéticas , MicroRNAs/análise , MicroRNAs/genética , Animais , Humanos , MicroRNAs/metabolismoRESUMO
Colorectal cancer (CRC) is one of the most malignant cancers in gastrointestinal tract. In China, there are increasing rates of morbidity and mortality for CRC. As the mortality is closely related to the stage of disease at time of diagnosis, early diagnosis of CRC is important. However, current techniques used for clinical diagnosis have limitations which made them difficult to achieve the early diagnosis. The detection of RNAs in stool is a newly developed noninvasive technique for early diagnosis of CRC at molecular levels. Compared with the techniques including colonoscopy, fecal occult-blood test and stool DNA-based mutation detection, diagnosis based on the detection of RNAs in stool has the advantages of low-cost and high sensitivity. Moreover, stool RNA-based techniques are able to analyze multiplexed gene expression simultaneously and monitor cancer progression dynamically. This paper introduced the feasibility of stool RNA analysis, and systematically reviewed the genes associated with stool RNA analysis, methods of RNA isolation from stool sample, and techniques for gene expression analysis in stool RNA. Finally, further applications of stool RNA-based techniques for early diagnosis of CRC were briefly discussed.
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Neoplasias Colorretais/diagnóstico , Detecção Precoce de Câncer , Fezes/química , RNA/análise , Neoplasias Colorretais/genética , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da PolimeraseRESUMO
To avoid sequencing error resulting from use of apyrase in conventional 4- enzyme pyrosequencing system, a non-apyrase 3-enzyme pyrosequencing system with a better performance of quantitative analysis was established. The method is to immobilize biotinylated DNA template, ATP sulfurylase and luciferase on streptavidin-coated magnetic beads for pyrosequencing. After pyrosequencing, ATP produced from the pyrosequencing reaction and excess dNTPs were removed by magnetic separation technique; another dNTP was then dispensed for sequencing reaction, and the components interfering with the next circle of pyrosequencing reaction were removed by the same way, achieving the circular sequencing. This new system can accurately measure base sequences of a target DNA template, and also can quantitatively determine the relative ratio of two alleles. The allele ratios in two SNPs (rs1042917 and rs4818219) having a higher heterozygote rate on chromosome 21 were successfully detected for 16 normal samples and 8 clinical samples from Down's syndrome patients. The results can accurately demonstrate whether or not the target sample has equal copies of chromosome 21 from mother and father. This paper established a non-apyrase 3-enzyme pyrosequencing method, which owns a good perform-ance of quantitative analysis. The method is especially suitable to allelic quantification of an SNP, enabling the rapid diagnosis of Down's syndrome by analyzing allele ratio of SNPs on chromosome 21.
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DNA/genética , Síndrome de Down/diagnóstico , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodos , Alelos , Apirase/metabolismo , Cromossomos Humanos Par 21/genética , DNA/química , DNA/metabolismo , Síndrome de Down/genética , Frequência do Gene , Humanos , Luciferases/metabolismo , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Sulfato Adenililtransferase/metabolismoRESUMO
OBJECTIVE: To establish a method to detect Down's syndrome through quantitative pyrosequencing of the heterozygous single nucleotide polymorphisms (SNPs) on the chromosome 21. METHODS: An improved allele-specific-amplification was used to screen heterozygous SNPs on the chromosome 21 from 84 normal samples. Pyrosequencing was used to quantitatively determine the ratio between the two alleles of a heterozygote, and the diagnosis of Down's syndrome was thus carried out based on the ratio. RESULTS: By genotyping 84 genomic DNA samples from normal Chinese population, 6 SNPs with a relatively high level of heterozygosity were screened out. Heterozygote coverage of 92.9% was achieved by using a panel of 6 SNPs on the chromosome 21. Ten clinical samples from Down's syndrome patients were quantitatively determined by pyrosequencing, and 9 samples were accurately diagnosed by comparing the ratio of the two alleles. The pyrosequencing results showed that the ratio of the two alleles were 2:1 or 1:2 for the Down's syndrome patients. CONCLUSION: The method has the advantage of a low cost, simple process, and time-saving operation and could be potentially applicable to the rapid diagnosis of Down's syndrome.
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Cromossomos Humanos Par 21 , Síndrome de Down/diagnóstico , Polimorfismo de Nucleotídeo Único/genética , Diagnóstico Pré-Natal/métodos , Alelos , Povo Asiático/genética , Clonagem Molecular , DNA/análise , Síndrome de Down/genética , Feminino , Testes Genéticos , Humanos , Cariotipagem/métodos , Gravidez , Diagnóstico Pré-Natal/economiaRESUMO
A new method of DNA adapter ligation-mediated allele-specific amplification (ALM-ASA) was developed for typing multiple single nucleotide polymorphisms (SNPs) on the platform of microchip electrophoresis. Using seven SNPs of 794C>T, 1274C>T, 2143T>C, 2766T>del, 3298G>A, 5200G>A, and 5277C>T in the interleukin 1B (IL1B) gene as a target object, a long DNA fragment containing the seven SNPs of interest was pre-amplified to enhance the specificity. The pre-amplified DNA fragment was digested by a restriction endonuclease to form sticky ends; and then the adapter was ligated to either end of the digested fragment. Using the adapter-ligated fragments as templates, a 7-plex allele-specific amplification was performed by 7 allele-specific primers and a universal primer in one tube. The allele-specific products amplified were separated by chip electrophoresis and the types of SNPs were easily discriminated by the product sizes. The seven SNPs in IL1B gene in 48 healthy Chinese were successfully typed by microchip electrophoresis and the results coincided with those by PCR-restriction fragment length polymorphism and sequencing method. The method established was accurate and can be used to type multiple SNPs simultaneously. In combination with microchip electrophoresis for readout, ALM-ASA assay can be used for fast SNP detection with a small amount of sample. Using self-prepared gel matrix and reused chips for analysis, the SNP can be typed at an ultra low cost.
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Povo Asiático/genética , DNA/análise , Eletroforese em Microchip/métodos , Marcadores Genéticos/genética , Alelos , Humanos , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNARESUMO
Non-small cell lung cancer (NSCLC) is one of the most fatal types of cancer with significant mortality and morbidity worldwide. MicroRNAs (miRs) have been confirmed to have positive functions in NSCLC. In the present study, we try to explore the role of miR-758 in proliferation, migration, invasion, and apoptosis of NSCLC cells by regulating high-mobility group box (HMGB) 3 (HMGB3.) NSCLC and adjacent tissues were collected. Reverse transcription quantitative PCR (RT-qPCR) was employed to detect expression of miR-758 and HMGB3 in NSCLC and adjacent tissues, in BEAS-2B cells and NSCLC cell lines. The targetted relationship between miR-758 and HMGB3 was identified by dual luciferase reporter gene assay. The effects of miR-758 on proliferation, migration, invasion, cell cycle, and apoptosis of A549 cells. MiR-758 expression was lower in NSCLC tissues, which was opposite to HMGB3 expression. The results also demonstrated that miR-758 can target HMGB3. The cells transfected with miR-758 mimic had decreased HMGB3 expression, proliferation, migration, and invasion, with more arrested cells in G1 phase and increased apoptosis. Our results supported that the overexpression of miR-758 inhibits proliferation, migration, and invasion, and promotes apoptosis of NSCLC cells by negative regulating HMGB2. The present study may provide a novel target for NSCLC treatment.